Advances in Virus Research, Volume 71

CHAPTER

1 The Pathogenesis of Poliomyelitis: What We Don’t Know Neal Nathanson

Contents

I. Introduction II. Sequential Steps in the Spread of Infection A. Questions unanswered: Cellular sites of replication B. Questions unanswered: Neural invasion from the blood III. Provocation Poliomyelitis A. Questions unanswered: The mechanism of the provoking effect B. Questions unanswered: Neural spread IV. PVR, Tropism, and the Localization of Lesions A. Questions unanswered: Receptor expression is necessary but not sufficient B. Questions unanswered: Localization within the CNS C. Questions unanswered: How poliovirus kills cells V. Host Innate and Immune Response to Infection A. Questions unanswered: The acquired immune response VI. Immune Defenses and Viral Clearance: Mechanisms of Vaccine-Induced Protection A. Primary infections B. Secondary infection in immune hosts C. Poliovirus serotypes

3 3 4 6 6 7 10 11 11 13 15 15 16 17 17 18 20

Departments of Microbiology and Neurology, School of Medicine, University of Pennsylvania, Philadelphia 19104 Advances in Virus Research, Volume 71 ISSN 0065-3527, DOI: 10.1016/S0065-3527(08)00001-8

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2008 Elsevier Inc. All rights reserved.

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VII. Animal Models of Human Poliomyelitis A. Questions unanswered: Determinants of primate susceptibility B. Questions unanswered: The mechanism of rodent adaptation C. Questions unanswered: PVR mice D. Questions unanswered: The tropism enigma VIII. Virulence of Polioviruses A. Questions unanswered: Mechanisms of neurovirulence B. Questions unanswered: Viremia and virulence C. Questions unanswered: Epidemiological properties of polioviruses IX. How Does Poliovirus Persist? A. Questions unanswered: Overt persistence of poliovirus B. The post-polio syndrome and covert persistence of poliovirus X. Eradication A. Questions unanswered: Why is it so difficult to complete the global eradication of wild polioviruses? XI. Vaccine-Derived Polioviruses and the Eradication Endgame A. Questions unanswered: What strategy should be followed if wild polioviruses are eradicated? XII. Reprise Acknowledgments References

Abstract

21 22 23 25 26 26 32 32 33 34 35 35 36

37 38 39 41 42 42

Poliomyelitis has long served as a model for studies of viral pathogenesis, but there remain many important gaps in our understanding of this disease. It is the intent of this review to highlight these residual but important questions, in light of a possible future moratorium on research with polioviruses. Salient questions include: (1) What cells in the gastrointestinal tract are initially infected and act as the source of excreted virus? (2) What is the receptor used by mouse-adapted strains of poliovirus and how can some polioviruses use both mouse and primate receptors? (3) What determines species differences in susceptibility of the gastrointestinal tract to polioviruses? Why cannot PVR transgenic mice be infected by the natural enteric route? (4) Why are neuroadapted polioviruses unable to infect nonneural cells? (5) What is the role of postentry blocks in replication as determinants of neurovirulence? (6) What route(s) does poliovirus take to enter the central nervous system and how does it cross the blood–brain barrier? (7) Why does

Pathogenesis of Poliomyelitis

3

poliovirus preferentially attack lower motor neurons in contrast to many other neuronal types within the central nervous system? (8) Does cellular immunity play any role in recovery from acute infection or in vaccine-induced protection? (9) In which cells does poliovirus persist in patients with g-globulin deficiencies? (10) Is there any evidence that poliovirus genomes can persist in immunocompetent hosts? (11) Why has type 2 poliovirus been eradicated while types 1 and 3 have not? (12) Can transmission of vaccine-derived polioviruses be prevented with inactivated poliovirus vaccine? (13) What is the best strategy to control and eliminate vaccine-derived polioviruses?

I. INTRODUCTION Poliomyelitis has served as a model for studies of viral pathogenesis, beginning soon after the virus was isolated by Landsteiner and Popper (Flexner, 1931; Landsteiner and Popper, 1909). Since 1990, the development of transgenic rodent models and the ready manipulation of the viral genome have provided new approaches to polio pathogenesis. Beginning about 2000, it was perceived that the world was on the verge of global eradication of poliovirus, leading to proposals for a permanent moratorium on poliovirus research (Dowdle et al., 2006; Thompson et al., 2006). Thus, it is timely to review our knowledge of pathogenesis while opportunity may still exist to conduct research with wild polioviruses. Surprisingly, there remain many important gaps in our understanding of the pathogenesis of poliomyelitis, and it is the intent of this review to highlight these residual but important questions (Minor, 2004). The interested reader may wish to consult several excellent recent discussions by leading researchers (Mueller et al., 2005; Ohka and Nomoto, 2001; Racaniello, 2006).

II. SEQUENTIAL STEPS IN THE SPREAD OF INFECTION The general outlines of the sequential events in an infection with poliovirus (Fig. 1) were delineated by Bodian, Sabin, and others in the 1950s (Bodian, 1955a; Sabin, 1956). Polio is an enterovirus that is ingested and travels through the gastrointestinal tract where it can initiate infection at several sites, including the tonsils and Peyer’s patches of the small intestine. From the initial sites of entry, the virus travels to the draining lymph nodes where it replicates further and spreads via the efferent lymphatic vessels and thoracic duct to enter the bloodstream. In some instances, virus spreads to the central nervous system (CNS) and rarely (estimated 1 case

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Ingested virus 1-Alimentary pathway of virus spread Tonsils

Peyer’s patches Virus in throat secretions

2-Lymphatic pathways

3-Blood vascular pathway

4-Neural pathways

Deep cervical lymph nodes

Virus in feces

Mesenteric lymph nodes

Parenteral injection

Invasion of blood stream and spread to susceptible “Target organs”

Brown fat Central nervous system Lymphatic structures (Including tonsils and Peyer’s patches) Nerve fiber spread within CNS and centrifugally to sensory ganglia

FIGURE 1 The sequential events in poliovirus infection in chimpanzees. Boxes indicate primary sites of implantation while the secondary and tertiary sites of multiplication are underlined [from Bodian D. (1955a). Emerging concept of poliomyelitis infection. Science 122, 105-108. Reprinted with permission from AAAS].

per 100–200 infections) leads to permanent flaccid paralysis. Infected humans shed poliovirus in the pharyngeal secretions and feces, usually for 2–8 weeks, implying that virus replicates in the intestine. Presumably, virus contaminates the hands of the infected person and is transmitted by hand to hand contact to the next person in the chain of infection. There is little published information on the relative importance in transmission of pharyngeal versus fecal shedding that could be relevant to the impact of polio vaccines on herd immunity (see below). Poliovirus can spread in a susceptible host by either of two different routes, viremia or the neural pathway. The dominant route of spread depends upon the strain of virus. All polioviruses are neurotropic and most primary isolates are also pantropic (enterotropic and viremogenic) as shown in Fig. 2. A few neuroadapted strains behaved as obligatory neurotropes, defined by experimental data of the kind shown in Table I (Nathanson and Bodian, 1961).

A. Questions unanswered: Cellular sites of replication Many significant details about the sequential steps in infection remain unanswered. There is considerable evidence that poliovirus invades the gastrointestinal tract by transcytosis via microfold (M) cells that express the poliovirus receptor (PVR) on their surface. Ex vivo fragments of human Peyer’s patches have been reported to endocytose poliovirus

Viremia Neutralizing antibody

2

2

1

1

5

Antibody (titer per 0.25 ml serum)

Viremia (log10 TCD50 per ml)

Pathogenesis of Poliomyelitis

Negative

0

0 0

2

4 6 8 Days after infection

10

12

FIGURE 2 Viremia in experimental poliomyelitis. In this model, cynomolgus monkeys were infected by intramuscular injection of Mahoney virus, a virulent strain of wild type 1 poliovirus [after Nathanson and Bodian (1961), with permission]. TABLE I Different tropism of two strains of poliovirus, the neuroadapted MV (mixed virus) and the viremogenic Mahoney virus Neuroadapted MV strain

Paralysis Site of initial paralysis Injected leg Other Incubation to paralysis (median)

Viremogenic Mahoney strain

Control

Nerve block

Control

Nerve block

25/26

0/11

19/19

18/20

24 1 5 days

– – –

3 16 7 days

5 13 7.5 days

After injection into the gastronemius muscle, the MV strain spreads only by the neural route, causes initial paralysis in the injected limb, and is impeded by a neural block, while the viremogenic Mahoney strain spreads by viremia, does not cause localized initial paralysis, and is not impeded by nerve block. Neural block was achieved just prior to virus injection by freezing the innervating sciatic nerve with dry ice proximal to the site of virus injection [after Nathanson and Bodian (1961), with permission].

and similar observations have been made in a human monolayer culture containing M-like cells (Iwasaki et al., 2002; Ouzilou et al., 2002; Siciski et al., 1990). Following transcytosis, one or more types of lymphoreticular cells are infected at the sites of primary infection. Although freshly isolated human blood monocytes are not very susceptible to infection (Eberle et al., 1995; Freistadt and Eberle, 1996; Freistadt et al., 1993), when cultured under conditions that promote differentiation into

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macrophages or dendritic cells, they become very susceptible to poliovirus (Wahid et al., 2005). If the ex vivo data can be extrapolated to in vivo infection, then it is likely that monocyte-derived cells play an important role in the replication and spread of pantropic polioviruses. The cellular source of virus shed in the enteric tract has not been demonstrated nor is it clear how the virus finds its way into the intestinal lumen. The gut epithelium over Peyer’s patches does express the PVR, making it a candidate for poliovirus shedding (Sabin, 1956), but PVR expression per se is not necessarily sufficient for poliovirus replication (Zhang and Racaniello, 1997).

B. Questions unanswered: Neural invasion from the blood There has been a longstanding debate how poliovirus spreads to the nervous system from its initial replication in the gastrointestinal tract. Bodian (1955a) hypothesized that blood-borne virus crossed the blood– brain barrier, while Sabin (1956) suggested that virus invaded the peripheral nervous system, perhaps via autonomic ganglia, and spread centripetally via the neural route. Table I shows that after intramuscular injection of monkeys, the pantropic Mahoney virus caused initial paralysis almost at random, while the neurotropic MV strain caused initial paralysis in the injected limb. These data document the ability of polioviruses to utilize two different routes to the CNS, but they do not distinguish between the two hypotheses regarding neural invasion following viremia.

III. PROVOCATION POLIOMYELITIS It has long been known that if a poliovirus infection was preceded by some type of tissue trauma, such as an intramuscular injection, there was an increased risk of paralysis that localized to the traumatized limb (Hill and Knowelden, 1950). This phenomenon has been called ‘‘provocation poliomyelitis.’’ Two striking examples of this phenomenon were the Cutter incident (Nathanson and Langmuir, 1963) and vaccine-associated provocation poliomyelitis in Romania (Strebel et al., 1995). In the Cutter incident, children were immunized intramuscularly with inactivated poliovirus vaccine (IPV) that was contaminated with residual infectious type 1 poliovirus, resulting in 51 cases of paralytic poliomyelitis (Table II). There was a striking correlation between the site of IPV injection and the limb that was initially and/or most severely paralyzed. In Romania, children who were fed oral poliovirus vaccine (OPV) exhibited a much higher risk of vaccine-associated paralysis (onset of paralysis within 4–30 days after OPV) if they had received an intramuscular injection (antibiotic or other medication) after OPV (and within 30 days before onset of

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TABLE II Localization of paralysis in 50 vaccineesa who received an intramuscular injection (buttock or deltoid) of poliovirus-contaminated lots of Cutter ‘‘inactivated’’ poliovirus vaccine

Injected limb Uninoculated limbs

Number of limbs

Percent with initial paralysis

Percent with final paralysis

50 150

82.0% 10.7%

91.3% 57.2%

a

Excludes one paralyzed case where data were incomplete. After Nathanson and Langmuir (1963), with permission.

paralysis). Absent records of the sites of injection it was not possible to look for a correlation with sites of paralysis. The localization characteristic of provocation poliomyelitis may provide insight about the mechanism whereby the virus invades the CNS. Provocation poliomyelitis has been reproduced experimentally in nonhuman primates (Bodian, 1954b, 1955b) and PVR transgenic mice (Gromeier and Wimmer, 1998), and these models have been used to dissect the mechanism of localization. There are at least two possible explanations for localization: the peripheral trauma may render the blood–brain barrier more permeable in the region of the CNS that innervates the traumatized site, or circulating virus may penetrate the peripheral site of trauma, cross the local neuromuscular junction, and travel along the neural route to the CNS.

A. Questions unanswered: The mechanism of the provoking effect In favor of direct invasion across the blood–brain barrier is evidence that peripheral injury can increase permeability of the corresponding area of the CNS. Trueta (1955) showed that intramuscular injections of inflammatory materials could actually cause gross hemorrhages in the spinal cord of mice or rabbits. Bodian (1954b, 1955b) conducted a large study in monkeys injected intravascularly with the Mahoney strain of type 1 poliovirus (the same strain used in Table I and involved in the Cutter incident shown in Table II). Some of these animals also received a ‘‘provoking’’ injection in the right calf with one of several materials (gelatin, penicillin, and DPT) and developed initial right leg paralysis at a much higher rate (23%) than intravascularly infected but uninjected control animals (4%). Relevant to the question in hand, two salient observations were made. First, the monkeys with localized ‘‘provoked’’

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paralysis had an average incubation period of 9.5 days, similar to intravascularly infected uninjected animals. (In contrast, a different group of monkeys that were infected by intramuscular virus injection and developed initial paralysis in the injected leg had an average incubation period of 12 days to paralysis, suggesting that the neural route was somewhat slower than the intravascular route to the CNS.) Second, when monkeys were infected by the intravascular route, given a right peroneal nerve block and a provoking injection in the right leg, there was no reduction in the frequency of localized paralysis. A similar result was seen by Nathanson and Bodian (1962), although they used a somewhat different experimental design (Table III). Monkeys were infected by intramuscular injection of Mahoney virus but there was no evidence of localization of paralysis to the injected leg, and nerve block provided no reduction in the high paralytic rate, suggesting that the virus spread to the CNS by viremia only (Table I). An increase in the frequency of localized paralysis was seen following passive administration of g-globulin containing poliovirus antibodies but neural block did not alter this enhanced localization (Table III). These experiments suggested that—in this model—the enhanced frequency of localized paralysis was due to the penetration of the blood–spinal cord barrier corresponding to the innervation site of the injected muscle. A different outcome was observed by Gromeier and Wimmer (1998) who conducted an incisive set of experiments in PVR transgenic mice. As summarized in Table IV, mice were infected intravenously with the TABLE III Nerve block does not alter the frequency of localized paralysis in monkeys infected by left calf injection of the Mahoney strain of type 1 poliovirus Initial left leg paralysis among all paralyzed animals Degree of g-globulin protection

Paralytic rate

Freeze of left sciatic nerve

No nerve freeze (sham controls)

Total protection Partial protection No protection Controls (no g-globulin)

0/19 11/27 7/7 44/46

3/5 1/4 7/22

4/6 1/3 5/22

11/31

10/31

Totals

Animals were pretreated with varying doses of g-globulin containing poliovirus-neutralizing antibodies; half the animals were given a block of the left sciatic nerve; and all of the animals were then infected by virus injection into the left calf. For this analysis, animals were grouped according to the degree of protection provided by g-globulin. The group that was partially protected with g-globulin showed an increased rate of localized paralysis (7/11) compared to animals that were not protected or controls not receiving g-globulin (2/7 and 12/44, respectively). However, there was no evidence that neural block reduced the frequency of localized paralysis because the frequency of localized paralysis was very similar in animals with and without nerve block (11/31 and 10/31, respectively) [after Nathanson and Bodian (1962), with permission].

TABLE IV In PVR transgenic mice, the provoking effect is mediated by intramuscular replication of poliovirus followed by spread to the CNS by the neural route Number of mice with initial paralysis Group

I II III IV

Left sciatic nerve block

Provoking injections left gastrocnemius muscle

Left lower extremity

Right lower extremity

Both lower extremities

Upper extremities

No Yes No (sham operation) Yes

No No Yes

1 0 13

0 2 0

13 13 3

2 1 0

Yes

0

1

15

0

Four groups of 16 mice each were infected by intravascular injection with the Mahoney strain of type 1 poliovirus. The provoking effect was produced in two groups by the repeated injection in the left gastrocnemius muscle of phosphate-buffered saline at four intervals from 2 to 48 h after infection. Left sciatic nerve transection was performed 7 days before injection [after Gromeier and Wimmer (1998), with permission].

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Mahoney strain of poliovirus, and a provoking effect was induced by repeated injections in the left gastrocnemius (calf) muscle. To determine whether the localization of paralysis was associated with neural spread, the left sciatic nerve was severed in some groups. The data indicate that provocation did induce localized initial paralysis that was mediated by neural spread since it was abrogated by sciatic transection. Detailed analysis showed that virus localized and replicated to high titer in the traumatized muscle, leading to neural invasion of the corresponding segment of the spinal cord. How should these disparate findings be reconciled? I believe that the data reflect true differences in the animal models used by different investigators, an issue discussed further in a subsequent section of this chapter. I would suggest several tentative conclusions. (1) Polioviruses vary in their relative ability to cause viremia or spread by the neural route. (2) Permissive animal models also vary in their ability to support viremia versus neural spread. (3) It is likely that polioviruses can invade the CNS either directly across the blood–brain barrier or via the neural route, depending on the tropism of the particular viral strain and the relative permissiveness of different cell types in the host under study. Clearly, these hypotheses and the molecular mechanisms that explain them remain to be tested. Relevant to this problem is the question how poliovirus might cross the blood–brain barrier? Using mice, Yang et al. (1997) injected 35Slabeled poliovirus intravenously and followed the accumulation of label in various tissues for 7.5 h. Label accumulated in the CNS to a level >100-fold that in the contained blood, but not in other tissues where the amount of label could be accounted for by the tissue-specific blood volume. Furthermore, 35S accumulation was similar in normal and PVR transgenic mice. No attempt was made to clear contained blood by perfusion prior to evaluation of the various tissues. It is unclear whether these data reflect virus binding to the vascular bed or transmission across the blood–brain barrier.

B. Questions unanswered: Neural spread Wild poliovirus is usually pantropic and viremogenic but serial passage in neural tissues can select for viral strains that are obligatory neurotropes. A typical example is the MV strain of type 2 poliovirus, which is illustrated in Table I; this virus was derived by serial intracerebral passages in monkeys (Flexner, 1931). The mechanism that distinguishes pantropic from obligatory neurotropic polioviruses has never been elucidated, although it is tempting to speculate that it relates to subtle differences in viral entry into neural and other cells.

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11

Based on experiments in monkeys and mice using neural blockade (Howe and Bodian, 1942; Ohka et al., 1998), it was estimated that the neurotropic MV strain of poliovirus travels within axons at approximately 2.4 mm/h (
6 cm/day). This is similar to the speed of fast axonal transport (
10 cm/day). More recently, Mueller et al. (2002) have begun to elucidate the molecular mechanism of axonal transport of poliovirus. They showed that the cytoplasmic tail of CD155 (the PVR) binds to Tctex-1, a protein that is a component of the dynein complex, which is responsible for fast axonal transport along microtubules (Ohka et al., 2004). Based on these biochemical findings, they suggested a tentative scheme whereby poliovirions undergo CD155-mediated endocytosis, CD155 associates with dynein, and the virus-containing endosomes are then transported from the periphery to the neuronal cell body where the virus is uncoated and replicates (Fig. 3). This scheme is similar to the mechanism that is responsible for axonal transport of alphaherpesviruses such as pseudorabies virus, although pseudorabies viruses are transported as cytoplasmic nucleocapsids rather than intact virions within endosomes (Feierbach et al., 2007; Smith et al., 2007).

IV. PVR, TROPISM, AND THE LOCALIZATION OF LESIONS It has long been known that poliovirus can infect chimpanzees and old world monkeys, but not new world monkeys and other nonprimate mammalian species (McLaren et al., 1959). Circumstantial evidence indicated that this correlated with the species-specific expression of a PVR (Crowell, 1983; Holland et al., 1959; Medrano and Green, 1973). The receptor was identified in elegant experiments in which a DNA fragment encoding the PVR was transfected into mouse cells, which were rendered susceptible to infection (Mendelsohn et al., 1986). The PVR gene was subsequently isolated, cloned, sequenced, and shown to be a member of the immunoglobulin superfamily, now named CD155 (Mendelsohn et al., 1989). Further confirmation was provided by the creation of transgenic mice expressing CD155 that developed paralytic poliomyelitis after intracerebral injection of wild poliovirus (Koike et al., 1991; Ren et al., 1990). The CD155 has three extracellular domains and poliovirus binds to the outermost of these domains (He et al., 1999; Selinka et al., 1991).

A. Questions unanswered: Receptor expression is necessary but not sufficient To correlate receptors and viral disease, many different parameters could be examined, including receptor-specific mRNA, receptor protein synthesis or its concentration on the cell surface, and the concentration of viral RNA,

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3 Virus replication and destruction of motor neuron

Nucleus Cytoplasm Spinal cord motor neuron

Axoplasm −end

2

Microtubule CD155

Retrograde axonal transport of virus/ receptor complex

Dynein complex

Tctex-1 +end

1

Virus/CD155/dynein-complex

Virus invasion of innervating motor axon at neuromuscular junction

Endocytic vesicle

CD155

Infected muscle

FIGURE 3 Schema to explain the molecular mechanism for retrograde axonal transport of poliovirus. Frame 1: The virus binds to CD155 (the poliovirus receptor) and is endocytosed. The cytoplasmic tail of CD155 then binds to Tctex-1, a protein that is part of the dynein complex. Frame 2: The complex then associates with microtubules and is transported by the dynein motor together with its cargo, endosomes containing poliovirions. Frame 3: On reaching the neuronal cell body, the virion is released from the endosome and uncoated, freeing the viral RNA to initiate replication [JOURNAL OF BIOLOGICAL CHEMISTRY by Mueller, et al., Copyright 2002 by American Soc for Biochemistry & Molecular Biology. Reproduced with permission of American Soc for Biochemistry & Molecular Biology in the format other book via copyright clearance center.]

viral antigens, or production of infectious virions. For various technical reasons, the published data do not include all of these measures, somewhat confounding interpretation. Detailed studies of the organ distribution of cells expressing mRNA specific for the PVR reveal that susceptible cells (such as anterior horn cells in the spinal cord) expressed high levels of receptor mRNA (Ren and Racaniello, 1992a). However, cells in many tissues that also expressed receptor-specific mRNA did not contain poliovirus RNA or pathologic lesions (Table V). Therefore, it

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TABLE V The expression of pvr MRNA is necessary but not sufficient to explain the distribution of poliovirus replication in PVR transgenic mice infected by intraperitoneal or intracerebral routes

Anatomical area

Brain Cortex neurons Cerebellum neurons Brain stem neurons Spinal cord Anterior horn cells Dorsal horn neurons Skeletal muscle Intestinal tract Many cell types Kidney Bowman’s capsule, tubules Lung Macrophages Bronchial epithelium

PVR mRNA

Poliovirus mRNA

Pathological lesions

4+ 4+ 4+

2+ 2+ 2+

Trace Trace 1–2+

4+ 4+ 1–2+

4+ 1–3+ 2–4+

4+ 1–2+ ?

1–2+

0

0

4+

0

0

2–3+ 2+

0 0

0 0

Estimates of the relative amounts of PVR mRNA, poliovirus mRNA, and poliovirus lesions, on a scale of 1–4+, reconstructed from a verbal description [after Ren and Racaniello (1992a), with permission]. The data for poliovirus replication and pathological lesions are consistent with observations on monkeys infected with poliovirus (Bodian, 1959).

appears that receptors are necessary but not sufficient for infection (Zhang and Racaniello, 1997). The other requirements for productive infection and the development of pathological lesions are only partially known. It is striking that kidney cells express the PVR but are resistant to poliovirus in vivo, while—following a few days of culture—they become susceptible, an observation that has been made both in primates and in PVR transgenic mice (Ren and Racaniello, 1992). One important determinant is host defense mechanisms such as interferons that can play an important role in the course of infection in animals that express the PVR (see below).

B. Questions unanswered: Localization within the CNS Different neurotropic viruses exhibit marked variation in the distribution of lesions that they produce in the nervous system (Nathanson et al., 1967). Paralytic poliomyelitis is striking in its localization to lower motor neurons, leading to a flaccid paralysis that is often limited to one

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or both legs. There is little impairment of sensory function or destruction of first order sensory neurons. Likewise, there is minimal involvement of the brain except for lower motor neurons in the brain stem (Fig. 4). Two factors have been invoked that might play a role in localization, selective spread of poliovirus within the CNS and intrinsic differences in susceptibility of different neuronal types. There is some evidence for the first of these hypotheses. If monkeys are infected by intranasal exposure to poliovirus (the preferred route with the MV strain), pathological lesions develop along the olfactory pathway to olfactory centers in the limbic system of the brain. However, the limbic system is not involved in human cases of poliomyelitis nor are olfactory lesions seen in monkeys inoculated by intrathalamic or intraspinal routes (Bodian, 1959; Howe and Bodian, 1942). In experiments with the mouse-adapted Lansing strain of Type 2 poliovirus, Jubelt et al. (1980a,b) observed that adult mice were more susceptible than suckling mice to intracerebral virus injection, but were equally susceptible to intraspinal injection. It appeared that the mature CNS was a better conduit from brain to spinal cord than the neural pathways in the suckling mouse. Ren and Racaniello (1992b) observed that intracerebral virus injection of PVR transgenic mice led to some replication in several centers in the brain, which was not seen after intraperitoneal injection even though all the mice were paralyzed. However, it is clear that monkeys infected either by intraspinal or by intrathalamic routes develop severe lesions of the spinal cord with

Precentral gyrus

Pons Reticular formation

Roof nuclei of cerebellum

FIGURE 4 The distribution of pathological lesions in the central nervous system (CNS) of patients who died of acute poliomyelitis [after Bodian (1959)].

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relatively few lesions in most centers of the brain. This implies that there is some innate difference in the susceptibility of different groups of neurons, which is yet to be elucidated.

C. Questions unanswered: How poliovirus kills cells How poliovirus infection kills cells has been studied for many years, and several mechanisms are operative in different cells and under different experimental conditions (Romanova et al., 2005). The ability of poliovirus to initiate apoptosis in cultured cells (both neurons and macrophages) has been well documented (Blondel et al., 2005; Couderc et al., 2002; Wahid et al., 2005). Apoptosis is likely induced by several of the poliovirus nonstructural proteins, such as 2A, 2C, and 3C, that activate the caspase cascade (Autret et al., 2007; Buenz and Howe, 2005; Calandria et al., 2004). In some cultured cells, nonstructural poliovirus proteins (L, 2B, and 3A) have an antiapoptotic activity. When apoptosis is suppressed, poliovirus-infected HeLa cells may undergo a necrotic cytopathic effect (Agol et al., 1998, 2000; Romanova et al., 2005). Polioviruses shut down host cell protein synthesis by several mechanisms [reviewed by Racaniello (2007)] resulting in necrosis. Which pathway to cell death operates in vivo? Human blood-derived dendritic cells and macrophages cultured ex vivo undergo apoptosis when productively infected with poliovirus (Wahid et al., 2005). In mice with acute poliovirus-induced paralysis, it appears that apoptosis accounts for the death of infected neurons (Girard et al., 1999). The transient inflammatory scars associated with destruction of anterior horn cells in acute poliomyelitis (Bodian, 1959) are typical of necrosis and suggest that necrotic cell death may also play a role in vivo. It should be noted that cell lines expressing certain mutant variants of the PVR were permissive for poliovirus replication and released high virus titers without undergoing an apparent cytopathic effect (Morrison et al., 1994). This suggests that—contrary to longheld belief—cell destruction is not required for release of infectious poliovirions and that the interaction between virus and receptor may be the trigger for the cytopathic effect, perhaps by initiating apoptosis?

V. HOST INNATE AND IMMUNE RESPONSE TO INFECTION Most studies of host responses to poliovirus infection were conducted long before the modern era of immunology. Therefore, there is considerable data about the neutralizing antibody response, but little known about cellular immune responses. Following experimental infection of nonhuman primates, there is a brisk immune response with the appearance of circulating neutralizing antibody about one week after infection (Fig. 2).

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Human responses have been best documented in seronegative children who were given OPV and then followed (Fig. 5). Serum IgG and IgM responses are brisk; the IgM response is transient while the IgG response is long-lasting. Serum IgA is also produced but the response is slower than for IgG (Ogra and Karzon, 1969; Ogra et al., 1968). Antibody can also be detected in pharyngeal secretions and in stool samples.

A. Questions unanswered: The acquired immune response Presumably, circulating antipoliovirus antibodies are produced by B cells in central lymphoid tissues and shed into the efferent lymph to travel to the circulation via the thoracic duct. The question has not been studied, however, and the source of antibody found in the gastrointestinal tract has not been documented in published studies. Based on studies of immune responses to other mucosal viral infections, it seems likely that plasma cells in the gastrointestinal tract (tonsil, Peyer’s patches) must also produce antibody, particularly IgA (Yewdell and Bennink, 2002). Mucosal IgA production would account for the relatively high titers of fecal IgA in

Neutralizing antibody titer

100

10

1

0.1 0

10

20

30

40

Days after OPV Serum IgG

Serum IgA

Serum IgM

Colon IgA

FIGURE 5 Antibody response of seronegative children following immunization with oral poliovirus vaccine (OPV) via the distal colon. Infants with a colostomy necessitated by an imperforate anus (but otherwise normal) were administered type 1 monovalent OPV in the distal colon; serum and colonic fluids were tested for three classes of poliovirus antibodies in a radioimmunodiffusion assay [Copyright 1949 The American Association of Immunologists, Inc., with permission].

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17

subjects who have had an enteric poliovirus infection (Fig. 5) and also for their relative resistance to oral challenge with polioviruses compared to IPV recipients (see below). Following polio infection or immunization, circulating antibodies can be detected for several years, and appear to stabilize 1–2 years after infection (Lennette and Schmidt, 1957). Studies of small isolated villages in remote areas such as Greenland strongly suggest antibody persistence for many decades perhaps lifelong postinfection (Paffenbarger and Bodian, 1961; Paul et al., 1951). Because these observations were made following active poliovirus infection, it was debated whether this was due to the persistence of poliovirus (Melnick, 1952). However, persistence of serum antibodies was also noted after administration of IPV (Salk, 1955), although the follow-up was not as long. Recent data on memory B cells in experimental mouse models have suggested that central memory B cells can be self-renewing in the absence of antigen (Crotty and Ahmed, 2004; Gourley et al. 2004). However, there are no published studies of the basis of persistent poliovirus antibodies in tractable models such as PVR mice. There are few if any published data on cellular immune or innate responses to poliovirus infection. Experiments in PVR transgenic mice imply that there is an interferon response to poliovirus that plays a role in the control and clearance of the infection (see below). These questions could productively be studied in the PVR mouse model, using current immunobiological methods.

VI. IMMUNE DEFENSES AND VIRAL CLEARANCE: MECHANISMS OF VACCINE-INDUCED PROTECTION Host defenses are best considered under two headings: the response to primary infection and the response in hosts that have been immunized as a result of prior natural infection or immunization.

A. Primary infections In primary infection of nonhuman primates, sequential studies of virus replication and host immune responses strongly suggest that the immune response plays a key role in clearance of the virus. The termination of viremia appears to be immediately followed by the first detection of serum-neutralizing antibody, implying that antibody is responsible for controlling viremia (Fig. 2). Virus excretion in pharyngeal secretions and feces continues for about one month after infection of nonhuman primates. Although it is not obvious why shedding persists for so long, there is strong circumstantial evidence that the termination of shedding is also immune mediated because patients with hypogammaglobulinemia

18

Neal Nathanson

often exhibit persistent shedding that may last for months to years (MacLennan et al., 2004). A search among patients with T-cell deficiencies such as HIV/AIDS has failed to reveal any persistent excretors of poliovirus (Asturias et al., 2006; Moss et al., 2003), implying that clearance is mediated by antibody.

1. Questions Unanswered: Recovery from Primary Infections The mechanism by which the immune response clears a primary poliovirus infection is only partially understood. The unknown identity of cells that are replicating virus in the gastrointestinal tract makes it hard to determine how shedding is terminated. It is possible that poliovirus spreads from cell to cell and that IgA interrupts intercellular transmission. Alternatively, antibody could mediate lysis of infected cells, leading to viral clearance. There are few published data to test these hypotheses. (Clearance is discussed further in the section on viral persistence below.)

B. Secondary infection in immune hosts Protection against paralysis in hosts that were immunized by prior exposure to poliovirus or to vaccines is well documented, and the data permit some inferences about immune mechanisms. Passive antibody given prior to infection confers protection against paralysis, and in monkeys the protective level has been found by titration to be a serum-neutralizing titer of about 1:10 (Fig. 6). IPV induces the development of circulating neutralizing antibodies, and in human subjects, there is a good correlation between the antibody titer and protection against paralysis (Table VI). Although IPV protects against paralysis, it provides only a modest degree of protection against infection, as shown when vaccinees are challenged with OPV (Fig. 7). These data imply that protection against paralysis is conferred by preventing viremia not by preventing infection. OPV induces both humoral and mucosal immunity. When OPV-immunized children are challenged with OPV, they can be reinfected but they shed less virus for shorter times compared with unimmunized control children. Furthermore, OPV has a much greater damping effect on fecal shedding that does IPV (Fig. 7). Since both vaccines induce similar levels of circulating antibodies, this indicates that mucosal immunity plays an important role in preventing spread of polioviruses in the community.

1. Questions unanswered: Secondary infection in immune hosts The mechanisms of vaccine-induced immunity are only partially elucidated. In particular, there are few published data to explain the basis of mucosal immunity. The relative contribution of circulating IgG that transudates to the gut versus locally produced mucosal IgA is not well documented. From animal models of other acute viral infections, it may be

Pathogenesis of Poliomyelitis

19

100

Percent paralysed

80

60

40

20

0 Control

3

6

11

22

Passive antibody titer (1/X)

FIGURE 6 Protective efficacy of passive antibody in experimental poliomyelitis. Monkeys were given injections of graded doses of pooled human g-globulin with neutralizing activity against poliovirus and one day later were tested for their serum antibody titer. They were then challenged intramuscularly with the Mahoney strain of poliovirus. Data of this type could be used to estimate the serum titer that conferred protection against paralytic poliomyelitis [after Nathanson and Bodian (1962), with permission].

TABLE VI A Low titer of serum neutralizing antibody (1:4) correlates with protection against paralytic poliomyelitis

Number of paralytic cases Percent seroconversions after vaccination (1:4)

Unvaccinated placebo children

Vaccinated children

Percent protection against paralytic poliomyelitis

40

14

65%

0%

65%

In 1954, a large-scale clinical trial was done to test whether a poliovirus vaccine (IPV) would protect against poliomyelitis. An equal number of children were assigned to the vaccinated and unvaccinated placebo groups (about 200,000 in each group) so the numerators can be directly compared; paralytic cases were reduced by 65% in the vaccinated group. To determine the level of antibody that correlated with protection, the proportion of vaccinated children with seroconversions at different titers were compared with the proportion protected by the vaccine. Of the children who were seronegative prior to immunization, approximately 65% had a titer of 1:4 after the full course of three doses of vaccine. Because this corresponded to the percent who were protected against paralytic poliomyelitis, it was concluded that a titer of 1:4 probably was sufficient to protect against paralytic disease. This analysis is limited to type 1 poliovirus, which was responsible for about 75% of all cases of paralytic poliomyelitis [After Francis et al. (1957), with permission].

20

Neal Nathanson

100

Percent excreting >104 TCD50 per gm feces

Naive controls 80 IPV immunized 60

40 OPV immunized 20

0 5

10

15

20

Days after feeding OPV

FIGURE 7 OPV provides substantial protection against fecal shedding of poliovirus while IPV provides only limited protection. Three groups of seronegative children were either immunized with OPV, IPV, or served as controls. All groups were then challenged with OPV and followed for shedding of poliovirus [after Henry et al. (1966), with permission from Cambridge University Press].

inferred that cellular immunity could play a role in vaccine-induced protection (Engle and Diamond, 2003; Shrestha and Diamond, 2004), but there are no data to test such a hypothesis for polio vaccines. Recently, the question has again arisen, whether IPV can—via transudation of circulating anti-polio IgG—be used to complement OPV in regions where it is difficult to eradicate wild polioviruses (see below). Epidemiological studies, in the period 1956–1961 before OPV was introduced, suggested that IPV did provide a considerable degree of herd immunity (Marine et al., 1962; Stickle, 1964). In the United States, the incidence of poliomyelitis was reduced well below the level expected based on the proportion of children who had received a course of IPV, suggesting that IPV had conferred considerable herd immunity (Table VII).

C. Poliovirus serotypes Polioviruses can be sorted into three serotypes, types 1, 2, and 3. The serotypes were originally defined by protection experiments in which monkeys were infected with one virus isolate and then challenged with another isolate (Bodian, 1949). Those viruses that conferred 100% crossprotection against paralysis were grouped in a single serotype. Similar results were obtained using cell culture neutralization where a panel of

Pathogenesis of Poliomyelitis

21

TABLE VII Epidemiological evidence suggesting that IPV can induce substantial herd immunity

Year

1958 1959 1960 1961 1958–1961 totals Percent of baseline

Baseline absent IPV

Cases expected based on the proportion of the population that received IPV

Cases observed

27,316 28,224 28,964 29,521 114,025

11,794 10,191 8,964 8,471 39,420

3,795 6,358 2,556 1,002 13,711

35%

12%

Data for the United States for 1951–1954 were used to project the numbers of cases of poliomyelitis in 1958– 1961, if there had not been an immunization program (baseline). Based on the proportion of the population that had been immunized, a calculation was made of the numbers of cases that would be expected. Observed cases were consistently much lower than the numbers expected, suggesting that IPV had reduced the spread of poliovirus to unimmunized children and adults [after Stickle (1964), with permission].

convalescent sera was tested against a panel of virus isolates (Salk et al., 1951). In these early studies, some degree of crossing was observed between type 2 and the other serotypes, but none between types 1 and 3. Because serotypes reflected cross protection in vivo, both IPV and OPV were formulated as trivalent immunogens. With the advent of monoclonal antibody and sequencing technology, the neutralizing antigenic sites on poliovirus were characterized in detail (Minor et al., 1986). Neutralizing sites were mapped to external residues on the surface of the viral capsid using the crystallographic structure (Hogle et al., 1985). Three major antigenic sites (not to be confused with the serotypes) were identified. Somewhat surprisingly, none of these sites served as epitopes for all three serotypes. Site 1 was antigenic for serotypes 2 and 3, and site 3b was antigenic for serotypes 1 and 3. The relative importance of different structural epitopes in protection against disease has not been determined.

VII. ANIMAL MODELS OF HUMAN POLIOMYELITIS Nonhuman primates: Chimpanzees and old world monkeys are susceptible to human polioviruses, and can be infected by oral administration of wild polioviruses, as well as by artificial routes such as intramuscular, intravenous, intraspinal, intracerebral, or intranasal administration. These species develop acute flaccid paralysis that closely mimics poliomyelitis

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Neal Nathanson

in humans. Even after intracerebral injection, there is a marked predilection for destruction of lower motor neurons with little evidence of encephalitis, resembling the differential neuronal susceptibility seen in humans. As in humans, only a proportion of infections result in overt paralysis; the frequency of paralysis depends upon the virus isolate, dose, and route of injection and the species of nonhuman primates. For instance, cynomolgus macaques appear to be more susceptible to paralysis than rhesus macaques (Bodian, 1956). Intramuscular injection of a virulent virus strain can cause close to 100% paralysis (Table I) that is convenient for certain experimental studies but is much higher than the estimated ratio in humans of 1 paralytic case per 100–200 primary infections.

A. Questions unanswered: Determinants of primate susceptibility As mentioned above, in general, new world monkeys appear resistant to human poliovirus infection (Hsiung et al., 1964; Ida-Hosonuma et al., 2003). Incomplete data indicate that this is due to variability in the first immunoglobulin domain of the PVR gene, but only limited comparisons of this gene in nonhuman primates have been published (Ida-Hosonuma et al., 2003). Recent studies of HIV/AIDS have made it clear that humans likely vary in their genetically programmed response to all infectious agents (Carrington and O’Brien, 2003; Martin and Carrington, 2005). However, there are no published data on genetic differences among individual humans or monkeys in their susceptibility to poliovirus. Mouse models using mouse-adapted strains of poliovirus: A few isolates of human polioviruses were successfully adapted to rodents by intracerebral passage (Paul, 1971). In particular, the mouse-adapted type 2 Lansing strain (Armstrong, 1939) has been used for studies of polio pathogenesis (Ford et al., 2002; Jubelt et al., 1980a,b; La Monica et al., 1986). In mice, the Lansing strain caused flaccid paralysis after intracerebral injection. Immunofluorescent staining of poliovirus antigens demonstrated selective infection of anterior horn cells in the spinal cord. A careful comparison of the pathological lesions in the CNS indicated that they closely resembled those seen in human and other primates (Ford et al., 2002). There was no evidence of extraneural virus replication. Following intracerebral injection of mouse-adapted Lansing virus, adult mice were more susceptible than suckling mice as judged by the percent paralyzed or the length of incubation period ( Jubelt et al., 1980a,b). This paradoxical finding was explained by the transmission of virus from brain to spinal cord, which was less efficient in the immature CNS of newborn mice. After intraspinal virus injection, suckling mice were more susceptible than were adult mice. Transection of the spinal cord in adult mice prevented intracerebral virus from reaching the

Pathogenesis of Poliomyelitis

23

lumbar spinal cord, providing further evidence that the Lansing strain behaved as an obligatory neurotrope in this model.

B. Questions unanswered: The mechanism of rodent adaptation Adaptation to mice was only accomplished for a few poliovirus strains, more frequently for type 2 than for other poliovirus types (Paul, 1971). Type 1 Mahoney virus cannot infect mice but its RNA can replicate if transfected into mouse cells, implying that the block is at viral entry into potential host cells (Detjen et al., 1978). The viral mutations associated with rodent adaptation of Lansing type 2 virus were mapped to a neutralizing antigenic site (antigenic site 1) within one of the capsid proteins, VP1 (La Monica et al., 1986; Murray et al., 1988). Replacing six amino acids of type 1 Mahoney virus with the corresponding amino acids from type 2 Lansing virus conferred on the chimeric virus the ability to produce fatal poliomyelitis in mice (Martin et al., 1988; Murray et al., 1988). The chimeric virus could replicate well in primate cells and was neutralized by type 2-specific neutralizing monoclonal antibodies but not type 1 antibodies directed against antigenic site 1. One interpretation of these observations is that the mouse-adapted virus utilizes the same domain on VP1 for attachment to both primate and mouse cells. Although the receptor protein on mouse cells is still unknown, this would imply a similarity in primate and mouse cellular binding sites. Alternatively, neutralizing antibody may not prevent attachment to the receptor on mouse cells but interfere with subsequent steps in viral uncoating. Structural studies could test these alternate hypotheses. It should be mentioned that there is one report that mouse-adapted strains of poliovirus produce diffuse encephalomyelitis not resembling poliomyelitis after intracerebral injection (Gromeier et al., 1995) but this was not confirmed by another laboratory (Ford et al., 2002). The explanation for this discrepancy is unclear. PVR transgenic mouse model: An important milestone in experimental studies of polio pathogenesis was the development of transgenic mice expressing PVR (Koike et al., 1991; Ren and Racaniello, 1990, 1992a,b). Following intracerebral injection with wild polioviruses, PVR mice develop flaccid paralysis. However, these animals differ from nonhuman primates in two significant respects. First, they cannot be infected by feeding of poliovirus. Second, when they are injected intramuscularly with a pantropic strain of poliovirus, the virus appears to spread exclusively by the neural route (Table VIII). In PVR mice, the pantropic Mahoney strain behaves like the neurotropic MV strain in nonhuman primates (see Table I).

TABLE VIII In transgenic pvr mice, poliovirus spreads by neural pathways

Mice

PVR transgenic

PVR transgenic Normal

Experimental procedure

Virus

Paralyzed/ infected

Site virus injection

Site of initial paralysis

40/40 36/36 14/14 16/16

LH RH LF LH

LH RH LF LH

0/12

LH (footpad)

None

Type 1 Mahoney

PBS in RH Left sciatic transection

Type 1 Mahoney Type 1 Mahoney

–

Type 2 Lansing

2/23 12/12

LH (footpad) LH

LH LH

–

Type 2 Lansing

4/4

LH

RH 1 RF 1 RLH 2

PVR transgenic or normal mice were injected with type 1 Mahoney virus (a pantropic strain) or with mouse-adapted type 2 Lansing virus, and were followed to determine the site of initial paralysis. A few groups received a sciatic nerve transection one day prior to virus injection. In PVR transgenic mice, type 1 Mahoney virus always produced initial paralysis in the injected leg and sciatic transection almost always protected mice from paralysis, implying that the virus traveled by a neural pathway from the injected muscle to the spinal cord. Type 2 Lansing virus exhibited a similar pattern in transgenic mice, but in normal mice, the variable distribution of initial sites of paralysis suggested that it spread by the viremia route. LH, left hind leg; RH, right hind leg; LF, left front leg [after Ren and Racaniello (1992b), with permission].

Pathogenesis of Poliomyelitis

25

Subsequently, other strains of PVR transgenic mice have been developed using a variety of promoters (Crotty et al., 2002; Ida-Hosonuma et al., 2002; Koike et al., 1991; Nagata et al., 2004). For the most part, the results were similar to those reported by Ren and Racaniello (1992a,b). It is possible to infect PVR transgenic mice by the intranasal route, and virus spreads along the olfactory pathway (Crotty et al., 2002; Nagata et al., 2004), closely resembling the behavior of the MV strain in monkeys (Flexner, 1931; Howe and Bodian, 1942). One line of PVR transgenic mice that used an ubiquitously strong promoter (based on the b-actin promoter) led to expression of the PVR in glial and ependymal cells as well as neurons (Ida-Hosonuma, et al., 2002). In these mice, poliovirus replicated mainly in glial and ependymal cells in the CNS and produced a pathological picture totally different from poliomyelitis. Perhaps the most striking finding was the demonstration that PRV mice that were crossed with Ifnar (interferon a-receptor) knockout mice to obtain PRV+/+/Ifnar/ mice were much more susceptible to type 1 Mahoney virus injected by intracerebral, intravenous, or intraperitoneal routes (Ida-Hosonuma et al., 2005). These mice could be infected by mouth with a large virus dose that produced paralysis in 50% of the animals. Poliovirus replicated to high titer in many visceral organs and there was a high titer viremia. However, these investigators did not determine the consequences of virus injection by the intramuscular route or the effect of nerve block, so the analysis is incomplete. The data do suggest that knocking out an important component of the innate immune response reveals a latent ability to produce viremia. However, the model may be somewhat artificial since poliovirus replicated to high titer in organs such as liver, pancreas, and kidney, which are not involved in human poliovirus infection. Furthermore, the observations do not completely explain the apparent reduced enterotropism or loss of viremogenicity in PVR mice infected with pantropic polioviruses.

C. Questions unanswered: PVR mice The behavior of pantropic polioviruses in PVR mice suggests that the virus can infect neural cells but not cells in the lymphoreticular system, and therefore cannot produce a viremia. It has been questioned whether this is due to localized expression of the PVR perhaps associated with the promoter used in the PVR mice. Zhang and Racaniello (1997) tested this hypothesis by creating PVR transgenic mice in which the transgene was under the control of an enterocyte-specific promoter. Although there was substantial binding of poliovirus to intestinal tissues, the mice were not rendered permissive for oral infection. Likewise, Crotty et al. (2002) used a b-actin promoter that led to expression of the PVR in intestinal homogenates, but did not render mice susceptible to oral infection.

26

Neal Nathanson

D. Questions unanswered: The tropism enigma Table IX summarizes data on nonhuman primate and mouse models for poliomyelitis, with a focus on tropism. Each model can be characterized as pantropic or obligatory neurotropic, based on several parameters. Obligatory neurotropic viruses cause localized paralysis after intramuscular injection, and protection is conferred by neural block proximal to an intramuscular injection site, while pantropic viruses produce initial paralysis at many different sites and are not inhibited by nerve block. Pantropic viruses cause a viremia and are infectious by virus feeding, while obligatory neurotropic viruses are not viremogenic and do not infect when administered by mouth. What determines tropism? Several observations can be made about the data. (1) Tropism appears to be a property of the virus–host combination. Some viruses (such as type 1 Mahoney) can behave as a pantropic agent in macaques but as an obligatory neurotrope in PVR mice. Conversely, in a single host such as the macaque, different viruses can behave as pantropic (type 1 Mahoney) or neurotropic (type 2 MV). (2) All polioviruses are neurotropic in that they can replicate in neurons and spread along neural pathways, but pantropic viruses have the additional ability to replicate in extraneural tissues, including cells of the monocytes–macrophage lineage and perhaps enteric epithelial cells. (3) It is likely that obligatory neurotropic viruses can be selected by serial passage in the CNS, judging by the passage history of the type 2 MV strain and of mouse-adapted polioviruses such as type 2 Lansing virus. At a cellular and molecular level what determines tropism? One hypothesis is that tropism is an entry-determined phenomenon. Under this hypothesis, an alternative entry pathway is used when poliovirus infects nonneural cells. This hypothetical pathway could involve an alternate cellular receptor or it could operate at a postattachment step. Pantropic viruses would be able to utilize both pathways, while obligatory neurotropic viruses would be restricted to the default neuronal pathway. Passage of pantropic viruses by CNS passage in monkeys or rodents could select for virus variants that can only utilize the neurotropic entry pathway. This hypothesis should be testable by several different approaches.

VIII. VIRULENCE OF POLIOVIRUSES For polioviruses, the overall virulence phenotype is probably best measured by the ability of a virus strain to cause paralysis in the human host following infection by the oral route. This phenotype reflects sequential steps in infection including replication in the oropharynx and intestine; the intensity of viremia; invasion of the CNS, spread to the spinal cord; and

TABLE IX A comparison of different experimental models of poliomyelitis, to contrast pantropic and neurotropic attributes of virus and host

Virus

Passage history

Host

Pantropic (route of infection)

Type 1 Mahoney

Primate tissue culture Intracerebral in monkeys

Monkey

YES (iv)

Type 2 MV

Obligatory neurotropic (route of infection)

References

Bodian, 1954a

Monkey

YES (im)

Nathanson and Bodian, 1961

Mice

YES (ic, is)

Ford et al., 2002; Jubelt et al., 1980a,b; La Monica et al., 1986 Ren and Racaniello, 1992b Crotty et al., 2002; Koike et al., 1991; Nagata et al., 2004; Ren and Racaniello, 1992a,b; Ren et al., 1990 Zhang and Racaniello, 1997

Type 2 Lansing Type 2 Lansing

Intracerebral in mice Intracerebral in mice

Type 1 Mahoney

Primate tissue culture

PRV mice (several promoters)

YES (ic, ip, iv, im, po, in)

Type 1 Mahoney

Primate tissue culture

PRV mice (enterocyte promoter)

YES (ic, po)

Mice

? YESa (im)

(continued)

TABLE IX

(continued)

Virus

Type 1 Mahoney

Passage history

Primate tissue culture

Host

PRV mice (Ifnar knockout)

Criteria for classification Localized paralysis after im infection Protection by neural block Viremia Infectious by mouth a

Pantropic (route of infection)

Obligatory neurotropic (route of infection)

b

Ida-Hosonuma et al., 2005

? YES (ic, ip, iv, po) NO NO YES YES

YES YES NO NO

Based on four mice with variable sites of initial paralysis. Replication in many visceral organs not involved in human poliomyelitis. Abbreviations: ic, intracerebral; ip, intraperitoneal; im, intramuscular; is, intraspinal; po, per os; in, intranasal.

b

References

Bodian, 1954a, 1955b; Nathanson and Bodian, 1961

Pathogenesis of Poliomyelitis

29

intrinsic neurovirulence. A variety of experimental studies have demonstrated that these parameters may vary independently in different poliovirus strains. Sabin’s extensive studies of attenuated polioviruses showed that his candidate vaccine strains retained robust enterogenicity (ability to replicate in the gastrointestinal tract and be shed in the feces) while having markedly reduced neurovirulence as determined by direct intrathalamic or intraspinal injection in macaques, illustrating the dissociation of different replication phenotypes (Sabin et al., 1954). Also, the host used in pathogenesis studies can influence relative virulence. Sabin (1956, 1965) reported a hierarchy, from humans to chimpanzees to cynomolgus to rhesus monkeys, of decreasing susceptibility of the gastrointestinal tract and increasing susceptibility of the spinal cord, based on titration by oral and intraspinal routes of infection. Much of the modern research on polioviruses has been directed to the genetic determinants of virulence, with a focus on the attenuated strains used in OPV. Following licensing of OPV in 1963, surveillance in the United States documented the occurrence of vaccine-associated cases of poliomyelitis in both vaccine recipients and in their close contacts. Epidemiological data indicated that the three OPV strains caused paralysis at a rate of about 2 cases per million primary vaccinations and were approximately 10,000-fold attenuated compared to wild polioviruses (Table X). The need to understand the molecular basis of vaccine-associated paralytic poliomyelitis (VAPP) led to a substantial body of research. Polioviruses have a genome of about 7500 nucleotides and a point mutation rate of about 104, so that each nascent virion will have about 1 point mutation (Kew et al., 2005). During a single human infection, there will be a large number of mutant virions among >108 virus particles that are shed, providing plentiful opportunity for rapid selection of variants with enhanced fitness. Although the OPV viruses are enterogenic, they are less fit than variant polioviruses that outgrow them during intestinal passage. Neurovirulence testing of virus shed by vaccinees compared to the neurovirulence of the parent strains of OPV show that OPV frequently reverts to greater neurovirulence on a single human passage. As expressed in George Dick’s epigram, ‘‘in like a lamb out like a lion.’’ Sequencing many revertant isolates of virus shed by recent OPV vaccinees has identified the most frequent mutations. Genetic chimeras between OPV and revertant viruses were used to pinpoint (for each of the three types of OPV) mutations that were associated with enhanced virulence (Table XI). The virulence (attenuation) phenotype was associated with 5–10 individual point mutations, both in the 50 nontranslated region of the genome and in some of the capsid proteins. Sequencing of a few viruses isolated from VAPP cases suggested that paralysis was caused by revertant viruses and was not an intrinsic property of unmutated OPV (Evans et al., 1985). However, when tested in PVR mice, revertant viruses isolated from VAPP cases were less virulent

TABLE X

Comparison of the virulence of wild poliovirus and OPV, based on data from the United States

Poliovirus

Wild-type infections OPV immunizations

Study period

Population (person-years)

Paralytic cases

Paralytic rate per 106 primary infections or immunizations

1931–1954

96  106 (total infections) 135  106 (primary vaccinations)

650,000

7,000


10,000

84

0.62

1

1961–1978

Relative rates

The paralytic rate for wild poliovirus is based on reported cases of paralytic poliomyelitis and an estimate of the number of infections that occurred during the same period [after Nathanson and Martin (1979) with permission]. The rate for OPV is based on VAPP in recipients of OPV, and the numbers of primary vaccinations during the same period [after Nkowane et al. (1987) and Schonberger et al. (1976), with permission].

TABLE XI

Analysis of genetic determinants of poliovirus virulence Nucleotide

Relative virulence

Virus Clone

Score

More virulent

P3/L SV1/L SP2/L S39/L SLR2

2.71 2.68 2.51 2.40 2.39

L472V3/S SCC/L SV3/L L472/S LV3/S ST/L

2.07 1.93 1.74 1.58 1.32 1.14

A A A A

A A

A A A

P3/S SLR1

0.41 0.28

A A

A A

A A

Intermediate

Less virulent

220

472

871

2034

3333

3464

4064

6127

7165

7432

A A

A A

A A A

A A

A

A

A

A A

A A

A A

A A

A A

A

A

A

A

A A

A A

A A

A A

A A

A A

A

A

A

A

A

A

This example compares virulent type 3 poliovirus (P3/Leon) with the attenuated type 3 OPV (P3/Sabin) derived therefrom. There are 10 nucleotide differences between the two viruses (in the whole genome of >7000 nucleotides) and a set of recombinant viruses were constructed to determine which divergent nucleotides influenced virulence. In this instance, it appeared that nucleotides 220 and 472 in the 50 nontranslated region and nucleotides 871 (VP4) and 2034 (amino acid 91 in VP3) were the most critical sites. The nucleotide found in the attenuated OPV is marked ‘‘A’’ while the nucleotide found in the virulent Leon type3 virus is blank. Boxes indicate that the five most virulent recombinant viruses had the ‘‘virulent’’ nucleotide at all four of these sites while the two most attenuated recombinant viruses had the ‘‘attenuated’’ nucleotide at these four sites. Virus clone: P3/L, virulent parental P3 Leon strain; P3/S, attenuated P3 Sabin strain; Score: neurovirulence score based on severity of spinal cord lesions after intracerebral injection of macaques [after Minor (1992), with permission].

32

Neal Nathanson

than some stool isolates from healthy children following OPV administration (Georgescu et al., 1994, 1997).

A. Questions unanswered: Mechanisms of neurovirulence The genetic determinants of virulence reversion (and attenuation) of OPV strains have been delineated in exquisite detail by the cumulative efforts of many investigators (Bouchard et al., 1995; Evans et al., 1985; Kawamura et al., 1989; La Monica et al., 1987; Macadam et al., 1993; Minor, 1992; Omata et al., 1986; Ren et al., 1991; Westrop et al., 1989). However, information about the comparative pathogenesis of attenuated viruses is incomplete. Revertant strains of OPV show marked increases in neurovirulence but little is known about the molecular mechanisms of neurovirulence or its attenuation. It appears likely that attenuated polioviruses are host range mutants because they replicate well in the enteric tract but not in the spinal cord, and are usually temperature sensitive. The sites of the mutations suggest that the structure of the capsid proteins is involved, perhaps due to nuances in entry of the virus. Important determinants in the 50 noncoding region are located at sites involved in the secondary structure of RNA, which may influence the initiation of translation at the internal ribosome entry site (IRES). One example of the potential role of the IRES in determining neurovirulence is a set of experiments in which the IRES of a virulent poliovirus was exchanged with that from other enteroviruses (Gromeier et al., 1996). A virulent poliovirus was compared with a recombinant bearing the IRES from human rhinovirus type 2. Both viruses grew equally well in HeLa cells but the recombinant virus scarcely replicated in human neuroblastoma cells and failed to paralyze PVR transgenic mice, while the virulent virus replicated well in neuroblastoma cells and had a high intracerebral LD50 titer in transgenic mice. These experiments indicate that virulence is often a host range phenomenon that can be influenced by both virus and host cell determinants, and the subtleties of their interaction. Viremia and virulence: Studies in nonhuman primates indicated that different isolates of human poliovirus varied in their viremogenicity following intravascular injection (Fig. 8). There was a correlation between high viremia level (Mahoney strain) and high frequency of paralysis, suggesting that viremogenicity could play a role in the virulence of poliovirus.

B. Questions unanswered: Viremia and virulence There is a paucity of published data on the comparative viremogenicity of OPV and revertant vaccine viruses so it is unclear whether virulence reversion is associated with an increase in viremogenicity. Limited studies in infants fed OPV showed that type 2 OPV was more viremogenic

Pathogenesis of Poliomyelitis

33

Log10 TCD50 per ml serum

4

3

2

1

0 0

1

2

3

4

5

6

7

8

Days after infection

Mahoney (80/167)

Y-SK (1/14)

Maclean (4/14)

Saukett (0/15)

FIGURE 8 Viremogenicity is a virulence determinant in poliomyelitis. Different strains of wild poliovirus cause different levels of viremia, following intravascular infection of monkeys. The Mahoney strain caused the highest titer viremia and also the highest rate of paralysis, shown in parentheses [after Bodian (1954a), with permission].

than types 1 or 3 (Horstmann et al., 1964). However, type 2 OPV caused vaccine-associated paralysis at a lower rate than did the other types (Table XII).

C. Questions unanswered: Epidemiological properties of polioviruses There are some striking epidemiological differences between the three types of polioviruses (Table XII). Of all paralytic cases of poliomyelitis (prior to the introduction of polio vaccine), about 75% were caused by type 1, 15% by type 2, and 10% by type 3 virus (Nathanson and Martin, 1979). Consistent with this, in recent years, most of the residual cases of paralytic poliomyelitis have been caused by wild type 1 poliovirus. Eradication programs have eliminated type 2 but not types 1 and 3 polioviruses. Presumably, these type-specific differences are due to subtle variation in the biological properties of the three types of viruses, differences that have never been elucidated.

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TABLE XII Epidemiological differences between the three types of poliovirus Parameter

Type 1

Type 2

Type 3

References

Proportion of paralytic cases caused by wild poliovirus in the prevaccine era Paralytic poliomyelitis in OPV recipients

75%

15%

10%

Nathanson and Martin, 1979

22

13

62

17/19

0/16

Nkowane et al., 1987; Schonberger et al., 1976 Horstmann et al., 1964

Viremogenicity 0/16 following OPV feeding to seronegative infants Monkey neurovirulence of OPV Mean lesion score 1.05 Percent severe lesions 0.05%

0.79 1.15%

Nathanson and Horn, 1992

Wild Type 1 strains were more paralytogenic in humans but type 3 OPV was more likely to cause vaccineassociated paralysis in children, probably associated with the higher frequency of virulent revertant viruses in the final batches of OPV.

The seasonality of poliovirus and its correlation with temperate climates are well documented (Yorke et al., 1979). It has been suggested that shed virus survives less well under conditions of low humidity in wintertime, but there is no experimental evidence for this hypothesis. Competition between enteroviruses could be due to innate immunity but there are no published data to support such a mechanism.

IX. HOW DOES POLIOVIRUS PERSIST? Available data indicate that poliovirus causes an acute self-limited infection in normal humans. As described above, studies in human or nonhuman primates infected with either wild polioviruses or OPV showed that virus could be isolated from the feces from 2 to 8 weeks after infection, but rarely thereafter (Bodian, 1959; Henry et al., 1966; Howe, 1957; Khan et al., 2006). Likewise, virus titers in the spinal cords of infected monkeys peak about the first day of paralysis and wane rapidly for the next week; infectious virus is rarely detected more than 4 weeks after the onset of paralysis (Bodian, 1959). However, these early studies did not use current

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methods for demonstration of persistence such as immunosuppression of nonhuman primates or PCR testing. Studies of vaccine-associated cases of poliomyelitis in recipients of OPV have identified a subset of patients who are hypo- or agammaglobulinemic (Martin, 2006; Minor, 2006). Strikingly, some of these patients have continued to shed fecal virus for months to many years. This suggests two inferences: first, poliovirus is potentially capable of producing a persistent infection in humans and, second, viral clearance and termination of acute infections is mediated by the acquired immune response. Parenteral or oral administration of g-globulin with high titers of poliovirus antibodies has failed to terminate virus shedding in these patients (Maclennan et al., 2004).

A. Questions unanswered: Overt persistence of poliovirus As mentioned above, the cells responsible for enteric shedding of poliovirus have not been identified, nor has the mechanism whereby poliovirus antibody terminates viral shedding. There are two plausible scenarios for overt persistence. (1) If poliovirus causes lytic infection in enteric cells, then the virus would have to continue to spread from cell to cell during persistence. Under these circumstances, antibody could act by preventing cell-to-cell dissemination; absent antibody, the virus could persist by continual extracellular transmission. (2) Less likely, antibody could terminate infection by mediating a lytic attack on persistently infected cells. Absent antibody, poliovirus could then persist in individual enteric cells. Persistent productive infection of cultured cells without cytopathic effect has been reported under rather artificial laboratory conditions (ColbereGarapin et al., 1989). It should also be noted that persistent shedding is not seen in many patients with hypogammaglobulinemia who have been infected with OPV, and persistent poliovirus shedding can terminate spontaneously in some patients with hypogammaglobulinemia. Also, there is no consistent relationship between fecal antipoliovirus IgA levels and termination of viral shedding. These observations imply that persistence may be a stochastic process, influenced by other factors in addition to antibody (Maclennan et al., 2004).

B. The post-polio syndrome and covert persistence of poliovirus Patients who survive paralytic poliomyelitis usually stabilize the extent of their paralysis within 2–3 months after onset, and may improve function with the aid of physical therapy. Following the postacute phase of illness, the extent of the handicap remains stable for a long time. However, many years

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later some of these patients experience a gradual loss of muscle strength, known as the ‘‘post-polio syndrome’’ (Dalakas et al., 1995; Mulder et al., 1972). It is generally agreed that this clinical syndrome is caused by a gradual deterioration of motor neurons that are stressed by maintaining large motor units for many years. However, it has been suggested that covert persistence of poliovirus could play a role in the pathogenesis of the post-polio syndrome. Investigators have looked at both antibodies (particularly poliovirus IgM) and viral footprints (RNA detected by PCR) in patients with the postpolio syndrome. Most—but not all (Leon-Monzan and Dalakas, 1995)— studies have failed to find convincing consistent circumstantial evidence for persistence of poliovirus ( Jubelt et al., 1995). One laboratory has reported a mouse model of poliovirus infection in which viral RNA and antigens persist (Destombes et al., 1997; Girard et al., 2002). These investigators employed a mouse-adapted strain of type 1 poliovirus that paralyzed mice but with a high survival rate. The initial stages of paralysis resembled other mouse models of paralytic poliomyelitis, with infectious virus isolated for about 10 days after paralysis but not thereafter. However, for at least 12 months after infection, there was persistence of viral RNA by PCR, but at a level 100,000-fold lower than at peak replication, and viral antigens could be detected in a few cells by immunofluorescence. Furthermore, there was histological evidence of a persistent low level of neuronal death and inflammation. Persistent poliovirus infections have not been reported by other investigators working with mouse models so this may represent an atypical biological variant. To date, the weight of evidence suggests that poliovirus does not cause persistent infections in immunocompetent humans or nonhuman primates.

X. ERADICATION In 1988, the World Health Organization announced a goal to eradicate wild polioviruses. This initiative was founded on several biological parameters: (1) humans are the only known natural host for polioviruses; (2) poliovirus causes self-limited acute infections and can only perpetuate itself by infecting new susceptible hosts with a generation time of about 10 days; (3) if the number or density of susceptible hosts falls below a critical level, poliovirus fades away; and (4) OPV immunization reduces the ability of vaccinated subjects to act as links in the chain of transmission. The plausibility of eradication as a goal was first provided by the unpredicted disappearance of wild polioviruses in the United States in 1972, 10 years after the introduction of OPV (Nathanson, 1984). The successful eradication of wild polioviruses in South America in the 1980s, following an aggressive immunization program, further validated the credibility of eradication (de Quadros et al., 1997). Since 1988, the eradication campaign

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has had a significant impact, reducing annual confirmed cases of poliomyelitis from an estimated 350,000 to about 1000–2000 by 2002. It is estimated that about one million cases of paralytic poliomyelitis occurred annually in the 1950s, prior to the introduction of poliovirus vaccines. Thus, the campaign has reduced the global incidence of paralytic poliomyelitis by about 99.9%. Notably, it appears that global eradication was achieved for type 2 poliovirus in 1999 (Kew et al., 2005).

A. Questions unanswered: Why is it so difficult to complete the global eradication of wild polioviruses? The eradication campaign has been stalled from about 2002 to 2007 (Pallansch and Sandhu, 2006), as shown in Fig. 9. Currently, there are two epicenters that have resisted virus elimination, one in South Asia (Afghanistan, Pakistan, northern India) and one in West Africa (centered in Nigeria). What explains the persistence of wild polioviruses in these two foci? There are at least three factors that can be identified: (1) in northern climates, poliomyelitis is a very seasonal disease, such that incidence in the peak months of August and September is up to 30% of annual total, while in trough months (March), it is 0.1% of annual total. Under these circumstances, poliovirus would ‘‘fade out’’ during the winter even in the presence of considerable numbers of unvaccinated susceptible children. By contrast, there is very little seasonality in tropical climates (Nathanson and Martin, 1979). (2) In contrast to injected IPV, OPV must infect a vaccinated person in order to produce an immune response. When a group of seronegative susceptible children are fed OPV, most (but not all) will be

Confirmed polio cases

8000

6000

4000

Nigeria stops OPV 1 year

2000

0

1998

2000

2002 Year

2004

2006

FIGURE 9 Global incidence of confirmed cases of paralytic poliomyelitis, 1997–2006, cumulated for Africa, Southeast Asia, and Eastern Mediterranean regions [after WHO (2007), with permission].

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infected and immunized; for this reason, most schedules call for 2–3 doses of OPV to maximize the percent of vaccinees who are successfully immunized. It has long been known that human enteroviruses will ‘‘interfere’’ with OPV. As a result, the proportion of successful immunizations is highest when children are fed OPV in the winter time in temperate climates (>95%) and can be much lower (40–80%) when children in tropical climates are fed OPV, particularly in settings where other enteroviruses are highly prevalent (Sabin et al., 1960). (3) It can be difficult to immunize a high proportion of children in developing countries that may lack a wellestablished health care delivery system for well child care. In some instances, national immunization days (NIDs) have been used to overcome this problem, but this strategy requires strong government support. Another impediment may be active resistance of a subset of the population to immunization, arising from cultural norms that can be exacerbated by political controversy (Aylward and Mayer, 2006). To some extent, each of these factors has operated in South Asia and West Africa, areas where enteroviruses are highly prevalent and seasonality is relatively muted (Aylward and Mayer 2006; Workshop at the National Institutes of Health, 2007). In northern Nigeria, rumors that the intense polio immunization campaign was intended to sterilize girls led to a one year moratorium on vaccination. In this area, the problem appears to be mainly under utilization of OPV, that is, ‘‘failure to vaccinate.’’ In northern India (Bihar and Uttar Pradesh states), a massive effort has been made to immunize children, but the seroconversion rate has been as low as 10–20%, indicating a ‘‘failure of the vaccine’’ (Workshop at the National Institutes of Health, 2007). In this area of India, enteric infections circulate at a very high level, so that infants under 6 months of age—who are difficult to access—play a role in maintaining wild polioviruses. The local and national governments are pressing forward to overcome these problems, mainly relying on a two-pronged strategy: the use of monovalent type 1 OPV (in place of trivalent OPV) and a commitment of additional human resources in the few remaining sites of indigenous wild polioviruses (Heymann et al., 2006). However, in 2007 (time of writing), the experts diverge in their views whether or not wild type 1 poliovirus will ever be eradicated (Aylward et al., 2005; Katz, 2006; Roberts, 2006; Thompson and Duinter Tebbens, 2007).

XI. VACCINE-DERIVED POLIOVIRUSES AND THE ERADICATION ENDGAME The development of methods for rapid sequencing of viral genomes has made it possible to distinguish vaccine-derived polioviruses (VDPVs) from wild poliovirus strains. The genomes of VDPV are much closer to

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their parental OPV strain than they are to wild poliovirus isolates. Because poliovirus genomes evolve at a fairly constant rate, the number of nucleotide differences between OPV and a VDPV isolate can be used to calculate the length of time that a VDPV isolate has been circulating in the human population. Tracking of individual virus strains through the population has shown that each year about 1% of nucleotides in the genome of VDPV is replaced (Kew et al., 2005). Because the poliovirus genome is about 7500 nucleotides, it appears that about 75 mutations are introduced in the course of a year, or one new mutation every 5 days. On human passage of OPV, there is a strong selection for mutants with a revertant phenotype, defined as an increase in monkey neurovirulence. Sequencing shows that reversion is either due to reversal of attenuating mutations or due to new compensating mutations (Agol, 2006). The selection for revertant strains implies that the attenuating mutations in OPV strains make them less fit than wild polioviruses for replication in the gut. Presumably, the revertant mutations also enhance neurovirulence, although selection does not involve passage in the CNS. The frequent and rapid reversion of OPV to viruses of enhanced virulence has raised the possibility that VDPV could spread into the community and cause cases of paralytic poliomyelitis. A search for polio outbreaks caused by VDPV has uncovered about 10 such outbreaks, occurring between 1990 and 2007 (Aylward et al., 2006; Kew et al., 2004, 2005; Martin et al., 2000). These outbreaks share several common features. They tend to occur in communities with relatively low OPV coverage (less than 50% of children have been immunized), providing a large population of susceptible hosts for the spread of VDPV. Outbreaks have in general been small, with less than 50 paralytic cases, and most have been self-limited. In the one instance where the outbreak was discovered in time for intervention, an aggressive mass immunization program with OPV brought the outbreak to a conclusion (Kew et al., 2002).

A. Questions unanswered: What strategy should be followed if wild polioviruses are eradicated? There are at least two options for countries where wild polioviruses have been eliminated, either shift from OPV to IPV or discontinue poliovirus immunization (Heymann et al., 2006). Many industrialized countries have already invoked the first option that eliminates the risks of VDPV outbreaks and maintains population-wide immunity. However, in developing countries, there are several substantial impediments to conversion to IPV: the costs of IPV (which is much more expensive than OPV), the lack of an adequate supply of IPV, and the absence of a

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health care system sufficient to insure administration of an injected vaccine to a high proportion of children. What are the epidemiological consequences of terminating OPV without shifting to IPV? Will this lead to outbreaks of VDPV? Several countries have adopted such a strategy. Best known is the experience in Cuba that has a strongly centralized public health system. Cuba has for many years relied on NIDs, when all young children and their parents were immunized with OPV during a single day. Tracking of excreted enteroviruses following NIDs indicated that OPV disappeared from communal sewage within several months following the mass immunization, presumably because of a paucity of susceptible children to maintain VDPV (Mas Lago et al., 2001). There have been no reported outbreaks associated with VDPV in Cuba. Another example is New Zealand, where the termination of OPV was carefully planned and monitored (Fig. 10). VDPV disappeared within about 5 months after converting from OPV to IPV (Huang et al., 2005). PCR testing identified a few virus genomes in the sewage and in fecal samples several months after the apparent 100

Feb 1, 2002 switch from OPV to IPV

% Positive for polioviruses

80

60

40

20

0 Jan 2002

Jan 2003 Months, November, 2001 to April, 2003

FIGURE 10 Disappearance of VDPV after termination of OPV immunization. Frequency of poliovirus isolates in sewage after switching from OPV to IPV in New Zealand, February, 2002. After terminating OPV, VDPV were isolated for about 5 months at decreasing frequency. There were five VDPV isolates at 6–12 months following the termination of OPV, but it was inferred that these were due to viruses imported by visitors from abroad because they were 99.7–100% homologous with OPV, which indicated that they had been shed by persons who had received OPV after February, 2002 [reprinted from Huang et al. (2005), with permission from Elsevier].

Pathogenesis of Poliomyelitis

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disappearance of VDPV. However, molecular analysis indicated that these viruses had been excreted by recent vaccinees, leading to the conclusion that they represented imported VDPV, possibly carried by visitors from Australia where OPV was still being used. These reports suggest that it may be possible to terminate the use of OPV without the occurrence of VDPV outbreaks, in settings where a high proportion (? >90%) of children have received OPV. The New Zealand data also indicates that it will be important to synchronize the termination of OPV in contiguous countries. However, recent discussions (Workshop at the National Institutes of Health, 2007) have led to a general—if not universal—consensus that it is not acceptable (epidemiologically or ethically) to terminate OPV without shifting to IPV because of the potential dangers of VDPV outbreaks. It remains to be seen whether there is the leadership and political will among the governments and nongovernment organizations of the northern countries to commission the production of large supplies of IPV and support the logistics of IPV administration in low-income countries. Many questions regarding the ‘‘endgame’’ in poliovirus eradication remain to be answered (Workshop at the National Institutes of Health, 2007). Will there be a shift from OPV to IPV in low-income countries (Fine et al., 2004; Workshop at the National Institutes of Health, 2007)? If OPV is terminated, will outbreaks of VDPV occur? If outbreaks do occur, can they be identified and controlled (Dowdle et al., 2006; Katz, 2006; Thompson and Duinter Tebbens, 2007)? Can VDPV outbreaks be controlled with IPV or will OPV be required (fighting fire with fire)? And, as a worse-case scenario, will the circulation of VDPV make global eradication of polioviruses impossible (Roberts, 2006)?

XII. REPRISE Most of the outstanding questions in the pathogenesis of poliomyelitis could be attacked with the vast array of experimental techniques now available (Racaniello, 2006). Salient questions include: (1) What cells in the gastrointestinal tract are initially infected and act as the source of excreted virus? (2) What is the receptor used by mouse-adapted strains of poliovirus and how can some polioviruses use both mouse and primate receptors? (3) What determines species differences in susceptibility of the gastrointestinal tract to polioviruses? Why cannot PVR transgenic mice be infected by the natural enteric route? (4) Why are neuroadapted polioviruses unable to infect nonneural cells? (5) What is the role of postentry blocks in replication as determinants of neurovirulence? (6) What route(s) does poliovirus take to enter the CNS and how does it cross the blood– brain barrier? (7) Why does poliovirus preferentially attack lower motor

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neurons in contrast to many other neuronal types within the CNS? (8) Does cellular immunity play any role in recovery from acute infection or in vaccine-induced protection? (9) In which cells does poliovirus persist in patients with g-globulin deficiencies? (10) Is there any evidence that poliovirus genomes can persist in immunocompetent hosts? (11) Why has type 2 poliovirus been eradicated while types 1 and 3 have not? (12) Can transmission of VDPVs be prevented with IPV? (13) What is the best strategy to control and eliminate VDPVs? Poliomyelitis has long served as a model for studies of viral pathogenesis, and it would be of great interest to fill in some of these remaining gaps in knowledge before the curtain is rung down by a future moratorium on research with polioviruses.

ACKNOWLEDGMENTS This chapter is dedicated to the memory of David Bodian, who was my mentor in viral pathogenesis. I thank Philip Minor and Frederick Murphy for their insightful comments and constructive suggestions.

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CHAPTER

2 Cutting the Gordian Knot-Development and Biological Relevance of Hepatitis C Virus Cell Culture Systems Judith M. Gottwein* and Jens Bukh*,†,‡

Contents

I. Introduction II. Genetic Heterogeneity of HCV—Genotypes, Subtypes, Isolates, and Quasispecies III. The HCV Genome and Its Encoded Proteins IV. Host Cell Factors Supporting the HCV Life Cycle V. Consensus HCV CDNA Clones—Infectious in Transfected Chimpanzees VI. The Replicon System—Autonomous HCV RNA Replication in Hepatoma Cell Lines A. Identification of adaptive mutations led to more efficient replicon systems B. The study of replicon systems led to identification of highly permissive Huh7 cell lines VII. Pseudo-Particles Expressing the HCV Envelope Proteins (HCVpp)—A System for the Study of Viral Entry and Neutralization VIII. The JFH1 Isolate—Generation of Cell Culture Derived HCV (HCVCC) in Full Viral Life Cycle Cell Culture Systems

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* Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research

Centre, Copenhagen University Hospital, Hvidovre, Denmark Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Denmark { Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland {

Advances in Virus Research, Volume 71 ISSN 0065-3527, DOI: 10.1016/S0065-3527(08)00002-X

#

2008 Elsevier Inc. All rights reserved.

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A. The original and adapted JFH1 cell culture system B. The J6/JFH1 cell culture system C. Analysis of HCV buoyant density suggests a role of lipoproteins for the viral life cycle D. Possible causes of special growth characteristics of JFH1 and J6/JFH1 E. Applicability of JFH1 and J6/JFH1 cell culture systems IX. Perspectives for Further Development of HCV Cell Culture Systems A. Adaptation of cell culture systems to yield higher viral titers B. Cell culture systems for other HCV genotypes C. Expansion of cell culture systems to different host cells X. Conclusion—Implications of Novel Cell Culture Systems Acknowledgments References

Abstract

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Worldwide
180 million people are chronically infected with hepatitis C virus (HCV). HCV isolates exhibit extensive genetic heterogeneity and have been grouped in six genotypes and various subtypes. Additionally, several naturally occurring intergenotypic recombinants have been described. Research on the viral life cycle, efficient therapeutics, and a vaccine has been hampered by the absence of suitable cell culture systems. The first system permitting studies of the full viral life cycle was intrahepatic transfection of RNA transcripts of HCV consensus complementary DNA (cDNA) clones into chimpanzees. However, such full-length clones were not infectious in vitro. The development of the replicon system and HCV pseudo-particles allowed in vitro studies of certain aspects of the viral life cycle, RNA replication, and viral entry, respectively. Identification of the genotype 2 isolate JFH1, which for unknown reasons showed an exceptional replication capability and resulted in formation of infectious viral particles in the human hepatoma cell line Huh7, led in 2005 to the development of the first full viral life cycle in vitro systems. JFH1-based systems now enable in vitro studies of the function of viral proteins, their interaction with each other and host proteins, new antivirals, and neutralizing antibodies in the context of the full viral life cycle. However, several challenges remain, including development of cell culture systems for all major HCV genotypes and identification of other susceptible cell lines.

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I. INTRODUCTION About 180 million people are infected with hepatitis C virus (HCV) worldwide, with an incidence of 3–4 million each year (Alter and Seeff, 2000; Wasley and Alter, 2000; World Health Organization, 1998). While the acute phase of infection is mostly asymptomatic, 50–80% of infected individuals develop chronic hepatitis and are therefore at increased risk of developing severe liver disease. Thus, 4–20% of persistently infected individuals develop liver cirrhosis after 10–30 years. In patients with liver cirrhosis the risk of developing hepatocellular carcinoma is 1–5% per year (Hoofnagle, 2002; Seeff, 2002). HCV induced end-stage liver disease is now the leading indication for liver transplantation in developed countries (Brown, 2005). HCV is a small enveloped virus, classified as a member of the Flaviviridae family. Its genome consists of a long (9.6 kb) single stranded (ss) RNA of positive polarity composed of 50 and 30 untranslated regions (UTR) and one long open reading frame (ORF) encoding the HCV structural proteins (Core, E1, E2), p7 and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (Bartenschlager et al., 2004; Lindenbach and Rice, 2005; Penin et al., 2004b), which are acting, supported by host cell proteins, at different steps of the viral life cycle. HCV isolates from around the world exhibit significant genetic heterogeneity; six major genotypes (genotype 1–6) and numerous subtypes (a, b, c, etc.) have been identified (Simmonds et al., 2005). The only approved therapy for HCV, combination therapy with interferon-a (IFN-a) and ribavirin, is costly and associated with severe side effects and contraindications (De Francesco and Migliaccio, 2005; Falck-Ytter et al., 2002; Feld and Hoofnagle, 2005; Manns et al., 2006). A sustained viral response can be achieved in only about 50% of infected patients in general with important genotype-specific differences in treatment outcome (Manns et al., 2001; Pearlman, 2004). There is no vaccine against HCV. Thus, there is an urgent need for improved antiviral drugs and a prophylactic or therapeutic vaccine (De Francesco and Migliaccio, 2005). Since its discovery in 1989, research on HCV has been hampered by the lack of a small animal model and appropriate cell culture systems. The only true animal model remain chimpanzees which are expensive and only available for studies in the United States (Bukh, 2004). An emerging promising small animal model, the uPA-SCID mouse grafted with human hepatocytes permits mainly infectivity studies (Mercer et al., 2001; Meuleman et al., 2005). These mice are difficult to generate and allow investigations only during a restricted time frame. Even more limiting for classical virus research has been the absence of a robust in vitro system. An important first advance in the quest for a HCV cell

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culture system was the development of full-length consensus complementary (cDNA) clones. RNA transcripts of cDNA clones of HCV strain H77 (genotype 1a) were initially shown to be infectious in the chimpanzee model (Kolykhalov et al., 1997; Yanagi et al., 1997). However, these clones as well as subsequently developed infectious cDNA clones of other genotypes did not produce viral particles in vitro (Bartenschlager, 2006; Bartenschlager and Sparacio, 2007). A great leap forward was the development of subgenomic and subsequently full-genome length replicons, which allowed the study of HCV RNA replication in the human liver hepatoma cell line Huh7 (Blight et al., 2000; Lohmann et al., 1999). The development of pseudo-particles bearing the HCV envelope glycoproteins (HCVpp) created an important tool for the study of viral entry and neutralizing antibodies (ntAb) in Huh7 cells (Bartosch et al., 2003b; Hsu et al., 2003). Thus, researchers had been provided with two different model systems, which employed the same cell line but mimicked different aspects of the HCV life cycle. However, certain aspects, such as assembly and release and the complexity of the full viral life cycle could still not be studied in cell culture. A breakthrough came with the identification of JFH1, the genotype 2a isolate of a Japanese patient with fulminant hepatitis (Kato et al., 2001). For unknown reasons, this isolate was capable of forming cell culture derived infectious viral particles (HCVcc) after transfection of RNA transcripts from a cDNA clone into Huh7 or derivative cell lines with improved permissiveness, such as Huh7.5 (Wakita et al., 2005; Zhong et al., 2005). Also an intragenotypic recombinant genotype 2a genome, in which the structural genes (Core, E1, E2), p7 and NS2 of JFH1 were replaced by the respective genes of strain J6 (genotype 2a) from another infectious clone pJ6CF (Yanagi et al., 1999a), produced infectious viral particles in Huh7.5 cells with accelerated kinetics (Lindenbach et al., 2005). Thus, eventually classical virological in vitro studies of HCV became possible, even though these studies were still restricted to genotype 2a and to a single hepatomaderived cell line. Therefore, besides the study of the HCV life cycle in this complete cell culture model, a current research focus is the expansion of the cell culture systems to the six major HCV genotypes and perhaps to other cell lines.

II. GENETIC HETEROGENEITY OF HCV—GENOTYPES, SUBTYPES, ISOLATES, AND QUASISPECIES HCV isolates from around the world exhibit significant genetic heterogeneity. Thus, at least six major HCV genotypes (genotypes 1–6) have been defined by phylogenetic analysis (Bukh et al., 1992, 1993, 1994; Simmonds et al., 1993). Their genomes differ by 31–33% on the nucleotide

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level (Bukh et al., 1995; Simmonds et al., 2005). In addition, numerous subtypes have been described (a, b, c, etc.) showing a difference of 20–25% at the nucleotide level, with genotype 5 being currently the only genotype featuring only one subtype. A subtype represents different isolates or strains derived from different infected individuals, such as the prototype isolates HCV-1 and H77, belonging to subtype 1a; their genomes typically differ by
2–4% at the nucleotide level. In each individual infected with a particular isolate, HCV circulates as a quasispecies which is a population of closely related genomes exhibiting 1–2% variation at the nucleotide level, clustered around a consensus sequence (Bukh et al., 1995; Forns et al., 1999; Martell et al., 1992; Weiner et al., 1991). The quasispecies distribution has been shown to influence the progression from acute to chronic HCV infection (Farci et al., 2000), the progression of liver fibrosis (Wang et al., 2007), as well as the response to IFN (Farci et al., 2002; Pawlotsky et al., 1998, 1999). The development of specialized HCV sequence databases [European HCV database, euHCVdb (Combet et al., 2007); Los Alamos hepatitis C sequence database, usHCVdb/LANL (Kuiken et al., 2005); Japanese Hepatitis Virus Database, jpHCVdb/ Nagoya (Kuiken et al., 2006b; Shin-I et al., 2008)], which organize sequences of the published HCV isolates and provide tools for their analysis, facilitate studies of HCV genetic heterogeneity. Importantly, to achieve consistent classification and naming of HCV genotypes, consensus guidelines were developed (Simmonds et al., 2005) and adapted by the databases. Furthermore, to permit straightforward comparisons between HCV isolates, a comprehensive system for consistent numbering of HCV sequences has been proposed (Kuiken et al., 2006a). HCV genotypes differ in prevalence and geographic distribution: In the Western World, genotype 1 is the most common, followed by genotypes 2 and 3. For example, in the United States
75% of HCV-infected individuals have genotype 1 (1a or 1b), while
15% are infected with genotype 2 (2a and 2b) and
5% with genotype 3 (predominantly 3a) (Bukh et al., 1995; Hoofnagle, 2002). However, genotype 3 is more prevalent in Europe infecting up to 50% of patients in certain countries (Corbet et al., 2003; Dal et al., 2002; Harris et al., 1999; Tamalet et al., 2003) with a high prevalence in specific risk groups, such as intravenous drug users (Berg et al., 1997; Bourliere et al., 2002; Pawlotsky et al., 1995). Genotype 3 has been shown to spread in Europe (van Soest et al., 2006) and, in addition, to be very prevalent in many highly populated countries in Asia (McCaughan, 2000) such as in India (Hissar et al., 2006) and Pakistan (Moatter et al., 2002), as well as in the former USSR (Shustov et al., 2005), Australia (Kaba et al., 1998), and Brazil (Campiotto et al., 2005). Genotypes 4–6 are more common in areas with high prevalence or even endemic levels of HCV infection. Genotype 4 is the most prevalent genotype in Egypt, where close to 20% of the population is infected (Kamel et al., 1992;

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Ray et al., 2000; Saeed et al., 1991), rendering it an interesting region for clinical vaccine trials. This endemic rate of genotype 4a infection was presumably the result of a nationwide parenteral antischistosomal therapy campaign in the 1970s (Arthur et al., 1995; Frank et al., 2000). Genotype 4 is also highly prevalent in central Africa and the Middle East (Bukh et al., 1993) but several recent reports also point to its spread in Europe (de Vries et al., 2006; Delwaide et al., 2006; Fernandez-Arcas et al., 2006; Katsoulidou et al., 2006; Mathei et al., 2005; Nicot et al., 2005; Toro et al., 2006; van de Laar et al., 2006). Genotype 5 has mainly been described in South Africa (Bukh et al., 1993) but has recently been spreading in European cities (Henquell et al., 2004; Jover et al., 2001). Genotype 6 is highly prevalent in Southeast Asia (Mellor et al., 1996; Nguyen and Keeffe, 2005). Interestingly, studies of HCV genetic heterogeneity in particular populations allow conclusions about the history of the epidemic and the routes of infection. For example, in the United States, Europe, and Japan the HCV epidemic apparently resulted from the relatively recent introduction of rather few strains from endemic countries (Simmonds, 1999), with viral spread predominantly occurring via transfusion of blood products and needle sharing by intravenous drug users (Alter, 1999). This apparently led to the presence of relatively few genotypes and subtypes (primarily 1a, 1b, 2a, 2b, and 3a) with limited genetic heterogeneity (Simmonds, 2001; Stumpf and Pybus, 2002). However, in certain populations in Africa and Southeast Asia, in which HCV has been circulating for a long time and has been transmitted by various routes including sexual and household transmissions, often a greater genetic heterogeneity is observed (Mellor et al., 1995; Simmonds, 2001, 2004; Stumpf and Pybus, 2002), making it difficult or impossible to classify such isolates within specific subtypes. Thus, for example, a broad genetic heterogeneity has been shown for genotype 2 isolates in Ghana (Candotti et al., 2003) and genotype 6 isolates in Southeast Asia (Doi et al., 1996; Mellor et al., 1996; Shinji et al., 2004). Determination of the HCV genotype is of great clinical relevance. The most striking clinical difference between genotypes is their differential sensitivity to combination therapy with IFN-a and ribavirin, currently the only licensed regimen for treatment of HCV infection. While a sustained viral response is achieved in
40–50% of patients infected with genotype 1 during a 48-week treatment course,
80–90% of genotype 2 and 3 infected patients can be cured by a treatment course of 24 weeks (Feld and Hoofnagle, 2005; Manns et al., 2001, 2006; Pearlman, 2004). Genotype 4 seems rather resistant toward treatment (El-Zayadi et al., 1999; Zylberberg et al., 2000), whereas genotype 5 appears to be relatively sensitive (LegrandAbravanel et al., 2004), and genotype 6 shows an intermediate sensitivity (Hui et al., 2003). In addition, particular clinical features have been attributed to different genotypes. For example, genotype 3 is suspected to be involved in more pronounced development of hepatocellular steatosis

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(Abid et al., 2005; Hourioux et al., 2007b; Jackel-Cram et al., 2007; Negro, 2006; Rubbia-Brandt et al., 2000). The association of genotype 1b with an aggressive course of liver disease, resulting in accelerated progression to liver cirrhosis and hepatocellular carcinoma (Pozzato et al., 1991; Prieto et al., 1999; Widell et al., 2000; Zein, 2000), has been controversial (Adinolfi et al., 2000; Wiese et al., 2000). Considering these differences among HCV genotypes, it is possible that different HCV genotypes would show differential responsiveness toward newly developed antiviral compounds (Pawlotsky et al., 2007) and that neutralizing antibodies might not cross-neutralize all genotypes (Meunier et al., 2005; Scheel et al., 2008). HCV genetic heterogeneity has so far been attributed to the lack of proofreading activity of the RNA-dependent RNA polymerase (NS5B) (Stumpf and Pybus, 2002). This leads to a high mutation rate, allowing HCV to collect mutations that are not detrimental for its life cycle and that in some cases permit escape from host immune responses. Interestingly, recent evidence points to an additional role of recombination events for genetic heterogeneity. Recombination occurs frequently among RNA viruses including members of the Flaviviridae family (Becher et al., 2001; Guillot et al., 2000; Holmes et al., 1999; Lai, 1992; Tolou et al., 2001; Twiddy and Holmes, 2003; Uzcategui et al., 2001; Worobey and Holmes, 1999; Worobey et al., 1999), but had not been thought to play a major role for HCV (Smith and Simmonds, 1997). However, detection of naturally occurring intergenotypic recombinants between genotypes 2k/1b (Kalinina et al., 2002; Moreau et al., 2006), 2b/1b (Kageyama et al., 2006), 2/5 (Legrand-Abravanel et al., 2007), and 2i/6p (Noppornpanth et al., 2006) as well as an intragenotypic (1b/1a) HCV recombinant (Colina et al., 2004) points to a greater role of recombination than previously anticipated (Fig. 1). In theory, several requirements have to be met for intra- or intergenotypic recombination to occur: The same patient and host cell must be infected with different HCV isolates, which is more likely to occur in multiexposed individuals living in endemic regions or in intravenous drug users. However, such mixed infections are thought to rarely occur in patients (Viazov et al., 2000) and it has recently been shown that mechanisms acting at the post-entry level might be able to counteract superinfection with different HCV viruses (Schaller et al., 2007; Tscherne et al., 2007). Further, genomes undergoing recombination must come into close proximity within the cell, which is a given for HCV, because replication is supposed to take place in a restricted specialized cellular compartment, designated the membranous web (see below). Recombination events are further determined by the properties of the viral polymerase. Different mechanisms might be involved in recombination of ssRNA genomes (Lai, 1992). The breakage-and-rejoining mechanism observed for double stranded (ds) DNA is, in contrast to most RNA recombination events, site specific. Therefore, the copy choice mechanism seems more

C

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25 8 27 0 69

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NS5A

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5⬘UTR

Putative function of encoded protein

3⬘UTR Capsid formation

Envelope glycoprotein (entry)

B Genotype 2k/1b St. Petersburg, Russia Kalinia 2002

N-terminus: C-terminus: Membra- Phosphoprotein serin protease RNA helicase/ nous web (replication) (polyprotein NTPase (replication) processing) (replication) NS2/3 cystein autoprotease (polyprotein processing) NS3 Ion channel cofactor (assembly)

RNA dependent RNA polymerase (replication)

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Genotype 2/5 France Legrand-Abravanel 2007

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Genotype 2b/1b Philippines Kageyama 2006

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p7 NS2

Genotype color codes

NS 4A

NS3 1a

1b

2b

2i

NS4B 2k

NS5A 2

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NS5B 6p

FIGURE 1 Genomic organization of hepatitis C virus (HCV) and the genetic composition of naturally occurring intra- and intergenotypic recombinants. (A) HCV genome organization of the reference isolate H77 (AF009606). Numbers indicate nucleotide positions at gene borders, with number 1 being the first nucleotide of the 50 untranslated region (UTR). C, Core; E, envelope; NS, nonstructural. Putative functions of the encoded proteins are indicated below. (B) Naturally occurring intra- and intergenotypic recombinant HCV isolates. The genotype names are indicated to the left of each genome together with the associated references (Colina et al., 2004; Kageyama et al., 2006; Kalinina et al., 2002; Legrand-Abravanel et al., 2007; Noppornpanth et al., 2006). Genotype-specific sequences are color-coded. Recombination crossover sites are indicated by arrows, but for the 2i/6p and 2/5 recombinants only a crossover region could be determined; numbers indicate the nucleotide position or genome region of recombination site according to the reference genome H77 (AF009606). In recombination analysis, the N-terminal part of the 2/5 recombinant was found to be related to genotype 2k (Legrand-Abravanel et al., 2007). On the basis of phylogenetic analysis of NS5B, the C-terminal part of this recombinant was identified as genotype 5. However, in contrast to 25 other genotype 5a isolates from the same region in France, which clustered with genotype 5a reference sequences, the C-terminal part of the 2/5 recombinant formed a distinct branch of the phylogenetic tree, apparently belonging to a new subtype of genotype 5 (Legrand-Abravanel et al., 2007).

likely. It employs template switching during RNA synthesis and probably requires pausing of the polymerase, shown to occur at regions with strong secondary structure. Intriguingly, most of the naturally occurring

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intergenotypic isolates exhibit a crossover point close to the NS2/NS3 junction (Kageyama et al., 2006; Kalinina et al., 2002; Legrand-Abravanel et al., 2007; Noppornpanth et al., 2006), a region involved in formation of a putative hairpin structure (Noppornpanth et al., 2006). Furthermore, prevalence of recombinant viruses should depend on their viability. Thus, a low prevalence could point to a low viability of such recombinant viruses; indeed, in vitro, several intergenotypic genomes showed low viability (Lindenbach et al., 2005; Pietschmann et al., 2006) and depended on the acquisition of adaptive mutations presumably enabling an interplay between proteins of different genotypes (see below) (Gottwein et al., 2007; Scheel et al., 2008; Yi et al., 2007). At last, recombinant genomes can only be readily identified if recombination occurred between different genotypes or subtypes showing a certain degree of genetic heterogeneity. The natural intra- and intergenotypic genomes so far detected are the result of homologous recombination events. Theoretically, nonhomologous recombination could be a mechanism contributing to the occurrence of truncated genomes, found to circulate in HCV-infected patients (Revie et al., 2006; Yagi et al., 2005). Naturally occurring recombinants are clinically relevant. The 2k/1b recombinant has been shown to spread in Europe (Kalinina et al., 2002; Moreau et al., 2006). Because most commercially available genotyping kits employ sequence analysis of the 50 UTR (Anderson et al., 2003; Germer et al., 1999; Halfon et al., 2001; Holland et al., 1998; Scott and Gretch, 2007), which is one of the most conserved regions of the HCV genome (Bukh et al., 1992, 1995; Simmonds et al., 1994), infection with recombinants might not be diagnosed and a suboptimal treatment regimen might be chosen. It is, however, not known which part of such recombinants would determine treatment outcome. For example, in the case of the 2k/1b variant, the 2k portion would predict a more favorable response, while the 1b portion would indicate requirement for a long-term treatment course. Such treatment studies could perhaps contribute to our understanding of the differential sensitivity of the different genotypes toward treatment. However, it is clear that for reliable genotyping the analysis of additional genome regions, such as Core/E1 and NS5B, should be recommended (Bukh et al., 1995; Robertson et al., 1998; Simmonds et al., 2005).

III. THE HCV GENOME AND ITS ENCODED PROTEINS In this chapter, we aim at giving an overview on HCV genome structures which provide the basis for the molecular approaches discussed. More detailed overviews of HCV gene and protein function and interaction have been published by others (Bartenschlager et al., 2004; Lindenbach and Rice, 2005; Penin et al., 2004b). The protein functions are summarized in Fig. 1.

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The HCV genome is composed of a ssRNA molecule consisting of
9600 nucleotides (Fig. 1). It is of positive polarity and can thus be directly translated upon introduction into the host cell. The genome features a 50 and 30 UTR and one long ORF coding for the
3000 amino acids long HCV polyprotein. The expressed polyprotein is co- and posttranslationally cleaved by cellular and viral peptidases yielding the HCV structural proteins (Core, E1 and E2), the p7 protein, and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Host peptidases are believed to be responsible for cleavage at the Core/E1, E1/E2, E2/p7, and p7/NS2 junctions (Bartenschlager et al., 2004). The nonstructural proteins are released by two different HCV proteases, with the NS2/ NS3 autoprotease mediating cleavage at the NS2/NS3 junction, and the NS3/NS4A serine protease being responsible for release of the remaining nonstructural proteins (Hijikata et al., 1993a). These cleavage processes occur with differential efficiency, thus giving rise to several intermediate proteins like E2-p7-NS2, E2-p7, and NS4B-5A (Bartenschlager et al., 2004). The significance of these intermediate proteins has only been partly elucidated (Jones et al., 2007). The HCV 50 and 30 UTR sequences form strong secondary structures (Blight and Rice, 1997; Honda et al., 1999; Schuster et al., 2002; Smith et al., 2002) and are essential for genome translation and replication. The 50 UTR is highly conserved among genotypes and essential for replication (Friebe et al., 2001; Kim et al., 2002; Luo et al., 2003; Reusken et al., 2003). It contains an internal ribosomal entry site (IRES) which reaches into the Core sequence and initiates cap-independent translation by directly binding to the 40S ribosomal subunit (Ji et al., 2004; Kieft et al., 2002; Otto and Puglisi, 2004; Pestova et al., 1998; Spahn et al., 2001; Tsukiyama-Kohara et al., 1992; Wang et al., 1993). The 30 UTR consists of (i) a variable region, the sequence of which differs greatly among isolates belonging to different genotypes, (ii) a poly(U/UC) tract of variable length and composition, and (iii) the highly conserved X-region (Kolykhalov et al., 1996; Tanaka et al., 1996; Yamada et al., 1996), which was predicted to feature three stem-loop structures (SL1, 2, 3) (Blight and Rice, 1997; Ito and Lai, 1997; Yi and Lemon, 2003b). A poly(U/UC) tract of a certain length and the entire conserved X-region are essential for viral replication (Friebe and Bartenschlager, 2002; Kolykhalov et al., 2000; Yanagi et al., 1999b; Yi and Lemon, 2003a,b). While the 30 UTR seems to govern RNA negative strand synthesis, 50 UTR sequences are apparently involved in positive RNA strand generation (Binder et al., 2007). Initiation of replication depends on the interplay of cis-acting RNA elements located in the UTRs and the ORF such as the ‘‘kissing loop interaction’’ between SL2 of the 30 UTR X-region and a conserved stem-loop structure within the NS5B-coding region (Friebe et al., 2005; You et al., 2004). In addition, NS5B seems to interact with the most 30 -terminal stem loop of the X-region (SL1) in a

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genotype-specific manner (Binder et al., 2007). The HCV 30 UTR, specifically the variable region, the poly(U/UC) tract and SL1 of the X-region, are implicated in stimulation of IRES-dependent translation in human hepatoma cells (Bradrick et al., 2006; Song et al., 2006). Hereby the 30 UTR seems to facilitate termination of translation and possibly ribosome recycling (Bradrick et al., 2006) and might mediate RNA 50 –30 interactions as reported for eukaryotic RNA (Pestova et al., 2001) and other viruses like dengue virus and rotavirus (Holden and Harris, 2004; Piron et al., 1998). Core monomers presumably compose the viral nucleocapsid required for genome packaging. Core maturation involves its cleavage from the C-terminally located signal sequence, which targets E1 to the ER (Santolini et al., 1994); this cleavage is thought to be mediated by cellular signal peptidase and signal peptide peptidase (McLauchlan et al., 2002). The mature Core protein consists of two domains. Domain I seems to be implicated in binding of RNA (Cristofari et al., 2004; Fan et al., 1999; Santolini et al., 1994; Shimoike et al., 1999) and Core–Core interactions, presumably leading to capsid assembly (Boulant et al., 2005; Klein et al., 2004, 2005; Kunkel et al., 2001; Matsumoto et al., 1996; Nolandt et al., 1997). These homotypic interactions might be supported by Core binding to RNA, thus leading to conformational changes of Core resulting in an increased stability (Kim et al., 2006). Additional oligomerization motifs seem to be located in domain II of Core (Yan et al., 1998a), which might be responsible for RNA-independent spontaneous Core multimerization at the early stages of viral assembly (Kim et al., 2006). Further, the domain II seems to target Core to the cytoplasmatic surface of the endoplasmatic reticulum (ER) or of cytoplasmatic lipid droplets (Barba et al., 1997; Hope and McLauchlan, 2000; McLauchlan et al., 2002; Moradpour et al., 1996; Pietschmann et al., 2002). Thus, certain deletions and several point mutations in domain II abolished association of Core with lipid droplets, thereby reducing Core stability and self-assembly capacity (Boulant et al., 2007; Hope and McLauchlan, 2000; Hourioux et al., 2007a), and led to loss of production of infectious viruses in the JFH1 cell culture system (see below) (Boulant et al., 2007). Core interacts with HCV E1 (Baumert et al., 1998; Lo et al., 1996; Nakai et al., 2006) and recently evidence for possible interactions with p7 and NS2 has been provided (Murray et al., 2007). Interaction of Core with a wide range of host cell proteins and cellular signaling pathways has also been described (Lai and Ware, 2000; McLauchlan, 2000; Ray and Ray, 2001; Tellinghuisen and Rice, 2002; Yan et al., 2007), and in vivo Core has been implicated in the development of steatosis and hepatocellular carcinoma (Lerat et al., 2002; Moriya et al., 1998; Negro, 2006). During translation of Core unusual translation-level events, such as ribosomal frameshifting, apparently result in the expression of a family of so-called alternate reading frame proteins (ARFPs). ARFPs seem to be

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of biological relevance, because their coding sequence is conserved between different genotypes and patient sera derived antibodies show reactivity toward ARFPs (Branch et al., 2005; Varaklioti et al., 2002; Walewski et al., 2001; Xu et al., 2001). Recent studies showed that ARFPs are not essential for HCV replication in cell culture or in vivo but that their coding region might contain a functionally important RNA element (McMullan et al., 2007). E1/E2 envelope glycoprotein heterodimers reside on the viral surface (Kaito et al., 2006) and mediate HCV entry. They are type I transmembrane proteins with N-terminal ectodomains, which upon intracellular translation translocate into the ER lumen. E1/E2 maturation involves the ER chaperone and glycosylation system (Choukhi et al., 1998; Dubuisson and Rice, 1996; Merola et al., 2001). Thus, glucosidase inhibitors led to misfolded envelope glycoproteins and reduced HCVpp infectivity (Chapel et al., 2007) and E2 glycosylation modulated binding to neutralizing antibodies and the HCV coreceptor CD81 (Falkowska et al., 2007; Helle et al., 2007). Further, E1 maturation seems to depend on E2 and Core coexpression, whereas E2 folding apparently occurs independent of other viral proteins (Merola et al., 2001; Michalak et al., 1997). The C-terminal hydrophobic transmembrane domains of E1 and E2 are multifunctional and implicated in membrane anchoring, ER retention (Cocquerel et al., 1998, 1999; Duvet et al., 1998; Flint and McKeating, 1999), and formation of the noncovalent heterodimeric E1/E2 complexes (Cocquerel et al., 2000, 2002; Deleersnyder et al., 1997; Op De Beeck et al., 2000, 2001; Selby et al., 1994). Additionally, heterodimer formation has been shown to be dependent on an E2 membrane-proximal heptad repeat sequence (Drummer and Poumbourios, 2004). Recently, a polytopic form of E1 has been found to span the ER membrane twice and exhibit a cytoplasmic loop associating with Core, which in the presence of RNA oligomerized to form capsid-like structures (Nakai et al., 2006), pointing to an important role of E1 in virus assembly. Further, a role of the transmembrane domains in viral fusion with the host cell membrane has been described (Ciczora et al., 2007). During entry, E2 apparently first interacts with the putative receptor complex including the tetraspanin CD81 (Drummer et al., 2002; Flint et al., 1999a; Pileri et al., 1998), SR-BI, physiologically involved in uptake of cholesteryl esters from high-density lipoprotein (HDL) (Scarselli et al., 2002), and the tight junction component claudin-1 (Evans et al., 2007). After receptor binding by E2, the E1 protein might be responsible for fusion of the virus with the cell membrane (Flint and McKeating, 1999; Garry and Dash, 2003; Perez-Berna et al., 2006). The E2 receptor binding domain (RBD) localizes to the N-terminal three fourth of E2; its CD81binding motifs are conserved (Drummer et al., 2006; Owsianka et al., 2006) and are interspersed by the three variable regions [HVR1 (hypervariable

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region 1), HVR2, and igVR (intergenotypic variable region)] of E2 which can be deleted without affecting E2 binding to CD81 (Kato et al., 1992; McCaffrey et al., 2007; Weiner et al., 1991). Sequences of HVR1, which localizes to the very N-terminus of E2 differ by as much as 80% at the amino acid level. While deletion of HVR1 from the HCV genotype 1a reference strain H77 led to attenuation but not loss of infectivity in the chimpanzee model (Forns et al., 2000) and deletion of all three E2 variable regions did not affect CD81 binding to H77 E2, infectivity of HCV genotype 1 HCVpp was enhanced by an HDL-mediated interaction of HVR1 with SR-BI (Bartosch et al., 2005). The amount of basic residues in HVR1 has been shown to influence infectivity in an HDL/SR-BIindependent manner (Callens et al., 2005). Beyond its influence on infectivity, the interaction of HVR1 with HDL might shield HCVpp from neutralizing serum antibodies (Bartosch et al., 2005). Further, HVR1 is supposed to play a not yet entirely clarified immunological role. It contains a neutralization epitope (Farci et al., 1996; Zibert et al., 1995) and in patients rapid genetic evolution of HVR1 has been shown to predict progression from acute to chronic hepatitis (Farci et al., 2000). On the contrary, HVR1 has been proposed to act as ‘‘immunological decoy’’ by stimulation of a strong immune response, which, however, is not able to clear the infection (Mondelli et al., 2001; Ray et al., 1999). HVR2 is supposed to modulate E2 receptor binding (Roccasecca et al., 2003; Scarselli et al., 2002). The function of the recently identified igVR (McCaffrey et al., 2007) which is conserved within but varies between HCV genotypes is so far unclear. The E2 PePHD domain (PKR/eIF2aplha phosporylation homology domain) maps downstream of the three variable regions and is similar to phosphorylation sites of PKR (a dsRNA activated, IFN-ainduced protein kinase). It has been shown to bind to and inhibit PKR (Taylor et al., 1999). The fact that PePHD of HCV genotype 1 shows the greatest similarity to the equivalent domain of PKR and that it is genetically conserved [in sequences retrieved from databases and during IFN treatment in patients (Polyak et al., 2000)], might point to a possible explanation for the reduced sensitivity of genotype 1 to IFN treatment. The envelope glycoproteins and HCV entry are further discussed in the section on the HCVpp system (see below) and in (Barth et al., 2006; Bartosch and Cosset, 2006; Cocquerel et al., 2006; Diedrich, 2006). p7, consisting of only 63 amino acids, has been shown to be a transmembrane protein with two transmembrane domains (TM1 and TM2) connected by a short basic loop, which is located in the cytoplasm. p7 oligomerizes to form a putative ion channel (Carrere-Kremer et al., 2002; Clarke et al., 2006; Griffin et al., 2003; Patargias et al., 2006; Premkumar et al., 2004), which upon assembly in artificial lipid membranes could be blocked with the antiviral drug amantadin (Griffin et al., 2003), however, apparently only at high concentrations cytotoxic for

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Huh7 cells (Steinmann et al., 2007b). Additionally, several other compounds have been described to block p7 (Pavlovic et al., 2003; Premkumar et al., 2004; Steinmann et al., 2007b; Stgelais et al., 2007). p7 is not critical for replication but for infectivity in the chimpanzee (Sakai et al., 2003) and cell culture model (Jones et al., 2007; Steinmann et al., 2007a). Employing so far unresolved tracking mechanisms, p7 seems to reside in both normal ER and specialized areas of ER, designated mitochondrion-associated membranes (Carrere-Kremer et al., 2002; Griffin et al., 2005; Haqshenas et al., 2007b; Pavlovic et al., 2003). ERassociated p7 appears to be a key determinant of viral assembly (Steinmann et al., 2007a) or an even earlier stage in viral morphogenesis (Jones et al., 2007), possibly in analogy to other viroporins (Gonzalez and Carrasco, 2003) by securing a stable pH, thus preventing the envelope glycoproteins from prematurely adopting a fusogenic conformation. This hypothesis is supported by the finding that HCV glycoprotein-mediated cell entry is pH dependent (Hsu et al., 2003) and that p7 can replace the influenza A virus M2 protein, implied in maintenance of the pH-sensitive conformation of hemagglutinin during viral release, in a cell-based assay (Griffin et al., 2004). The NS2/NS3 autoprotease is responsible for cleavage at the NS2/ NS3 junction of the HCV polyprotein (Grakoui et al., 1993; Hijikata et al., 1993a; Reed et al., 1995). The revelation of the crystal structure of its catalytic domain confirmed it to be a cysteine protease (Gouttenoire et al., 2006; Lorenz et al., 2006). NS2/NS3 activity has been shown to be zinc dependent (Pallaoro et al., 2001; Tedbury and Harris, 2007). Released NS2 is an integral membrane protein of unclear function (Santolini et al., 1995). NS2 seems not to be required for HCV RNA replication but production of infectious viruses, probably acting at an early stage of virus morphogenesis (Jones et al., 2007). Further, a role in modulation of host cell gene expression and apoptosis has been described (Kaukinen et al., 2006; Welbourn and Pause, 2007). NS3 is a multifunctional protein: The N-terminal serine protease domain is, in association with its cofactor NS4A, responsible for the polyprotein cleavage events downstream of NS3, which occur in a preferential order (Bartenschlager et al., 2004). While the central part of NS4A seems to be necessary for NS3 stabilization (Kim et al., 1996; Lin et al., 1995; Tanji et al., 1995; Yan et al., 1998b), its hydrophobic N-terminal part appears to anchor the NS3/NS4A complex to the ER and mitochondrial outer membrane (Nomura-Takigawa et al., 2006; Wolk et al., 2000). The C-terminal domain of NS3 presents an RNA helicase/NTPase domain (Tai et al., 1996), which mediates cyclic, ATP-dependent unwinding of RNA (Dumont et al., 2006; Frick, 2007; Levin et al., 2003, 2005; Serebrov and Pyle, 2004). Helicase activity might be required for unwinding of strong secondary RNA structures before RNA replication, for separation

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of nascent from template RNA strands during replication, and/or for liberation of RNA binding proteins from RNA. NS3 helicase activity is augmented by NS4A (Kuang et al., 2004; Pang et al., 2002) and NS4A has been shown to be important for HCV RNA replication (Lindenbach et al., 2007). A naturally occurring truncated form of NS3 appears to be the result of internal NS3 cleavage mediated by NS4A-dependent NS3 protease activity in a genotype-specific manner and might act as an oncogen (Kou et al., 2007). Recently evidence for a role of NS3 in evasion of the innate host defense by inhibition of the IFN response has been provided (Feld and Hoofnagle, 2005; Gale and Foy, 2005; Meylan et al., 2005). NS4B is an integral membrane protein (Hugle et al., 2001) apparently inducing the formation of a specialized, presumably ER derived membranous compartment, designated the membranous web, which probably provides a scaffold for assembly of the HCV replication complex (Egger et al., 2002; El-Hage and Luo, 2003; Gosert et al., 2003; Moradpour et al., 2004b; Shi et al., 2003). NS4B shows a dual membrane topology: Either its N- and C-terminus are oriented toward the cytosol or the N-terminal end of NS4B translocates into the lumen of the ER (Lundin et al., 2003, 2006). It has recently been reported that induction of the membranous web depended on the latter topology (Lundin et al., 2006). NS5A is anchored to ER membranes by an amino-terminal amphipathic alpha-helix (Brass et al., 2002, 2007; Penin et al., 2004a). Structural analysis shows the zinc-binding domain (Moradpour et al., 2005; Tellinghuisen et al., 2005) to exhibit conserved external surfaces which presumably interact with cellular proteins (Macdonald and Harris, 2004; Tellinghuisen and Rice, 2002) and other components of the HCV replication complex (Gosert et al., 2003). This region also contains a highly basic channel, which could serve as an RNA-binding pocket during replication (Huang et al., 2005; Tellinghuisen et al., 2005). NS5A is of critical importance for replication. Mutation of four conserved cysteine residues moderating zinc binding (Tellinghuisen et al., 2004), as well as of the N-terminal amphipathic helix mediating NS5A membrane localization (Elazar et al., 2003), inhibited replication. NS5A replication efficiency was influenced by its phosphorylation state: While a hyperphosphorylated form (58 kDa) induced by the action of cellular kinases seemed to reduce replication (Neddermann et al., 2004; Quintavalle et al., 2006, 2007), the basally phosphorylated form (56 kDa), which apparently is furthered by adaptive mutations selected in the replicon system (Bartenschlager et al., 2004) (see below), enhanced replication (Appel et al., 2005; Huang et al., 2007b). Recently, the C-terminal part of NS4A was shown to be important for NS5A hyperphosphorylation (Lindenbach et al., 2007). The existence of a putative NS5A interferon sensitivity determining region (Enomoto et al., 1995, 1996) involved in modulation of the clinical response toward IFN is controversial (Brillet et al., 2007; Katze et al., 2002; Pawlotsky, 2000).

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In vitro studies yielded inconsistent results regarding correlation of mutations in the NS5A PKR-binding domain with IFN sensitivity (Podevin et al., 2001; Tan and Katze, 2001; Taylor, 2001). Recently, an alternative NS5A mediated molecular mechanism possibly determining IFN sensitivity has been proposed, because NS5A inhibited IFN-a signaling through suppression of STAT1 phosphorylation (Lan et al., 2007) which is involved in activation of IFN-a/b-stimulated genes (ISGs) (Platanias, 2005). NS5B, the key component of the HCV replicase, is anchored to the ER membrane through its C-terminal transmembrane domain (Ivashkina et al., 2002; Moradpour et al., 2004a; Schmidt-Mende et al., 2001). This RNA-dependent RNA polymerase (RdRp) has a right-hand structure, which is typical for polymerases (Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., 1999). The thumb and finger domains create a channel for a ssRNA template positioning it toward dNTPs bound at the catalytic sites in the palm domain (Bressanelli et al., 2002). Replication probably occurs de novo (unprimed) (Luo et al., 2000; Oh et al., 1999; Zhong et al., 2000) involving a multiprotein complex, the replicase (van Dijk et al., 2004). Minus stranded RNA intermediates are generated to serve as templates for amplification of nascent plus stranded HCV genomes. HCV apparently assembles a replication complex localizing to a specialized virusinduced compartment, the membranous web (see above) (Egger et al., 2002; El-Hage and Luo, 2003; Gosert et al., 2003; Moradpour et al., 2003; Schwartz et al., 2002). This replication complex is thought to consist of the viral nonstructural proteins, replicating RNA, and host cell proteins. Thereby, HCV proteins could be attached to the ER or specialized intracellular, presumably ER derived membranes of the membranous web, bringing them in close proximity to each other. While various interactions between the nonstructural proteins have been described (Dimitrova et al., 2003) the precise modes of these interactions are largely unknown. NS5A sequences involved in its interaction with NS5B and regulation of replication have been mapped (Shirota et al., 2002). Additionally, NS3 helicase, NS5A and NS5B were described to interact with the 50 UTR during positive strand RNA synthesis in a genotype-specific manner (Binder et al., 2007). Furthermore, several cellular proteins are supposed to be recruited to the replication complex (see below). Formation of a specialized compartment containing the HCV replication complex could provide several advantages: All components critical for replication can be physically organized and brought into close proximity to each other enabling smooth interaction. Limiting factors, such as lipid components, could be enriched and provided in the optimal concentration. At last, this compartmentalization might shield dsRNA intermediates from cellular innate antiviral defense mechanisms (Gale and Foy, 2005; Kawai and Akira, 2006).

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IV. HOST CELL FACTORS SUPPORTING THE HCV LIFE CYCLE The HCV life cycle seems to depend on a wide range of cellular proteins. Such host cell factors could serve as therapeutic targets (He et al., 2007). By targeting host cell proteins with a critical impact on the viral life cycle, rapid development of resistance toward therapy, as observed during treatment with antivirals targeting HCV proteins, might be avoided. Additionally, certain host cell proteins might also support the life cycle of other viruses; thus, targeting such factors, might enable development of broad spectrum antivirals. However, inhibitors of host cell proteins might be associated with more undesired side effects than drugs inhibiting specific viral proteins. Even though various cellular proteins were described to interact with HCV RNA or proteins, the impact of host cell proteins on HCV replication could not be studied until after the development of HCV replicon systems. Thus, vesicle associated proteins VAP-A/B (Hamamoto et al., 2005; Zhang et al., 2004b), polypyrimidine tract binding protein 1 (PTBP1) and the putative transcription termination factor SSB (La) (Zhang et al., 2004b), the proteasome component PSMA-7 and the mRNA-associated protein ELAVL1 (HuR) (Korf et al., 2005) as well as RAF-1, a component of the MAP-Kinase pathway (Burckstummer et al., 2006), were shown to support HCV RNA replication. Additionally, replication was found to depend on the geranylgeranylated cellular protein FBL2 interacting with NS5A (Wang et al., 2005) and cyclophylin B interacting with NS5B (Watashi et al., 2005). Cyclophylin B inhibitors have been developed and have been shown to lower HCV viral load in clinical trials (Pawlotsky et al., 2007). By screening a library of small interfering RNAs (siRNAs) targeting
4000 genes in Huh7-derived EN5-3 cells containing a subgenomic genotype 1b replicon, the G-protein-coupled receptor TBXA2R, the membrane protein LTb, the adapter protein TRAF2, transcription factors RELA and NFkB2, protein kinases MKK7 and SNARK and transporter proteins SLC12A4 and SLC12A5 were identified to support HCV replication (Ng et al., 2007). HCV pseudo-particles enabled studies of the effect of cellular proteins, especially the putative HCV coreceptors, such as CD81, SR-BI, and claudin-1, on HCV entry (see below) (Bartosch et al., 2003c). However, only after development of a full viral life cycle cell culture system (see below), did studies of the impact of putative HCV interacting host factors on the complete viral life cycle become possible. In a comprehensive study by Randall et al. (2007), using the genotype 2a J6/JFH1 cell culture system (see below), silencing of 31/68 host proteins decreased HCV infectivity titers greater than threefold, whereas silencing of only one protein increased infectivity titers (Gottwein and Bukh; 2007). A summary of these proteins including their putative role for host cell and HCV life cycle, interacting HCV genome regions/ proteins, as well as

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the impact of silencing on HCV infectivity titers is given in Table I. Several proteins, such as DDX3X, ELAVL1, PTBP1, HNRPC, and PCBP2, presumably play a role in host cell RNA processing and trafficking and are thought to interact with the HCV UTRs, thus modulating HCV RNA replication. Cellular translation initiation factors such as eIF2S3 and eIF4E, but also proteasome components, such as PSMA7, might modulate HCV translation. Host proteins involved in vesicle trafficking, such as vesicle-associated membrane protein-associated protein A (VAP-A), possibly aid formation of the HCV replication complex by membrane targeting of its components and regulate a switch between translation and replication, probably by interaction with NS5A dependent on the NS5A phosphorylation state (see below) (Evans et al., 2004). Additionally, components of the cytoskeleton, such as ACTN1, presumably interacting with NS5B, could be involved in intracellular targeting of HCV proteins. Furthermore HCV NS5A seems to transactivate nuclear gene expression by interaction with cellular transcription factors (SRCAP, RELA, and STAT-3) and modulate cellular proliferation and differentiation by interaction with components of the MAP-Kinase pathway (GRB-2, RAF-1). Interestingly, HCV infectivity titers were reduced by silencing of shared components of the microRNA biogenesis and RNA interference (RNAi) pathway such as Drosha, Dicer and main components of the RNAinduced silencing complex (RISC), the Argonaute proteins eIF2C1–4 (Randall et al., 2007). This could be explained by a dependence of HCV on the liver-specific microRNA miR-122 (Jopling et al., 2005; Randall et al., 2007). However, a direct effect of components involved in miRNA/RNAi pathways on the HCV life cycle cannot be ruled out. The fact that RNAi components exert a proviral effect on the HCV life cycle further supports the hypothesis that mammalian cells might not employ RNAi as an innate antiviral defense mechanism (Cullen, 2006a), as described for plants and invertebrates (Bartel, 2004; Cullen, 2002, 2006b; Voinnet, 2005). In addition to a range of host cell proteins, the HCV life cycle appears to depend on the liver-specific lipid environment (Ye, 2007) and the liver-specific microRNA miR-122 (Jopling et al., 2005; Lagos-Quintana et al., 2002). Both topics are reviewed below in conjunction with possible determinants of HCV tissue tropism. Theoretically, HCV might have evolved mechanisms to regulate cellular pathways according to its need, and induce overexpression of factors with impact on its life cycle. It appears, that expression of some of the cellular proteins supporting the J6/JFH1 life cycle, specifically the RNAbinding protein HNRPC, the cytoskeleton component ACTN1, and the vesicle trafficking protein VAP-A, was upregulated in Huh7 cells containing a genotype 1b replicon (Fang et al., 2006). Furthermore, PTBP1, possibly involved in HCV replication, was also detected by proteomic analysis of HCV-infected human liver biopsies (Jacobs et al., 2005), and microarray

TABLE I Host proteins with influence on HCV infectivity titers in the J6/JFH1 cell culture model identified by silencing experiments by Randall et al. (2007)

Putative function in host cell

Role of interaction for HCV life cycle

RNA processing/trafficking

Modulation of replication

Translation initiation

Modulation of translation

Vesicle trafficking

Modulation of replication/ translation; membrane targeting of replication complexes Modulation of translation

Proteasome, protein degradation Protein processing

Nucleocytoplasmatic transport

Cytoskeleton

Protein processing

Modulation of nucleocytoplasmatic transport Targeting of viral components (e.g., replication complex)

Host cell protein

HCV interacting domain

/+ n, fold change in viral infectivity

Core 50 and 30 UTR 50 and 30 UTR 50 and 30 UTR 50 UTR 50 UTR ? NS5A/B

42 9 5 4 3 30 4 9

DDX3X ELAVL1 PTBP1 HNRPC PCBP2 EIF2S3 EIF4E VAP-A, VAP-B VPS35

NS5A

4

PSMA7

30 UTR

5

HM13 signal peptidase HSPBP1 co-chaperone RANBP5

Core

6

?

6

NS5A

4

ACTN1

NS5B

4 (continued)

TABLE I

(continued)

Putative function in host cell

Membrane protein (tetraspanin) Innate immune defense, dsRNA sensing, IFN pathway Transcription factors

ER stress—unfolded protein response (UPR) MAP-kinase pathways, transduction of mitogenic signals from membrane to nucleus Carbohydrate Metabolism miRNA biogenesis

Role of interaction for HCV life cycle

Host cell protein

HCV interacting domain

Coreceptor

CD81

E2

Escape from innate immune defense

EIF2AK2

E2, NS5A

Transactivation of nuclear gene expression

SRCAP RELA STAT-3 ATF-6

NS5A NS5A NS5A NS4B

MAPK1 GRB-2 RAF-1

? NS5A Core, NS5A

GADPH Drosha DGCR8 Dicer EIF2C1 EIF2C2 EIF2C3 EIF2C4

3 0 UTR ? ? ? ? ? ? ?

Induction of unfolded protein response Modulation of cellular proliferation/differentiation

Modulation of replication Effects mediated of miR-122 or direct, unknown role

/+ n, fold change in viral infectivity

11 6 5 3 13 +7 3 6 6 5 3 2 8 2 2 2 10

From left to right the following parameters are given: putative function in the host cell; possible role for the HCV life cycle; name of the cellular protein; putative HCV interacting genome region or protein (?, unknown); maximal change in infectivity titer following silencing with experimental RNAi, measured by a TCID50 assay. Thus, a reduction in infectivity titer following silencing indicates a supporting role for the HCV life cycle. References describing putative interactions of the respective proteins with HCV proteins are provided in supplementary materials by Randall et al. (2007).

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analysis of acutely infected chimpanzee livers showed an upregulation of proteasome and cytoskeleton components (Bigger et al., 2001), however, different ones than identified by Randall et al. (2007). Thus, regulation by HCV might partly account for the differential RNA/protein expression pattern observed in HCV-infected cells or clinical specimens.

V. CONSENSUS HCV CDNA CLONES—INFECTIOUS IN TRANSFECTED CHIMPANZEES After the discovery of HCV, there was an urgent need for robust animal and cell culture systems employing HCV with a defined genome sequence, thus allowing reverse genetic studies. Therefore, a main research focus was the development of infectious HCV clones. However, the first cloned HCV genomes were not functional, mainly because they were missing the 30 terminal sequence of the 30 UTR (Choo et al., 1989; Kato et al., 1990; Okamoto et al., 1991; Takamizawa et al., 1991). However, even after identification of this sequence (Kolykhalov et al., 1996; Tanaka et al., 1995, 1996; Yamada et al., 1996), fatal mutations resulting from viral replication with an error prone RNA polymerase and experimental reverse transcription polymerase chain reaction (RT-PCR) amplification rendered cloned HCV genomes nonfunctional (Yanagi et al., 1997). Therefore, it became evident that the consensus sequence of a particular isolate—the sequence featuring the most common nucleotide at each position—had to be determined and cloned. This approach had previously been used to exclude randomly occurring fatal mutations from an infectious pestivirus consensus cDNA clone (Moormann et al., 1996). Technically, the consensus sequence is determined by direct sequencing of overlapping PCR fragments obtained by RT-PCR or by analyzing several (at least 3) clones of each subcloned overlapping PCR fragment (Fig. 2) (Forns et al., 1999). The latter strategy has been greatly facilitated by the development of long-range PCR technologies with high fidelity enzymes reducing the number of PCR fragments that need to be generated in order to cover the full-length HCV genome (Tellier et al., 2003). H77, a prototype genotype 1a isolate, was used to construct the first full-length cDNA clones of HCV (Kolykhalov et al., 1997; Yanagi et al., 1997). After linearization at the 30 end, RNA in vitro transcripts are generated from these cDNA clones with T7 polymerase employing the bacteriophage T7 promotor, which is positioned immediately upstream of the HCV sequence (Fig. 2); the cDNA clone developed by Yanagi et al. (1997) is linearized with XbaI at the very 30 end of the HCV sequence, providing in vitro transcripts with the authentic 30 end of the HCV genome. RNA transcripts of cDNA clones of HCV strain H77 were shown to be infectious after direct inoculation into the liver of

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+ssRNA HCV genome

C

E1

E2

p7 NS2

NS3

NS NS4B 4A

NS5A

NS5B

Reverse transcription cDNA

C

E1

E2

p7 NS2

NS3

NS NS4B 4A

C

NS5A

E1

E2

p7 NS2

NS3

NS NS4B 4A

NS5A

PCR amplification

NS5B

NS5B

C

E1

E2

p7 NS2

NS3

NS NS4B 4A

NS5A

NS5B

Subcloning and sequencing of Direct sequencing of overlapping ≥3 clones of each fragment PCR products Determination of consensus sequence and construction of cDNA clone Consensus cDNA clone

XbaI T7

+ssRNA in vitro transcripts Full-length genomes of genotypes 1a, 1b and 2a Intrahepatic inoculation into chimpanzee

C

E1

E2

p7 NS2

NS3

NS NS4B 4A

NS5A

NS5B

In vitro transcription C

E1

E2

p7 NS2

NS3

NS NS4B 4A

NS5A

NS5B

?

Intrahepatic inoculation into uPA-SCID mouse engrafted with human liver

JFH1 (genotype 2a) and JFH1 derived recombinants

Transfection into human hepatoma cells (Huh7 or Huh7 derived cell lines)

FIGURE 2 Development of infectious cDNA consensus clones of hepatitis C virus (HCV). Serum-derived HCV single stranded RNA genomes of positive polarity (+ssRNA) were reverse transcribed using reverse transcriptase and a gene-specific primer or random hexamers. The resulting cDNA served as template for polymerase chain reaction (PCR) amplification of overlapping DNA fragments. These PCR products were either sequenced directly (left) or subcloned followed by sequencing of 3 clones of each fragment (right). The consensus sequence—defined as the sequence featuring the most common nucleotide at each position—was determined and a cDNA clone containing this consensus sequence was constructed. In some cases, the infectious cDNA clone contained noncoding nonconsensus nucleotide sequences. After linearization of the cDNA consensus clone at the 30 end [e.g., by XbaI as described by Yanagi et al. (1997), indicated by a black arrow], in vitro transcripts were generated with a DNA-dependent RNA polymerase under control of a T7 promoter located immediately upstream of the

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chimpanzees (Kolykhalov et al., 1997; Yanagi et al., 1997). Subsequently, cDNA clones of other genotypes, such as 2a (J6 isolate) (Yanagi et al., 1999a) and 1b (J4 isolate) (Yanagi et al., 1998), were developed (Bartenschlager, 2006; Bartenschlager and Sparacio, 2007). Thus, it had become possible to induce monoclonal HCV infections in the only available animal model, the chimpanzee. Using this model, monoclonal virus pools could be generated and valuable insights in the natural history of acute and chronic HCV infection, viral kinetics and host responses, including innate, humoral, and adaptive immune responses, were achieved (Bukh, 2004). Compared to polyclonal HCV infections induced by inoculation with serum-derived HCV, results obtained in a monoclonal system are less prone to confounding factors caused by the quasispecies nature of the inoculum. Additionally, serum-derived HCV might be of limited availability and, due to the presence of neutralizing antibodies and lipoproteins, of variable infectivity. Most importantly, infectious clones allowed reverse genetic studies of HCV. Thereby targeted manipulations on the cDNA level allowed functional studies of the manipulated sequences in vivo. Thus, HCVencoded enzymatic activities of the NS2/3 and NS3/4A proteases, the NS3 NTPase/helicase, and the NS5B polymerase were confirmed (Kolykhalov et al., 2000) and a critical role of the 30 UTR (Kolykhalov et al., 2000; Yanagi et al., 1999b) and p7 (Sakai et al., 2003), but not E2 HVR1 (Forns et al., 2000) for HCV infectivity was demonstrated. Unfortunately, RNA transcripts derived from various cDNA clones did not replicate in liver cell lines including Huh7.5 cells (Bartenschlager, 2006; Bartenschlager and Sparacio, 2007). However, the developed consensus cDNA clones defined functional HCV genomes and thus became the foundation for the subsequent development of cell culture systems. The first such system was the subgenomic replicon system.

VI. THE REPLICON SYSTEM—AUTONOMOUS HCV RNA REPLICATION IN HEPATOMA CELL LINES In 1999, development of the subgenomic replicon system (Lohmann et al., 1999) enabled in vitro studies of HCV RNA replication. Previously, subgenomic replicon systems had successfully been developed for other

HCV cDNA sequence (indicated by red arrow). In vitro transcripts equivalent to +ssRNA HCV genomes were used for intrahepatic inoculation of chimpanzees and could theoretically also be used for intrahepatic transfection of uPA-SCID mice engrafted with human liver, as well as for transfection into human hepatoma cells, such as Huh7 cells and derived cell lines.

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viruses (Behrens et al., 1998; Kaplan and Racaniello, 1988; Khromykh and Westaway, 1997). Based on the hypothesis, that replication of RNA transcripts derived from the developed HCV cDNA clones (see above) might occur at low levels in cell culture, a selectable marker was used to identify cells containing highly replication competent viral RNA. Further, the fact that a truncated ‘‘minigenome’’ presumably replicated more efficiently than a full-length genome was exploited. Thus, in the prototype subgenomic HCV replicon a neomycin resistance gene and the heterologous IRES of the encephalomyocarditis virus (EMCV) replaced the structural proteins (Core, E1, E2), p7 and NS2 of the HCV genome (Lohmann et al., 1999). From this bicistronic RNA the neomycin resistance gene was expressed under control of the HCV IRES while HCV NS3—NS5B proteins were translated under control of the EMCV–IRES. After transfection of Huh7 cells with RNA transcripts of these replicon constructs, cell clones with high RNA replication level could be selected by treatment with neomycin (Lohmann et al., 1999) (Fig. 3). Subsequent analysis of these cell clones and the replicons they contained implied that their increased replication capacity was caused by adaptive mutations of the replicating RNA and by increased host cell permissiveness.

A. Identification of adaptive mutations led to more efficient replicon systems Soon after the development of the original genotype 1b (Con1 isolate) subgenomic replicon (Lohmann et al., 1999), its dependence on adaptive mutations became evident (Blight et al., 2000; Lohmann et al., 2001). While HCV NS3, NS4B, and NS5A appeared to be hot spots for adaptive mutations, these were found to occur in all nonstructural proteins contained in the Con1 and subsequently developed subgenomic replicons. Subsequent studies focused on the detailed characterization of the detected adaptive mutations. Interestingly, certain mutations were found to act cooperatively, while others were incompatible (Krieger et al., 2001; Lohmann et al., 2001, 2003). Specifically, the combination of strongly adaptive mutations present in NS5A (also in NS4B and NS5B) seemed to reduce replication efficiency, whereas their combination with weakly adaptive mutations found in NS3 resulted in an increased replication efficiency (Bartenschlager et al., 2003; Bartenschlager and Sparacio, 2007; Krieger et al., 2001; Lohmann et al., 2001, 2003). On the basis of detailed characterization of adaptive mutations, highly adapted subgenomic (Krieger et al., 2001; Lohmann et al., 2003) and selectable full-genome length (Pietschmann et al., 2002) Con1 replicons could be developed. At the same time efficient selectable subgenomic and full-length replicon systems for another genotype 1b isolate, HCV-N, a

Hepatitis C Virus Cell Culture Systems

Replicon system

HCVpp system

Transfection with in vitro Infection with pseudotranscripts of subgenomic or particles bearing the HCV full-length genome envelope glycoproteins

Receptor complex

75

HCVcc system Transfection with in vitro transcripts of JFH1 or JFH1 based intra- and intergenotypic recombinants

Receptor complex

Huh 7 cells

Expression of HCV protein

Replication of RNA

Release of GFP indicator construct

Unpacking release of viral RNA

Expression of HCV protein Expression of GFP

Virus assembly Replication of RNA

FIGURE 3 In vitro systems developed in Huh7 and derived cell lines for specific isolates of hepatitis C virus (HCV). (Left) HCV replicon systems. Several subgenomic as well as full genome length replicons with or without selectable marker genes have been developed. Transfection of in vitro transcripts of these replicon constructs (in the examples shown, HCV sequences are blue and heterologous sequences are orange) into Huh7 or derived cell lines led to expression of HCV proteins and HCV RNA replication. However, the culture system yielded no or very low levels of (for some full-length replicons) infectious viral particles. (Middle) HCV pseudo-particle systems. Pseudo-particles displaying the HCV envelope glycoproteins E1 and E2 assembled on virus-like structures containing lentiviral or retroviral capsid proteins and a green fluorescent protein (GFP) marker construct can be used to infect Huh7 and derived cell lines. The resulting intracellular GFP expression can be detected by fluorescent activated cell sorting (FACS) analysis and is a convenient measure for entry efficiency. (Right) HCV full viral life cycle cell culture systems. In vitro transcripts derived from the JFH1 or intra- and intergenotypic JFH1-based recombinant cDNA clones are transfected into Huh7 or derived cell lines. In these systems, expression of HCV protein and HCV RNA replication results in assembly and egress of infectious viral particles. Thus, these systems mimic the complete viral life cycle of HCV.

strain that was only poorly infectious in the chimpanzee model (Beard et al., 1999), were described (Ikeda et al., 2002). In vitro, replication of HCV-N depended on a naturally occurring four amino acid insertion in NS5A, but apparently not on additional adaptive mutations. However,

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introduction of adaptive mutations identified in the Con1 replicon system into the HCV-N system led to a clear increase of its efficiency. Similarly, adaptive mutations were essential for the development of subgenomic (Blight et al., 2003; Grobler et al., 2003) and full-length (Blight et al., 2003; Yi and Lemon, 2004) replicon systems for the genotype 1a isolate H77. With increasing efficiency of the described replicon systems, due to the employment of favorable combinations of adaptive mutations (Yi and Lemon, 2004) and the development of highly permissive Huh7derived cell lines (see below) (Blight et al., 2002), the use of a selectable marker ceased to be an absolute requirement (Blight et al., 2003; Yi and Lemon, 2004). Thus, heterologous elements such as the neomycin resistance gene and the EMCV–IRES, that potentially reduced replication efficiency (Blight et al., 2003), could be removed from the replicons. In this way unmodified HCV genomes could be studied in cell culture, making it possible to determine whether such genomes could promote virus particle formation (see below). The mechanisms by which adaptive mutations increase RNA replication capacity are not known. The fact that most adaptive mutations do not occur naturally, suggests that they might be specific adaptations to Huh7 hepatoma cells and derived cell lines. Because most of these mutations are localized on the surface of the respective HCV protein, they might modify interactions of viral with cellular proteins. For example, most NS3 mutations mapped to the solvent accessible surface of the helicase domain, being accessible to interacting proteins (Blight et al., 2003; Krieger et al., 2001; Lohmann et al., 2003). However, other NS3 mutations (Yi and Lemon, 2004) mapped to residues close to or part of the protease active site. Because the NS3/4A protease was shown to be capable of blocking the transcription factor IFN regulatory factor-3 (IRF-3) (Foy et al., 2003), it was hypothesized that these adaptive mutations might enhance NS3/4 ability to cleave a cellular protein involved in IRF-3 signaling (Yi and Lemon, 2004). Adaptive mutations in NS5A presumably reduce hyperphosphorylation of this protein, which was found to increase its interaction with the cellular protein VAP-A and HCV RNA replication (Appel et al., 2005; Evans et al., 2004; Neddermann et al., 2004). Since VAP-A seems to play a critical role for localization of the HCV replication complex to intracellular membranes (Gao et al., 2004), adaptive mutations in NS5A could lead to a more efficient assembly of the replication complex (Huang et al., 2007b). After development of full-length replication competent HCV genomes, there was great hope that these systems would yield cell culture derived infectious viruses, because, in contrast to the subgenomic replicon system, all viral components required for the full viral life cycle were expressed. However, even though genome replication and expression of HCV proteins could be detected, no robust release of viral particles

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was recorded. Therefore, even though these adapted full-length HCV genomes were highly replication competent, they seemed to be unable to promote formation of infectious viral particles. This observation was supported by the following in vivo study: Introduction of an in vitro optimized combination of highly adaptive mutations (Krieger et al., 2001) into the Con1 full-length genome abrogated its ability to productively infect chimpanzees (Bukh et al., 2002). Interestingly, in this study the genome with a single adaptive mutation in NS5A led to infection, but the recovered viral genomes had reverted to the Con1 wild-type sequence. In line with these findings, Pietschmann and Bartenschlager observed that the Con1 full-length genome without adaptive mutations replicated poorly but led to release of viral RNA and Core into the supernatant; this release was abrogated by the introduction of adaptive mutations, which in turn resulted in efficient RNA replication (Bartenschlager and Sparacio, 2007). These observations indicated that cell culture adaptive mutations, though conferring favorable replication characteristics in vitro, precluded formation of infectious viruses in vitro and in vivo. Because the described adaptive mutations were selected for high replication capability in subgenomic replicons, the viral life cycle might have shifted toward optimization of replication at the expense of viral assembly and release. The only study, which did not fit this paradigm, reported formation of viral particles in cell culture after transfection of in vitro RNA transcripts of a strongly replication adapted genotype 1a full-length cDNA clone (H77-S) (Yi and Lemon, 2004) into Huh7.5 cells (Yi et al., 2006). However, this system yielded only low levels of infectious virus (see below) and the viability of H77-S in vivo has so far not been examined. The described replicon systems proved valuable tools for studies of the role of different HCV genome segments and proteins for HCV RNA replication, of the intracellular localization of HCV proteins, of virus–host interactions, and for testing of therapeutic compounds interfering with HCV replication. Thus, replication was shown to depend on the 50 UTR (Binder et al., 2007; Friebe et al., 2001; Kim et al., 2002; Luo et al., 2003; Reusken et al., 2003) and 30 UTR (Binder et al., 2007; Friebe and Bartenschlager, 2002; Yi and Lemon, 2003a,b) as well as on cis-acting RNA elements localized in the 30 UTR and NS5B (Binder et al., 2007; Friebe et al., 2005; You et al., 2004). Another major achievement was the identification of NS5A as a strong regulator of replication. Reverse genetic studies demonstrated, that the NS5A N-terminal amphipathic domain, which anchors this protein to ER membranes (Elazar et al., 2003; Penin et al., 2004a), as well as four conserved cysteine residues, localizing to the NS5A zinc-binding site (Tellinghuisen et al., 2004), were critical for replication. Further, replication was demonstrated to be regulated by the NS5A hyperphosphorylation state (Appel et al., 2005; Neddermann

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et al., 2004; Quintavalle et al., 2006, 2007), which is apparently modulated by NS4A (Lindenbach et al., 2007). Employing replicon systems, the intracellular localization of HCV proteins could be studied in a more physiological setting compared with studies using expression of isolated proteins from plasmids. Thus, in full-genome length replicon systems, HCV Core was shown to localize to intracellular lipid droplets, while the envelope glycoproteins were found to be associated with the ER (Pietschmann et al., 2002). Additionally, localization of the HCV replication complex to intracellular membranous compartments, the membranous web, was reported (see above) (El-Hage and Luo, 2003; Gosert et al., 2003; Moradpour et al., 2004b; Shi et al., 2003). Studies in the replicon system also led to insights into different aspects of virus–host interactions. Several studies elucidated the impact of host cell proteins on HCV replication (see above) (Burckstummer et al., 2006; Evans et al., 2004; Hamamoto et al., 2005; Korf et al., 2005; Ng et al., 2007; Wang et al., 2005; Watashi et al., 2005; Zhang et al., 2004b); others focused on studies of the host cell response to HCV RNA replication employing microarray and proteomics analysis (Abe et al., 2005; Fang et al., 2006; Jacobs et al., 2005). Finally, HCV replicons provided the first in vitro tool for testing of therapeutic compounds acting at the replication level, such as NS3/4A protease and NS5B polymerase inhibitors (Bartenschlager, 2002; De Francesco and Migliaccio, 2005; Pawlotsky et al., 2007).

B. The study of replicon systems led to identification of highly permissive Huh7 cell lines Replication levels in the replicon system are not only determined by adaptive mutations but also by the permissiveness of the host cell. Therefore, investigators reasoned that neomycin selected replicon containing Huh7 cell clones might be characterized by heightened permissiveness for viral replication. Indeed, after neomycin selected cell clones with high levels of replication had been cured with IFN, these cells were found to be more permissive for HCV replication (Blight et al., 2002; Lohmann et al., 2003; Murray et al., 2003). Thus, the now widely used Huh7.5 (Blight et al., 2002) and Huh7-Lunet cell lines were developed (Friebe et al., 2005). In the case of Huh7.5 cells, the improved permissiveness appears to be the result of a defect in the IFN signaling pathway being of major importance for innate cellular antiviral immunity. This cell clone exhibits a point mutation in the RNA helicase retinoic acid-inducible gene-I (RIG-I) (Sumpter et al., 2005) which seems to be implicated in the anti-HCV host response, because its expression is upregulated in chronically infected chimpanzees (Bigger et al., 2004). RIG-I senses intracellular dsRNA, as occurring during viral replication, and transduces an activation signal for the transcription factors IRF-3 and NF-kB (Yoneyama et al., 2004). IRF-3 and NF-kB in turn

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lead to IFN-b expression, which by involvement of the JAK/STAT pathway results in expression of IFN-stimulated genes (ISGs) that are main players of antiviral defense. Intracellular antiviral host defense and HCV evasion strategies have been reviewed by Gale and Foy (2005). Despite an intact RIG-I pathway, Huh7-Lunet cells appear to support JFH1-replication levels with comparable efficiency as Huh7.5 cells (Bartenschlager and Sparacio, 2007); however, due to lower CD81 expression, viral spread has been described to be less efficient (Koutsoudakis et al., 2007).

VII. PSEUDO-PARTICLES EXPRESSING THE HCV ENVELOPE PROTEINS (HCVpp)—A SYSTEM FOR THE STUDY OF VIRAL ENTRY AND NEUTRALIZATION The replicon system played a central role for the in vitro study of HCV RNA replication. Another major research focus has been the development of systems for the study of HCV entry events, mediated by its envelope glycoproteins (E1 and E2). Initial models employed truncated E2 proteins (Flint et al., 1999b; Pileri et al., 1998), identifying CD81 as a key HCV receptor, and liposomes reconstituted with E1/E2 glycoproteins (Lambot et al., 2002). More complex approaches were based on the production of HCV virus-like particles in insect cells (Baumert et al., 1998, 1999; Clayton et al., 2002; Owsianka et al., 2001; Triyatni et al., 2002; Wellnitz et al., 2002). An alterative approach was the construction of pseudotyped vesiculo virus (Buonocore et al., 2002; Lagging et al., 1998; Matsuura et al., 2001; Takikawa et al., 2000) or influenza virus (Flint et al., 1999b) particles featuring HCV E1/E2 with chimeric transmembrane domains, which targeted these otherwise ER localized proteins to the plasma membrane. However, chimeric E1/E2 exhibited impaired functionality (Hsu et al., 2003). Although some of these systems have contributed significantly to research on HCV entry, some systems showed low infectivity and inconsistent results (Bartenschlager et al., 2004). In 2003, highly infectious HCVpp featuring unmodified HCV E1/E2 glycoproteins assembled on lenti- or retroviral Core particles were developed. The presence of a packaged marker gene enabled the convenient detection of infection (Bartosch et al., 2003b; Hsu et al., 2003). In detail the following constructs were cotransfected in the packaging cell line 293-T (human embryonic kidney cells): (i) an HCV E1/E2 expression construct encoding the HCV envelope glycoproteins (E1 and E2); (ii) a packaging construct encoding retroviral Core, specifically, gag and pol proteins of murine leukemia virus (MLV) or human immunodeficiency virus (HIV); (iii) a packaging competent transfer vector construct encoding green fluorescent protein (GFP) as a marker. After transfection into 293-T cells,

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pseudo-particles were secreted into the cell culture supernatant and could be used for infection assays. With HCVpp, robust infection could be achieved in Huh7 cells and in primary human hepatocytes. The percentage of infected cells could be conveniently measured by flow cytometry analysis for GFP (Bartosch et al., 2003b) (Fig. 3). The system developed by Hsu et al. (2003) employed in addition to an HCV E1/E2 expression construct an envelope-defective HIV-1 proviral genome expressing a luciferase reporter, which allowed estimation of infection efficiency by measurement of luciferase activity. Employing the HCVpp system HCV entry events could be studied in a more physiological way (Barth et al., 2006). Thus, the importance of the C type lectins DC-SIGN and L-SIGN (Gardner et al., 2003; Lozach et al., 2003) as HCV capture receptors was verified. L-SIGN, expressed on endothelial cells in liver sinusoids, probably targets HCV to the liver, increasing the likelihood of interaction with its specific entry receptors on adjacent hepatocytes (Cormier et al., 2004a; Pohlmann et al., 2003). Further, the central role of the tetraspanin CD81 (Flint et al., 1999a; Pileri et al., 1998) as HCV receptor was confirmed and refined to that of a postattachment entry coreceptor (Cormier et al., 2004b; Zhang et al., 2004a). Reverse genetic studies in the HCVpp system led to identification of the E2 regions involved in CD81 binding (Drummer et al., 2006; McCaffrey et al., 2007; Owsianka et al., 2006) and the influence of E2 glycosylation on this binding to CD81 (see above). In addition, SR-BI which is physiologically responsible for the uptake of cholesteryl ester from HDL (Connelly and Williams, 2004) and which has been shown to bind soluble E2 (Scarselli et al., 2002), was verified to mediate infection with HCVpp (Bartosch et al., 2003c) in an HDL dependent manner. Hereby HDL apparently acted as the matchmaker between the HVR1 of HCV E2 and SR-BI (Bartosch et al., 2005; Dreux et al., 2006; Meunier et al., 2005; Voisset et al., 2005) and crosslinkage of CD81 and SR-BI seemed to occur (Heo et al., 2006). In addition to the enhancement of HCVpp (and HCVcc) infectivity by HDL, inhibition of their infectivity by oxidized low-density lipoprotein (LDL) and oxidized HDL was recently demonstrated to be SR-BI dependent (von Hahn et al., 2006). The important role of these coreceptors for HCV infection has recently been confirmed in the HCVcc system (Catanese et al., 2007; Kapadia et al., 2007), where HCV infection was shown to be modulated by the expression levels of CD81 (Akazawa et al., 2007; Koutsoudakis et al., 2007), SR-BI, and SR-BII (Grove et al., 2007). Studies in the HCVpp (and HCVcc) system have also led to the identification of claudin-1, a tight junction component expressed to high levels in the liver (Van Itallie and Anderson, 2006), as a HCV coreceptor required for late steps in entry (Evans et al., 2007). As already suggested by the pH dependence of HCVpp infection (Bartosch et al., 2003c; Hsu et al., 2003), HCV entry was recently confirmed to be dependent on endocytosis

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(Smith and Helenius, 2004) mediated by clathrin in the HCVpp (Blanchard et al., 2006; Codran et al., 2006; Meertens et al., 2006) and HCVcc system (Blanchard et al., 2006). HCVpp provided the first robust in vitro system for identification of ntAb against HCV. NtAb derived from serum of chronically infected patients, directed against several neutralization epitopes, could abrogate or reduce infectivity of HCVpp (Bartosch et al., 2003a; Yu et al., 2004). However, despite high titers of ntAb, chronically infected patients and chimpanzees apparently were unable to clear HCV infection (Bartosch et al., 2003a; Logvinoff et al., 2004; Meunier et al., 2005; Schofield et al., 2005) which is probably due to specific viral escape mechanisms (Barth et al., 2006). Thus, serum of patient H, being chronically infected with the genotype 1a strain H77, failed to neutralize HCVpp bearing autologous envelope glycoproteins with sequences dominating at the same timepoint, at which serum for neutralization was obtained. However, serum obtained at a given time could neutralize HCVpp bearing envelope glycoproteins with sequences identified at previous timepoints during infection, indicating that HCV outcompeted the humoral immune response by rapid evolvement of escape mutants (von Hahn et al., 2007). In contrast, ntAb were rarely observed during the acute phase of HCV infection (Logvinoff et al., 2004; Meunier et al., 2005; von Hahn et al., 2007); however, their development during this phase was associated with viral clearance (Lavillette et al., 2005). The development of HCVpp systems for the six major HCV genotypes enabled the identification of serum derived broadly reactive ntAb (Meunier et al., 2005), of major interest for passive immunotherapy and vaccine development. Specifically, antibodies raised during genotype 1a infection cross-neutralized HCVpp of genotypes 4a, 5a, and 6a but were only minimally efficient against genotype 2a and 3a (Meunier et al., 2005). These findings were confirmed in the HCVcc system employing intergenotypic JFH1-based recombinants containing the envelope glycoproteins of the six major genotypes (Scheel et al., 2008). The HCVpp system was also employed to study monoclonal antibodies (mAbs), which could be used for post exposure immunoprophylaxis and immunotherapy, primarily in order to prevent reinfection of HCV-infected liver transplant recipients. For these aims human mAbs would be preferable, because they can be better characterized, are more easily accessible and are expected to have less undesired effects than hyperimmune sera derived from HCV-infected individuals. Further, mAbs allow a more detailed investigation of the targeted epitopes and mechanisms involved in neutralization than polyclonal antibody preparations. In comparison to mouse mAbs, human mAbs show superior pharmacokinetics and fewer undesired effects, because they are less prone to formation of immune complexes and other inappropriate interactions with the human immune system, and are thus preferable for treatment

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purposes. Employing human mAbs, three immunogenic conformational domains were identified on HCV E2 of which two were shown to be neutralizing and able to block the interaction of E2 with CD81 (Hadlock et al., 2000; Keck et al., 2004); these results were recently confirmed in the HCVcc system (Keck et al., 2007). Cross-neutralizing human mAbs derived from a genotype 1a-infected patient were able to also neutralize genotype 1b and 2a HCVpp (Schofield et al., 2005). Additionally, mAbs derived from a HCV-infected patient whose genotype is unknown were able to immunoprecipitate HCV particles from sera of individuals infected with genotype 1, 2, and 3 (Eren et al., 2006). These antibodies have, with moderate success, already been used in clinical trials (Pawlotsky et al., 2007). Notably, the mouse monoclonal antibody AP33, directed against a broadly neutralizing E2 epitope, efficiently inhibited infection of all HCV genotypes as determined in the HCVpp system (Owsianka et al., 2005) and confirmed in the HCVcc system for genotype 2a (JFH1) (Tarr et al., 2006). Recently, several human mAbs, derived from a genotype 2b-infected patient, were shown to cross-neutralize HCVpp of all genotypes as well as JFH1 HCVcc by binding to conformational, across genotypes conserved epitopes which are located in E2 and which are important for E2–CD81 interaction (Johansson et al., 2007). Thus, it seems possible to identify serum antibodies and to raise mAbs able to cross-neutralize most or all HCV genotypes. Identification of such antibodies might further vaccine design (Chanock et al., 1993), and human mAbs could be employed in passive immunization strategies.

VIII. THE JFH1 ISOLATE—GENERATION OF CELL CULTURE DERIVED HCV (HCVCC) IN FULL VIRAL LIFE CYCLE CELL CULTURE SYSTEMS A. The original and adapted JFH1 cell culture system The history of the HCV isolate, which would provide a breakthrough in the quest for a full viral life cycle cell culture system, began rather unspectacularly in 2001, when Kato and colleagues published the sequence of a genotype 2a isolate of a Japanese patient suffering from fulminant hepatitis and termed it JFH1 (Kato et al., 2001). Sequence comparison with other genotype 2a isolates, derived from chronically infected patients, revealed major sequence deviations, especially in the 50 UTR, Core, NS3, and NS5A. In 2003, a JFH1 subgenomic replicon was shown to replicate in Huh7 cells without necessity of adaptive mutations yielding high HCV RNA titers (up to 20-fold higher than highly adapted Con1 replicons) (Kato et al., 2003). The exceptional replication capabilities of this isolate were underscored by its ability to replicate in otherwise

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unsusceptible host cell lines, such as 293-T and HeLa (human cervix carcinoma) cells (Date et al., 2004; Kato et al., 2005). Because adaptive mutations required for replication of all previously developed replicons inhibited formation of viral particles (Bartenschlager and Pietschmann, 2005; Bukh et al., 2002), it became an interesting task to test the viability of a full-length cDNA clone of the naturally replication competent JFH1 isolate in cell culture. Thus, in 2005 Wakita et al. (2005) were able to report the first HCV recombinant in vitro system supporting the full viral life cycle. In this system, transfection of full-length JFH1 in vitro transcripts into Huh7 cells resulted not only in RNA replication and HCV protein expression but also in formation of viral particles with a diameter of 50– 60 nm (Figs. 3 and 4), in accordance with previous size estimates of HCV particles (He et al., 1987; Kaito et al., 1994; Takahashi et al., 1992). Viral particles released from transfected cultures were able to infect naı¨ve Huh7 cells and a chimpanzee. In cell culture, JFH1 infectivity could be blocked by anti-CD81 antibodies and serum-derived ntAb, confirming the relevance of the system for studies of HCV-specific entry events and related therapeutics (Table II). However, infectivity of this original JFH1 virus appeared to be attenuated, because it did not spread in transfected and infected Huh7 cultures and also resulted in an attenuated course of infection in the chimpanzee model. Similarly, Zhong et al. (2005) observed, after transfection of highly permissive Huh7.5.1 cells, at first inefficient spread of JFH1 viruses. However, following this eclipse phase, rapid viral spread occurred in the transfected cell culture and immediate spread was observed in an indicator culture, suggesting that JFH1 might have acquired adaptive mutations allowing more efficient growth in culture. Such adaptive mutations, specifically a particular amino acid change in E2, were identified in a subsequent study (Zhong et al., 2006) and shown to result in accelerated growth kinetics and improved peak infectivity titers of
105–106 focus forming units (FFUs)/ml compared to
103–105 FFU/ml for the acute phase virus (Table II). Infectivity titers were determined by infection of cell culture replicates with serial dilutions of virus containing supernatants and subsequent immuno-stainings for HCV antigens revealing HCVpositive cell foci; calculation of FFU/ml was based on the number of such cell foci at the highest supernatant dilution used. Similar studies have been performed by others (Delgrange et al., 2007; Kaul et al., 2007), and eventually it will be important to identify the adaptive mutations conferring the most favorable growth kinetics to JFH1 and to determine the mechanism by which these mutations improve the JFH1 culture system. In the study by Zhong et al. (2006), JFH1 virus infection was described to exert a cytopathic effect on its host cells, leading to pronounced cell death after the virus had spread to the entire culture. Cell death was accompanied by a decrease in infectivity titer and percentage of infected

Judith M. Gottwein and Jens Bukh

Huh7

84

JFH1/GND

JFH1

4 12 24 48 72 4 12 24 48 72

Replication of HCV RNA genome (Wakita et al., 2005)

HCV RNA 28 S

Expression of HCV proteins (Lindenbach et al., 2005) Core

E2

NS5A

Visualization of HCV viral particles (Wakita et al., 2005)

48 h after transfection with RNA transcripts

48 h after transfer of supernatant to naive cells

Infectivity of cell culture supernatant (Lindenbach et al., 2005)

FIGURE 4 Evidence for the complete viral life cycle in JFH1-based cell culture systems. After transfection of RNA transcripts of the JFH1 (Wakita et al., 2005) or J6/JFH1 (Lindenbach et al., 2005) cDNA clones into Huh7 or derived cell lines, hepatitis C virus (HCV) RNA replication (first row) and HCV protein expression (second row) could be detected. This system resulted in the formation of viral particles, which were visualized by electron microscopy (third row). Viral particles released from transfected cell cultures were proven to be infectious, because supernatants derived from these cultures could be used to infect naive cultures (last row, staining for HCV NS5A protein expression).

cells and was followed by the emergence of cell populations with resistance toward JFH1 infection. Such a cytopathic effect was also observed for J6/JFH1 (Gottwein et al., 2007) and other JFH1-based intergenotypic recombinant viruses containing the Core-NS2 sequence of prototype strains of HCV genotypes 1a, 3a, and 4a (Gottwein et al., 2007; Scheel et al., 2008). This finding is in contrast to the perception of HCV as a noncytopathic virus, which resulted from studies in infected humans and experimentally infected chimpanzees. However, it remains to be

TABLE II

Characteristics of HCV full viral life cycle cell culture systems
Reciprocalspecific
Peak titers (culture supernatant) infectivitya Cell line

Log10 (HCV RNA)

Log10 (Infectivity)

Log10

Huh7

7–7.5 copies/ml

nr

nr

Huh7.5.1

7–8 GE/ml

3–5 FFU/ml

3

Huh7.5.1

7–8 GE/ml

5–6 FFU/ml

2

Huh7.5

7–7.5 IU/ml

4–5 TCID50/ml

3 (cells) 1 (animals)

RNA transcripts cDNA constructs

Huh7-Lunet

nr

5–6 TCID50/ml

3

Huh7, Huh7.5

7–7.5 copies/ml

4–5 FFU/ml

3

cDNA ribozyme constructs RNA transcripts RNA transcripts

Huh7, Huh7.5, Huh7.5.1

6–7 copies/ml

3–4 FFU/ml

3

Anti-E1; anti-E2; anti-CD81; IFN-a IFN-a

Huh7.5

nr

nr

nr

nr

Huh7, Huh7.5, FT3–7

nr

3–4 FFU/ml

nr

ntAb; anti-CD81

Transfected agent

Virus

Study

JFH1

(Wakita et al., 2005) (Zhong et al., 2005, 2006) (Zhong et al., 2006) (Lindenbach et al., 2005, 2006)

RNA transcripts RNA transcripts RNA transcripts RNA transcripts

J6/JFH1 (J6/C3 = Jc1) JFH1

(Pietschmann et al., 2006) (Cai et al., 2005)

JFH1

(Kato et al., 2007)

1a/JFH1 (H77/JFH1) 1a/JFH1 (H77/JFH1)

(McMullan et al., 2007) (Yi et al., 2007)

JFH1 Adapted JFH1 J6/JFH1

Interfering agents ntAb; anti-CD81 Anti-E2; anti-CD81 Anti-CD81 Anti-E2, IFN-a; NS3 and NS5B inhibitors Anti-E1

(continued)

TABLE II

(continued)
Reciprocalspecific
Peak titers (culture supernatant) infectivitya

Virus

Study

1a/JFH1 (H77/JFH1) 1a/JFH1 (H77/C3) 1b/JFH1 (Con1/C3) 1b/JFH1

(Scheel et al., 2008) (Pietschmann et al., 2006) (Pietschmann et al., 2006) (Haqshenas et al., 2007a) (Pietschmann et al., 2006) (Gottwein et al., 2007) (Scheel et al., 2008) (Yi et al., 2006)

3a/JFH1 (452/C6) 3a/JFH1 (S52/JFH1) 4a/JFH1 (ED43/JFH1) H77-S H77-C

a

(Kanda et al., 2006)

Transfected agent RNA transcripts RNA transcripts RNA transcripts RNA transcripts RNA transcripts RNA transcripts RNA transcripts RNA transcripts RNA transcripts

Cell line

Log10 (HCV RNA)

Log10 (Infectivity)

Log10

Interfering agents

Huh7.5

7–7.5 IU/ml

3–4 TCID50/ml

3–4

ntAb

Huh7-Lunet

nr

2–3 TCID50/ml

nr

Anti-E1

Huh7-Lunet

nr

2–3 TCID50/ml

nr

Huh7

7–7.5 copies/ml

2–3 FFU/ml

3–4

Anti-CD81; anti-E1 Amantadine

Huh7-Lunet

nr

1–2 TCID50/ml

nr

Anti-E1

Huh7.5

7–7.5 IU/ml

4–5 TCID50/ml

2–3

Anti-CD81

Huh7.5

7–7.5 IU/ml

3–4 TCID50/ml

3–4

Huh7.5

7–8 copies/mlb

2–3 FFU/mlb

4–5

Immortalized human hepatocytes

7–8 copies/mlc

4–5 FFU/mlc

3

ntAb; anti-CD81 ntAb; anti-CD81 ntAb

Specific infectivity: number of infectious viral particles related to overall number of viral particles (calculated as infectivity titer/HCV RNA titer or number of particles estimated by amount of HCV Core released into the supernatant). Values are reciprocal and given as Log10; for example, a specific infectivity of 1:1000 is given as 3, indicating that RNA titers were three orders of magnitude higher than infectivity titers. b Values measured in peak fraction of sucrose gradient of supernatant. c Values measured in 10–20 times concentrated supernatant. Ab, antibody; FFU, focus forming unit; GE, genome equivalents; IFN, interferon; nr, non reported; ntAb, neutralizing serum antibodies; TCID50, 50% tissue culture infectious dose

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87

determined whether this is a general feature of HCV or just a peculiarity of the JFH1 isolate and/or the cell culture conditions. Supporting the inherent properties of JFH1 to form viral particles in cell culture, Cai et al. (2005) reported the release of viral particles from Huh7 cells stably transfected with a cDNA expression construct, from which JFH1 genomes exhibiting the authentic 50 and 30 ends were transcribed under control of a cytomegalovirus (CMV) promotor. Cell culture supernatants derived from stably transfected cell lines showed HCV RNA and infectivity titers comparable to the JFH1 culture systems developed previously (Table II) (Wakita et al., 2005; Zhong et al., 2005). Secreted virus was able to infect Huh7.5 cells; however, its ability to persist and spread in this recipient cell culture was not studied. A drawback of this system might be expression of the HCV genome under control of a CMV promotor providing an unphysiological starting point; in stably transfected cells plasmid-based expression systems lead to nuclear transcription, whereas HCV replicates in the cytoplasm. This could result in intron excisions and other undesired mRNA editing events. Plasmid-based expression of the HCV envelope proteins has been shown to lead to splicing of a cryptic intron in E1 mRNA resulting in a partially deleted E1 glycoprotein (Dumonceaux et al., 2003). Finally, transfection of RNA transcripts resembles more closely the chimpanzee model, in which RNA transcripts are transfected directly into the liver. Thus, RNA transcripts generated in exactly the same manner could in parallel be tested in cell culture and in the chimpanzee model. Another cDNA expression system, containing HCV cDNA flanked by two self cleaving ribozymes, also yielded JFH1 but not genotype 1a (H77) and 1b (CG1b) viruses capable of infecting naı¨ve Huh7 cell cultures (Heller et al., 2005; Kato et al., 2007).

B. The J6/JFH1 cell culture system In parallel with the development of the original JFH1 cell culture system, Lindenbach et al. (2005) constructed an intragenotypic recombinant genotype 2a cDNA clone in which the HCV structural genes (Core, E1, E2), p7 and NS2 of JFH1 were replaced by the respective genes of the infectious clone pJ6CF (Yanagi et al., 1999a) (Fig. 5). After transfection or infection of Huh7.5 cells, J6/JFH1 viruses (Lindenbach et al., 2005) exhibited accelerated kinetics compared to nonadapted JFH1 viruses (Zhong et al., 2005, 2006) (Fig. 4). Furthermore, J6/JFH1 viruses showed higher specific infectivities than nonadapted JFH1 viruses, with peak infectivity titers of almost 105 50% tissue culture infectious doses (TCID50)/ml (Table II). Here infectivity titers of virus containing cell culture supernatants were determined by infecting replicate cell cultures with serial dilutions of such supernatants. The TCID50 values indicate the dilution of supernatant at which 50% of replicate cell cultures become infected

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Judith M. Gottwein and Jens Bukh

5⬘UTR H77 reference (genotype 1a) JFH1 (genotype 2a) Wakita 2005 Zhong 2005; 2006 J6/JFH1 (2a/2a) Lindenbach 2005

3⬘UTR C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

1a/JFH1 (H77/JFH1) Yi 2007

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

1a/JFH1 (H77/C3) Pietschmann 2006

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

1b/JFH1 (Con1/C3) Pietschmann 2006

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

1b/JFH1 Haqshenas 2007

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

3a/JFH1 (452/C6) Pietschmann 2006

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

3a/JFH1 (S52/JFH1) Gottwein 2007

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

4a/JFH1 (ED43/JFH1) Scheel 2008

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

4a/JFH1 (ED43/JFH1) Scheel 2008

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

H77-S Yi 2006

C

E1

E2

p7

NS2

NS3

NS 4A

NS4B

NS5A

NS5B

J6/JFH1 (J6/C3 = Jc1) Pietschmann 2006 1a/JFH1 (H77/JFH1) McMullan 2007 1a/JFH1 (H77/JFH1) Yi 2007 Scheel 2008

Genotype color codes

1a

1b

2a-JFH1

2a-J6

3a

4a

FIGURE 5 JFH1 and JFH1-based intra- and intergenotypic recombinants found to be infectious in Huh7 or derived cell lines. The genome of the reference isolate H77 (AF009606) is shown on top. Genotype-specific sequences are color-coded. The genotype and the strain names are indicated to the left of each genome together with the associated references (Gottwein et al., 2007; Haqshenas et al., 2007a; Lindenbach et al., 2005; McMullan et al., 2007; Pietschmann et al., 2006; Scheel et al., 2008; Wakita et al., 2005; Yi et al., 2006, 2007; Zhong et al., 2005, 2006). For specific recombinant names see associated publications. In genomes with the C3 NS2 junction, the C-terminal intergenotypic junction was localized between the first and second transmembrane domain of NS2. In other genomes, the C-terminal intergenotypic junction was located in the NS2 protease domain, equivalent to the junction identified by Kalinina et al. (2002) in the 2k/ 1b naturally occurring intergenotypic recombinant (see Fig. 1). In the remaining intergenotypic recombinants the C-terminal intergenotypic junction is localized at the NS2/NS3 junction. Several intergenotypic recombinants exhibit the nucleotide

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(Reed and Muench, 1938). Currently both TCID50 /ml and FFU/ml are used as measurement of infectivity titers, but it remains to be determined how these two measurements compare, in order to facilitate comparison of efficiencies of the HCV cell culture systems that are being developed. The robust growth characteristics of J6/JFH1 in cell culture indicated that this genome did not require adaptive mutations. This hypothesis was strengthened by the observation, that
50% of transfected cells produced infectious supernatant as determined by limiting dilution assays, indicating that infectivity of the supernatant derived from the transfected cell culture did not depend on rare virus variants (Lindenbach et al., 2005). Eventually, it has been demonstrated that viruses with unaltered J6/JFH1 genomes could be recovered after viral passage in Huh7.5 cells (Gottwein et al., 2007; Scheel et al., 2008). The biological relevance of the J6/JFH1 system is supported by its robust in vivo infectivity, in the chimpanzee and the uPA-SCID mouse model (Bukh and Purcell, 2006; Lindenbach et al., 2006). In chimpanzees, cell culture derived J6/JFH1 viruses induced a similar course of infection to that observed after intrahepatic transfection of RNA transcripts of infectious clones (Kolykhalov et al., 1997; Yanagi et al., 1997, 1998, 1999a). Similarly, J6/JFH1 viruses induced sustained infection in uPA-SCID mice whose serum could be used to infect naive mice. Animal-derived J6/JFH1 viruses could infect naı¨ve Huh7.5 cells, showing that in general it might be possible to yield robust infection of a permissive cell line with an infection-competent clinical isolate. Interestingly, specific infectivities of animal-derived J6/JFH1 were greatly improved compared with cell culture derived J6/JFH1 (Table II), pointing to its association with infectivity-enhancing factors present in vivo but not in vitro (see following section).

C. Analysis of HCV buoyant density suggests a role of lipoproteins for the viral life cycle Intriguingly, Lindenbach et al. (2006) found that specific infectivities of animal-derived J6/JFH1 viruses were almost 100-fold higher than that of HCVcc (mean specific infectivity of
1:20 for J6/JFH1 derived from

change C301T in the JFH1 50 untranslated region (UTR). Efficient culture of most intergenotypic recombinants depended on compensatory (or adaptive) mutations. H77-S is a highly replication-adapted genome with five adaptive mutations in NS3, NS4A, and NS5A (Yi and Lemon, 2004), thus differentiating it from the original H77C infectious clone sequence (Yanagi et al., 1997). After transfection of in vitro transcripts in Huh7.5 cells, production of viral particles was observed, however, with extremely low efficiency (Yi et al., 2006).

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different animal experiments compared to
1:1200 for HCVcc) (Bukh and Purcell, 2006; Lindenbach et al., 2006). However, after reculture of animalderived J6/JFH1 in Huh7.5 cells, the specific infectivity was typical for HCVcc, indicating that the observed difference might be caused by a different association of viral particles with hepatocyte- or serum-derived infectivity-enhancing factors such as lipoproteins. Association with such factors would also explain the differential buoyant densities observed for viral particles produced in vivo and in vitro, respectively. Whereas the majority of animal derived highly infectious HCV particles exhibited buoyant densities of
1.10 g/ml, the majority of cell culture derived HCV particles showed densities of
1.14 g/ml; however, also in HCVcc the highest infectivity was found in a minor virus fraction with a density of
1.10 g/ml (Lindenbach et al., 2006). These observations have been further elucidated by detailed analysis of JFH1 cell culture derived viruses (Gastaminza et al., 2006; Zhong et al., 2006) for which the main infectivity was also found in density fractions of
1.10 g/ml (and below
1.03 g/ml), whereas density fractions of
1.14 g/ml seemed to contain precursor viral particles, which also accounted for the majority of viruses detected intracellularly. Thus, intracellular HCV might prevail as high-density precursor and during egress acquire factors conferring lower density, such as host lipoprotein or apolipoproteins components. Association of lipoproteins and apolipoproteins to HCV particles has been implicated by studies in the HCVpp system (Bartosch et al., 2005; Meunier et al., 2005; Voisset et al., 2005) as well as by analysis of HCV particles derived from infected patients (Andre et al., 2002; Nielsen et al., 2006; Thomssen et al., 1993; Trestard et al., 1998). Further, HCV particle production by human hepatocytes has been shown to depend on the assembly and secretion of very low-density lipoprotein (VLDL) (Huang et al., 2007a). However, despite convincing evidence for this mechanism, an association between secreted HCVcc and VLDL could not be demonstrated, possibly due to lower stability of HCVcc–VLDL complexes in comparison to HCV–VLDL complexes derived from clinical specimens (Huang et al., 2007a). The association of HCV with lipoproteins is thought to be mediated by the HCV envelope glycoproteins (Bartosch et al., 2005; Monazahian et al., 2000). A point mutation in E2 has been shown to lower the buoyant density and to improve the infectivity of JFH1 virus particles, potentially by enhancement of the association with lipoprotein components (Zhong et al., 2006). In addition to a role in modulation of viral infectivity, lipoproteins could be of importance for viral egress by targeting HCV to the VLDL secretion pathway (Gastaminza et al., 2006). The density profile of HCV particles recovered from chimpanzees, uPA-SCID mice and cell cultures (Gastaminza et al., 2006; Lindenbach et al., 2005, 2006; Wakita et al., 2005; Zhong et al., 2006) is in accordance with that of HCV particles derived from patient sera (Hijikata et al., 1993b;

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Trestard et al., 1998), even though in the latter a broader density range was observed (Gastaminza et al., 2006). Analysis of patient sera also indicated that clinical HCV isolates with high specific infectivities might show low buoyant densities, further supporting the link between infectivity and density described above (Bradley et al., 1991). In summary, the buoyant density and specific infectivity of HCV particles seems to be modulated by yet unknown mechanisms which might involve lipoprotein pathways and appear to be different in vivo and in human hepatoma cell lines.

D. Possible causes of special growth characteristics of JFH1 and J6/JFH1 So far it has not been elucidated, why the JFH1 isolate, in contrast to all other HCV isolates, exhibits extraordinary capabilities to replicate in cell culture, and why it is able to produce infectious viral particles. The JFH1 isolate was derived from a patient suffering from fulminant hepatitis which is a rare course of HCV infection. However, unfortunately not all isolates rescued from patients with fulminant hepatitis seem to possess the same capability in cell culture. The infectious clone of the TN isolate (genotype 1a), which was derived from a patient with fulminant hepatitis and also caused severe hepatitis in an inoculated chimpanzee (Farci et al., 1999), did not replicate in cell culture (Sakai et al., 2007). A recent study of JFH1/Con 1 intergenotypic recombinant replicons pointed to an important role of JFH1 NS5B (Binder et al., 2007). Furthermore, in a detailed study of various intragenotypic J6/JFH1 recombinants JFH1 replication capabilities could not be localized to a single defined genome region. The JFH1 NS3 helicase, NS5B polymerase, and 30 UTR X-region together conferred replication capability to J6 subgenomic and full-length replicons; infectious viruses were produced after transfection of such full-length recombinants, however, with a greatly reduced efficiency compared to JFH1 (Murayama et al., 2007). These genome regions also conferred replication capability to subgenomic replicons of other genotype 2a but not genotype 1a and 1b isolates, pointing to the fact that the HCV nonstructural proteins interact in a genotype-specific manner (Murayama et al., 2007). JFH1, and specifically its NS3 helicase, NS5B polymerase and/or the 30 UTR X-region, could be less dependent on host cell factors, whose absence might limit growth of other HCV isolates in the examined host cell lines. Thus, genotype 1 but not JFH1 RNA replication seemed to depend on cyclophilin (Ishii et al., 2006; Watashi et al., 2005). In future studies, it would be interesting to narrow down the minimal genome regions responsible for JFH1 replication, to elucidate the mechanisms by which these confer cell culture viability to the JFH1

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isolate, and to identify possible host cell factors involved in this interplay. Such insights might then further the development of cell culture systems for other HCV genotypes. It was somewhat surprising that the intragenotypic recombinant J6/JFH1 performed better than the original JFH1 virus, which seemed to be attenuated in vitro and in vivo (Bartenschlager and Pietschmann, 2005; Lindenbach et al., 2005, 2006; Wakita et al., 2005; Zhong et al., 2005, 2006). In both cDNA clones, the genomic regions coding for the nonstructural proteins NS3, NS4A, NS5A, and NS5B are identical, whereas in the J6/JFH1 cDNA clone the genome regions encoding Core through NS2 of JFH1 were replaced by the equivalent sequence of the infectious clone pJ6CF (Yanagi et al., 1999a) (Fig. 5). The J6CF genome is known to induce robust infection in the chimpanzee model but is unable to replicate in cell culture including Huh7 cells. Thus, Core, E1, E2, p7, and/or NS2 of J6 seems to confer a higher capacity to mediate either viral assembly and release or entry. J6/JFH1 and JFH1 produced similar amounts of Core protein; however, JFH1 Core protein seemed to be captured intracellularly, because only about 1% of produced Core was released into the cell culture supernatant, whereas 10–30% was released in the case of J6/JFH1 (Pietschmann et al., 2006). This pointed to superior assembly and release capacities of J6/JFH1, which could at least partly be attributed to the J6 p7 protein, because replacement of JFH1 p7 by J6 p7 increased JFH1 peak infectivity titers up to 20-fold (Steinmann et al., 2007a). Furthermore, differences in infectivity mediated by the envelope glycoproteins—for example, by modulation of lipoprotein binding—might account for the better performance of J6/JFH1. Thus, a point mutation in JFH1 E2 has been shown to significantly improve infectivity of this virus (Zhong et al., 2006). These differences could be intrinsic features of the respective proteins of the two isolates or due to a suboptimal consensus sequence in the case of JFH1.

E. Applicability of JFH1 and J6/JFH1 cell culture systems Whereas studies in the replicon and HCVpp systems were limited to certain aspects of the viral life cycle (Fig. 3), with the identification of the JFH1 isolate the HCV research field finally had a highly efficient cell culture system mimicking the complete viral life cycle. As the replicon system, the JFH1 or J6/JFH1 systems were based on transfection of Huh7 cells or more permissive Huh7-derived cell lines with RNA transcripts. Whereas the primary J6/JFH1 genome led to efficient infection and seemed to be genetically stable, the JFH1 genome apparently required adaptive mutations for optimal efficiency. Upon transfection/infection, J6/JFH1 or adapted JFH1 viruses spread to the entire cell culture, yielding peak HCV RNA titers of
107.5 IU/ml and infectivity titers of
105 TCID50/ml or
106 FFU/ml

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(Table II). Furthermore, JFH1-based genomes containing reporter genes are being developed. Bicistronic JFH1 constructs containing the luciferase reporter gene facilitated convenient assessment of the HCV RNA replication level (Koutsoudakis et al., 2006; Wakita et al., 2005). Additionally, viable JFH1 genomes have been developed, in which luciferase was inserted in the C-terminal part of HCV NS5A (Kim et al., 2007). Furthermore, GFP could be inserted (Kim et al., 2007) at the same position as originally identified in the subgenomic replicon system (Moradpour et al., 2004b), enabling convenient detection of NS5A, for example, microscopically or by flow cytometry analysis. Development of these full viral life cycle in vitro systems will accelerate HCV research tremendously. They permit reverse genetic studies addressing the function of certain HCV genome regions and proteins. Furthermore, localization studies of HCV proteins as well as studies of HCV–host interactions can now be carried out in the context of the complete viral life cycle. Finally, new therapeutics, such as protease and polymerase inhibitors, can be tested in vitro. These systems not only allow confirmation of the relevance of results obtained in the replicon and HCVpp system for the full viral life cycle but also for the first time enable studies on so far poorly understood aspects of the viral life cycle, such as viral assembly and release, in a true cell culture system. Thus, reverse genetic studies have suggested a role for p7 and NS2 during early viral morphogenesis and assembly ( Jones et al., 2007; Steinmann et al., 2007a). Studies of the subcellular localization of HCV proteins in HCVcc-infected cells will refine results previously obtained in cells transfected with HCV protein expression—or replicon constructs. Thus, in JFH1, J6/JFH1 and 3a/JFH1 (see below)-infected cells HCV Core was associated with lipid droplets (Gottwein et al., 2007; Rouille et al., 2006) as previously described in Core expression (McLauchlan et al., 2002) and replicon systems (Pietschmann et al., 2002). Such studies might also be able to identify in which cellular compartments different steps of the viral life cycle occur. Whereas the membranous web is thought to be the site of HCV replication (Egger et al., 2002; El-Hage and Luo, 2003; Gosert et al., 2003; Moradpour et al., 2004b; Shi et al., 2003), by electron microscopy of JFH1-infected Huh7 cells, no virus-like particles could be identified in such membranous structures, suggesting that viral assembly might take place in a different cellular compartment (Rouille et al., 2006). A comprehensive study by Randall et al. (2007) identified Huh7.5 host cell factors of importance for the J6/JFH1 life cycle (see above); it will be interesting to further investigate the roles of these proteins. Additionally, the host cell response to HCV infection can now be studied in a full viral life cycle cell culture system and can be compared to results obtained by studies in the replicon system (Abe et al., 2005; Fang et al., 2006) and in vivo (Bigger et al., 2001, 2004; Jacobs et al., 2005; Su et al., 2002).

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The JFH1 and J6/JFH1 culture systems are valuable tools for testing of new therapeutics. A current research focus is the development of small molecule inhibitors of the NS3/4A protease and the NS5B polymerase (De Francesco and Migliaccio, 2005; Pawlotsky et al., 2007). In order to account for the complex interplay of HCV proteins with each other and cellular factors, these compounds should be tested in JFH1-based cell culture systems as exemplified by Lindenbach et al. (2005), even though in the current HCVcc systems NS3/4A and NS5B are of genotype 2a origin, which can in contrast to genotype 1 be treated well by the already approved combination therapy with IFN-a and ribavirin. Further, it is of importance to confirm and extend studies of ntAb, previously carried out in the HCVpp system, in the HCVcc system. In the HCVpp system, HCV E1/E2 proteins are assembled on lenti-/retroviral capsid proteins, which could lead to conformational changes compared to E1/E2 heterodimers physiologically assembled on HCV particles; in addition, HCVpp E1/E2 might show altered glycosylation patterns or associations with lipoproteins, because they are expressed in a kidney cell line, whose glycosylation and lipoprotein machinery differs from that of liver cells. Several groups have now demonstrated, that HCVcc can be efficiently neutralized with antibodies of different origins ( Johansson et al., 2007; Keck et al., 2007; Tarr et al., 2006) (Table II). Recently, it was shown that serum of patient H (genotype 1a infected) cross-neutralized a panel of JFH1-based intergenotypic recombinant viruses as efficiently as previously shown for HCVpp bearing the envelope proteins of the same prototype strains (Scheel et al., 2008; see below). Furthermore, the importance of several components of the putative HCV receptor complex for HCV entry, first elucidated in the HCVpp system, has been confirmed for HCVcc (Akazawa et al., 2007; Catanese et al., 2007; Evans et al., 2007; Grove et al., 2007; Kapadia et al., 2007; Koutsoudakis et al., 2007) (Table II), thus supporting the biological relevance of the HCVpp system. Additionally, the impact of antivirals targeting viral assembly and release can now for the first time be investigated in a cell culture system before testing them in chimpanzees or humans. Thus, it was recently demonstrated that amantadine, a putative p7 ion channel blocker, had no antiviral effect in the HCVcc system at noncytotoxic concentrations (Steinmann et al., 2007b), which might help explain the controversial results of clinical trials (Manns et al., 2006). However, even though JFH1-based cell culture systems mimic the full complexity of the viral life cycle, the replicon as well as the HCVpp system will also in the future be valuable tools to study the effect of interventions, for example, reverse genetics, on defined aspects of the viral life cycle.

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IX. PERSPECTIVES FOR FURTHER DEVELOPMENT OF HCV CELL CULTURE SYSTEMS A. Adaptation of cell culture systems to yield higher viral titers Although the development of the first cell culture systems allowing the study of the full viral life cycle was a major breakthrough, there is a strong need for further developments. The peak viral RNA and infectivity titers (Table II) are low when compared to many other virus systems. It would be desirable to achieve titers several orders of magnitude higher in order to facilitate the development of a cell culture grown inactivated virus vaccine, as was developed for another hepatitis virus, the hepatitis A virus (Peetermans, 1992). Adaptation of the described systems could be achieved by prolonged or serial passage in cell culture, as already carried out for JFH1 (Zhong et al., 2006), where a point mutation in E2 was shown to increase infectivity titers. Employing reverse genetics adaptive viral mutations identified as described above could be introduced in the original infectious cDNA clones (JFH1 and J6/JFH1) and could be tested for their effect in vitro. During viral passage to naı¨ve cells, the dose of the viral inoculum is thought to influence viral adaptation and fitness. Inoculation with big virus populations increases the chance of transfer of rare viral variants with increased fitness, which might then outcompete less fit viruses, thus increasing the overall fitness of the viral population present in the recipient cell culture. In contrast, a low-dose inoculum is at a greater risk of containing nonfunctional or less fit viruses resulting in a reduction of overall fitness of the virus population in the recipient cell culture (Domingo et al., 1997). In the case of the intragenotypic recombinant J6/JFH1, viral titers could be improved by mapping the optimal site of the C-terminal intragenotypic junction. Thus, relocalization of this junction between the first and second putative transmembrane domain of NS2 led to a
5–10-fold increase in viral titers compared to the original J6/JFH1 employing a C-terminal intragenotypic junction at the precise C-terminus of NS2 (Table II, Fig. 5). This improved junction was termed C3 and the resulting J6/JFH1 construct J6/C3 or Jc1 (Pietschmann et al., 2006). It is possible that changes of the N-terminal intragenotypic junction could also result in improved growth characteristics of the J6/JFH1 recombinants.

B. Cell culture systems for other HCV genotypes 1. Development of intra- and intergenotypic JFH1-based recombinants After development of the original JFH1-based cell cultures described above, in vitro studies of HCV were still limited to HCV genotype 2a. Because neutralizing antibodies are not expected to cross-neutralize all

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genotypes (Meunier et al., 2005) and specific antiviral drugs could have differential efficiencies against various genotypes (Pawlotsky et al., 2007), it is of importance to develop cell culture systems for the six major genotypes of HCV, preferably by use of prototype strains already characterized (Bukh et al., 1998). Because HCV in vivo infectious cDNA clones other than JFH1 and J6/JFH1 were not replicating in cell culture (Bartenschlager, 2006; Bartenschlager and Sparacio, 2007), one approach is the development of intergenotypic recombinant cDNA clones which would retain the extraordinary replication kinetics of JFH1. Intragenotypic (Colina et al., 2004) and intergenotypic (Kageyama et al., 2006; Kalinina et al., 2002; Legrand-Abravanel et al., 2007; Moreau et al., 2006; Noppornpanth et al., 2006) recombinant HCV genomes are occurring naturally (see above). In addition, intergenotypic replicons have been shown to be functional (Binder et al., 2007; Graham et al., 2006; Lanford et al., 2006). Since the intragenotypic recombinant J6/JFH1 showed even more favorable growth kinetics than the original JFH1 it seemed a logic starting point to replace, in analogy to J6/JFH1, the structural (Core, E1 and E2), p7, and NS2 genes with the respective genes of prototype HCV strains. With such intergenotypic recombinant genomes HCV entry events and related therapeutics (Meanwell, 2006; Pawlotsky et al., 2007), ntAb and the functions of the structural proteins, p7 and NS2 could be studied in a genotype-specific manner, while the replication characteristics of JFH1 would be retained. After primary reports indicated that an 1a/JFH1 intergenotypic recombinant, containing Core-NS2 of strain H77, replicated but did not lead to production of viral particles (Lindenbach et al., 2005), Pietschmann et al. (2006) showed intergenotypic JFH1-based recombinants of genotypes 1a (H77), 1b (Con1), and 3a (HCV-452) to be viable in cell culture (Fig. 5). Since the NS2/3 autoprotease relies on a functional cleavage site between NS2 and NS3, a focus of this study was the determination of the optimal intergenotypic C-terminal junction, which apparently depended on the isolate. Thus, a junction between the first and second transmembrane domain of NS2 (C3 junction) showed superior performance for the H77, Con1 and J6 recombinants. However, in the case of HCV-452, the NS2/NS3 junction (C6 junction), also employed in the original J6/JFH1 genome (Lindenbach et al., 2005), performed best (Fig. 5). These intergenotypic recombinants replicated as efficiently as JFH1 but yielded relatively low infectivity titers (Table II), possibly due to suboptimal interactions of proteins of different genotypes. In theory, such limitations might be overcome by the acquisition of compensating mutations. However, in this initial study of intergenotypic JFH1-based recombinants it was not investigated whether adaptive mutations were required for viability or if selection of such mutations, for example, by serial passage in cell culture, was able to improve growth characteristics of these intergenotypic viruses (Pietschmann et al., 2006).

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Such studies were subsequently carried out by Yi et al. (2007). Viability of a H77/JFH1 recombinant with the intergenotypic NS2/NS3 junction (Fig. 5) primarily depended on a mutation in NS3, and infectivity titers could be further improved by combination with another mutation in E1. Different mutations were acquired when an alternative C-terminal intergenotypic junction was used. Thus, an H77/JFH1 recombinant with a naturally occurring junction in NS2 (Kalinina et al., 2002) (Fig. 5) was dependent on mutations in p7 and NS2, either singly or in combination. Reverse genetic studies showed, that these H77/JFH1 recombinants with adaptive mutations yielded infectivity titers comparable to the original non-adapted JFH1 virus (Table II). However, a third H77/JFH1 recombinant with the C-terminal intergenotypic junction localized between p7 and NS2 could not be adapted to growth in cell culture, further underlining the importance of the localization of the intergenotypic junctions. Interestingly, a JFH1-based intergenotypic recombinant with the p7 protein of an Australian genotype 1b isolate (Fig. 5), thus also employing a C-terminal intergenotypic p7/NS2 junction, was viable and did not acquire adaptive mutations in the p7 sequence (Haqshenas et al., 2007a). However, comparison of its growth kinetics with that of JFH1 suggested dependence on adaptive mutations, which might be located in other, not sequenced, genome regions. Less effort has been undertaken to optimize the N-terminal intergenotypic junction. Most intergenotypic recombinants developed so far contain the JFH1 50 UTR and expressed the complete Core protein of the respective genotype (Fig. 5). However, it has been shown that the H77 50 UTR could be included in a H77/JFH1 recombinant with the C-terminal intergenotypic NS2/NS3 junction (McMullan et al., 2007). Similar to the equivalent construct with the JFH1 50 UTR (Yi et al., 2007), viability of this virus apparently also depended on adaptive mutations in E1 and NS3 (McMullan et al., 2007). Recent studies of our group focused on establishing cell culture systems for all major HCV genotypes. We constructed intergenotypic JFH1based recombinants containing Core-NS2 of prototype strains of different genotypes (Fig. 5). Interestingly, intergenotypic recombinants acquired mutations during passage in cell culture. In reverse genetic studies, we showed dependence of 1a/JFH1 on mutations in NS3 (Scheel et al., 2008). Two single mutations in p7 and NS3, respectively, and several combinations of mutations in p7 with mutations in NS3 or NS5A were able to confer viability to 3a/JFH1 (Gottwein et al., 2007). For 4a/JFH1 we also investigated the effect of different C-terminal intergenotypic junctions. While 4a/JFH1 with the junction after the first putative transmembrane domain of NS2 [equivalent to the C3 junction (Pietschmann et al., 2006)] could not be adapted to grow in cell culture, 4a/JFH1 with a naturally occurring junction in NS2 (Kalinina et al., 2002) depended on one mutation in the 4a part of NS2. Interestingly, 4a/JFH1 with the entire 4a

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NS2 depended on an additional mutation in the 4a NS2 sequence specific for this recombinant (Scheel et al., 2008). It will be interesting to investigate the mechanisms by which mutations identified in intergenotypic recombinants confer adaptation. They might either optimize HCV protein function to support critical steps of the viral life cycle or optimize the interaction of HCV proteins with cellular proteins or other HCV proteins derived from different genotypes. Interestingly, as adaptive mutations observed in the replicon system, many of the changed amino acids do not occur naturally at the observed position, thus raising the question if such adapted viruses might be viable in vivo. It will be also of interest to determine if and how the identified mutations can be combined and whether they can confer viability to other HCV isolates. Employing intergenotypic recombinants, our group demonstrated that infection of Huh7.5 cells with genotype 3a and 4a depended on CD81, as previously shown for genotype 2a (Wakita et al., 2005; Zhong et al., 2005), 1a (Yi et al., 2007), and 1b (Pietschmann et al., 2006). Thus, CD81 seems to be important for entry of most HCV genotypes. We also investigated the neutralization capacity of chronically infected patient sera showing that 1a and 4a intergenotypic recombinants could be neutralized with homologous serum. Furthermore, we demonstrated that antibodies with broadly neutralizing activity against different genotypes can be derived from patient serum; in cross-neutralization experiments chronic phase serum from patient H, infected with the genotype 1a strain H77, neutralized 1a, 4a, 5a, and 6a but not 2a and 3a intergenotypic recombinants (Scheel et al., 2008). These observations are in agreement with results obtained with serum of patient H in the HCVpp system (Meunier et al., 2005). Results obtained by Pietschmann et al. (2006) show that murine antibodies raised against genotype 1a (H77), were able to neutralize intergenotypic recombinants of genotypes 2a and 3a at least as efficiently as recombinants of genotype 1a (H77). These findings are encouraging, because they demonstrate that it might be possible to identify broadly cross-neutralizing antibodies, which now can be studied in complete cell culture systems of intergenotypic recombinants containing the envelope proteins of all major HCV genotypes.

2. Development of cell culture infectious full-length clones of other genotypes Intergenotypic recombinant cDNA clones will provide valuable information on the interaction of HCV proteins and a starting point for the development of full-genome length cell culture models of the major HCV genotypes. The genome regions conferring the unique growth characteristics of JFH1 apparently reside in its NS3 helicase domain and the NS5B to 30 UTR region (Murayama et al., 2007). It would be desirable to

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further confine these regions and the mechanisms facilitating JFH1 replication in order to be able to transfer such elements to cDNA clones of other genotypes. However, primary results obtained by Murayama et al. were slightly discouraging, because introduction of the JFH1 NS3 helicase and the NS5B to 30 UTR region could restore replication of other genotype 2a, but not of genotype 1a and 1b replicons (Murayama et al., 2007). The replication adapted H77-S genome, that had five mutations in NS3, NS4A, and NS5A (Yi and Lemon, 2004) compared with the H77C infectious clone from which it originated (Yanagi et al., 1997), yielded infectious viral particles in Huh7.5 cells (Yi et al., 2006). This system was characterized by a very low specific infectivity (
1:50,000) due to low infectivity titers (
102 FFU/ml) and relatively high RNA titers in fractions from gradients loaded with concentrated supernatants from transfected cells (Table II). Low infectivities could result from H77-S being adapted for HCV RNA replication, because it was previously shown that replication adaptive mutations could be detrimental for virus formation in vivo (Bukh et al., 2002) and probably also in vitro (Bartenschlager, 2005). It would be interesting to determine, if the replication adapted H77-S could also be further adapted to support the complete viral life cycle, for example, by serial passage in cell culture, as carried out for intergenotypic recombinants (see above). Another group reported generation of infectious HCV particles after transfection of in vitro RNA transcripts of the H77 infectious cDNA clone (Kolykhalov et al., 1997) in human hepatocytes, which were immortalized by transfection of the HCV Core genomic region of genotype 1a (Kanda et al., 2006). Concentrated cell culture supernatant was determined to yield an infectivity titer of 104–105 FFU/ml and a genome titer of
107–8 RNA copies/ml (Table II), while viral spread in the cell culture was not examined. Immortalized human hepatocytes might be more susceptible to full-length H77 replication than Huh7-derived cell lines. On the contrary, formation and release of viral particles might be enhanced by transexpression of genotype 1a Core, particularly, because Yi et al. (2006) reported a strongly reduced amount of Core expression from the H77-S genome when compared to JFH1. A different approach for the development of efficient full-length HCV cell culture systems could be systematic testing of clinical isolates in cell culture, because it was found, that serum samples derived from chimpanzees infected with J6/JFH1 viruses could be used to infect Huh7.5 cells. However, serum-derived HCV particles might show variable infectivity. On one hand, serum-derived factors such as lipoproteins might cause enhancement of infectivity (Lindenbach et al., 2006; Meunier et al., 2005). On the other hand, infectivity might be reduced due to association with ntAb. Evidence for such effects has previously been reported. After introduction of a multiantigen screening for anti-HCV

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antibodies (without screening for HCV RNA), an outbreak of acute hepatitis C occurred in patients treated with thus screened intravenous immune globulin preparations (Gammagard) in 1993/1994. However, patients, who had received unscreened preparations, were not affected. Most probably all preparations contained HCV viruses. However, apparently unscreened preparations additionally contained anti-HCV antibodies, which neutralized the coadministered HCV viruses and thus protected patients against infection (Bresee et al., 1996; From the Centers for Disease Control and Prevention, 1994; Healey et al., 1996).

C. Expansion of cell culture systems to different host cells Currently, HCV cell culture systems are restricted to one cell type, the human hepatoma cell line Huh7 and derived cell lines. For several reasons, it would be desirable to identify additional host cell lines. As a carcinoma cell line, Huh7 cells can be expected to substantially differ from primary hepatocytes. Thus, low expression levels of the dsRNAsensing Toll-like receptor 3 have been reported for Huh7 cells, and in Huh7.5 cells, additionally, an impairment of the dsRNA-sensing helicase RIG-I/IFN pathway (see above) has been described (Sumpter et al., 2005). Both factors are thought to contribute to the heightened permissiveness of Huh7 cells for HCV. Therefore, it would be of importance to confirm the biological relevance of findings achieved in Huh7 cultures in primary human hepatocytes. Even though primary hepatocytes derived from humans, chimpanzees (Bartenschlager et al., 2004), and tree shrews (Barth et al., 2005) could be infected with serum-derived HCV, replication levels were low, requiring PCR-based techniques for virus detection. Additionally, primary human hepatocytes are derived from surgical specimen resulting in limited access and, depending on the handling, in varying quality. At last, given the short life time of primary hepatocytes in vitro, a given study might have to be carried out on genetically heterogeneous hepatocyte populations derived from different individuals. Recently another, presumably more physiological cell culture system was proposed (Sainz and Chisari, 2006). In this study, growth arrest and differentiation of Huh7 cells were induced by DMSO treatment. Thus,
80% of cells in monolayer cultures could be infected with JFH1 for up to 9 weeks. This is in contrast to rapidly dividing original Huh7 and derived cell lines; in these cultures HCV infection induced a cytopathic effect followed by preponderance of Huh7 cells with nonpermissive phenotypes (Zhong et al., 2006). Research on HCV has been hampered by the absence of a small animal model, and the recently developed uPA-SCID mouse engrafted with human liver (Mercer et al., 2001; Meuleman et al., 2005) is rather

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complicated and provides a relatively short time frame in which studies can be performed, making it primarily suitable for infection studies. In addition, host adaptive immune responses cannot be studied due to the SCID genetic background. The first step toward a robust and widely available HCV mouse model could be adaptation of JFH1-based cell culture systems to growth in mouse hepatocytes. While the reasons for the HCV species barrier have not been elucidated, a Con1 subgenomic replicon was able to replicate in the mouse hepatoma cell line Hepa1-6 after transfection with total RNA derived from HeLa cells containing this replicon. Thus, replication in HeLa cells might have induced the acquisition of cell type specific adaptive mutations, subsequently enabling replication in Hepa1-6 cells (Zhu et al., 2003). Further, it was demonstrated that JFH1 but not several genotype 1a and 1b subgenomic and full-length replicons were able to replicate in mouse hepatocyte (MMH, AML12) and fibroblast (NIH3T3) cell lines; apparently no adaptive mutations were required for replication in MMH cells. However, no infectious virus was generated and these cell lines were not permissive to infection with JFH1 viral particles rescued from infected Huh7 cells, indicating that assembly or egress as well as entry events might be species specific (Uprichard et al., 2006). Once cell culture grown HCV could be adapted to yield higher titers, development of inactivated virus vaccines would become a feasible approach to explore. It would be advantageous to identify other, also nonhepatic, possible HCV host cell lines already approved for vaccine development (Huang et al., 2004). Apart from hepatocytes and hepatic cell lines, HCV was described to infect B cells and dendritic cells (Goutagny et al., 2003; Lerat et al., 1998; Navas et al., 2002; Sung et al., 2003). Subgenomic Con1 RNA could be adapted to replicate in the nonhepatic HeLa cell line, however, not in several other cell lines including Vero (African green monkey kidney epithelial) cells commonly used for vaccine development (Huang et al., 2004; Zhu et al., 2003). The JFH1 subgenomic replicon was also shown to replicate in HeLa and additionally in 293-T cell lines (Kato et al., 2005), but JFH1 virus did not infect HeLa cells (Wakita et al., 2005). A detailed review of cell lines supporting HCV RNA replication was previously provided by Bartenschlager and Sparacio (2007). Knowledge about the determinants of HCV tissue tropism would facilitate the identification of nonhepatic host cells. In the HCVpp system, all cells permissive to HCVpp expressed CD81 and SR-BI and were liver derived. However, certain nonhepatic cell lines (e.g., HeLa cells) expressed these putative HCV coreceptors but were still not permissive to HCVpp infection (Bartosch et al., 2003c). The tight-junction component, claudin-1, which is highly expressed in the liver, could contribute to organ tropism, because it appears to act as an HCV coreceptor during late entry and could confer infectivity to otherwise unsusceptible CD81 +

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SR-BI + 293-T cells (Evans et al., 2007). Hepatic glycosaminoglycans have a unique composition (Vongchan et al., 2005) and could thus contribute to tissue tropism on the entry level, because glycosaminoglycans are implicated in primary attachment of viruses in general and specifically HCV (Koutsoudakis et al., 2006; Morikawa et al., 2007; Sieczkarski and Whittaker, 2005). Also intracellular factors supporting different steps of the viral life cycle might be involved in HCV tissue tropism: MicroRNA 122 (miR-122) is specifically expressed in human, rat, and mouse liver, in Huh7 and Huh7.5 cells, but not in HeLa and HepG2 cell lines (Chang et al., 2004; Jopling et al., 2005; Lagos-Quintana et al., 2002; Randall et al., 2007). HCV RNA contains putative miR-122-binding sites in the 50 and 30 UTR. Direct interaction of miR-122 with the 50 UTR seems to be important for HCV genotype 1 replication (Jopling et al., 2005), and depletion of miR-122 led to an up to fourfold reduction of infectivity titers in the J6/JFH1 cell culture system (Randall et al., 2007). Alternatively to a direct role for HCV replication, miR-122 could be sequestered from its targets by physical interaction with HCV genomes. Indeed, miR-122 has recently been shown to be a key regulator of hepatic cholesterol and fatty acid metabolism and could as such have an impact on the HCV life cycle (Esau et al., 2006; Krutzfeldt et al., 2005). On the contrary, dependence of HCV on the liver-specific lipid environment might in itself contribute to liver tropism. HCV depends on the liver-specific cholesterol and lipid metabolism (Kapadia and Chisari, 2005; Sagan et al., 2006; Su et al., 2002; Ye, 2007) as well as lipoprotein components (Andre et al., 2002, 2005; Meunier et al., 2005; Nielsen et al., 2006). Recently, membrane vesicles containing the HCV replication complex have been shown to harbor proteins required for VLDL assembly, mainly apoB, apoE and microsomal triglyceride transfer protein (MTP) (Huang et al., 2007a). However, in the same study HCV replication was shown to be independent of VLDL synthesis, which instead seemed to play an important role for HCV assembly and release. Finally, stimulation of IRES-dependent translation of HCV RNA by the HCV 30 UTR (Bradrick et al., 2006; Song et al., 2006) was shown to be restricted to human hepatoma cells (Song et al., 2006). Such RNA 50 -30 interactions usually require cellular RNA-binding proteins (Sachs et al., 1997), which might only be present in human hepatoma cells. Theoretically, liver-specific expression levels of any host cell factor of importance for the HCV life cycle (see above) could contribute to HCV liver tropism. Knowledge about the main determinants of liver tropism would allow a more targeted search for alternative HCV host cell lines or could be used to supplement advantageous cell lines, for example, cell lines licensed for vaccine development, with factors required for HCV growth and thus conferring HCV permissiveness.

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X. CONCLUSION—IMPLICATIONS OF NOVEL CELL CULTURE SYSTEMS The newly developed cell culture systems will accelerate hepatitis C research tremendously, because they for the first time allow studies of the full viral life cycle of HCV in a robust, reproducible, and widely available model. Before the identification of the JFH1 isolate which apparently possesses unique replication characteristics, and the development of JFH1-based cell culture models, the full viral life cycle could only be studied in chimpanzees and since recently in the uPA-SCID mouse engrafted with human liver. Research in cell cultures, however, was restricted to certain aspects of the HCV life cycle. The replicon system could be employed for studies of HCV RNA replication, while HCV pseudo-particles allowed the investigation of HCV entry events. Both systems employed the human hepatoma cell line Huh7, and the studies in the replicon system led to identification of Huh7-derived cell lines with heightened permissiveness for HCV, such as Huh7.5 cells. After discovery of the unique replication capacity of JFH1 in the replicon system, insights gained from previous research, especially the knowledge about construction of cDNA clones and the availability of highly permissive Huh7 host cell lines, led to the rapid development of the first full viral life cycle JFH1based cell culture systems. Employment of cDNA clones allows reverse genetic studies, which enable studies of the effect of specific genetic manipulations on the HCV life cycle. It is advantageous that the same HCV genomes, generated as RNA in vitro transcripts from consensus cDNA clones, can be transfected into both chimpanzees and cell cultures. Thus, results obtained in vitro and in vivo could be compared directly. Full viral life cycle cell culture systems will lead to rapid growth of our knowledge of the function of HCV genomic regions and proteins as well as the interaction of HCV proteins with each other and with host cell proteins. Further, it will be possible to study the host response to HCV infection in vitro, for example, with proteomics and microarray analysis, and compare the results to previous data generated in vivo. Since the currently available therapy for HCV, combination therapy with IFN-a and ribavirin, can cure only about 50% of the treated patients and is expensive as well as associated with severe side effects, a main field of application of the new cell culture systems will be the study of potential new therapeutics. Currently, a major research focus is the development of small molecule antivirals, predominantly targeting the NS3/4A protease and NS5B polymerase. Even though the effect of such compounds on replication could already be studied in the replicon system, in this system the complex interplay of the targeted proteins with other HCV proteins and cellular factors during the complete HCV life cycle could not be taken

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into account. The new JFH1-based culture systems will also be used to confirm and expand studies of neutralizing serum antibodies, as carried out in the pseudo-particle system. Thus, the role of the host humoral immune response against HCV might be further elucidated and identification of broadly reactive neutralizing antibodies and conserved neutralization epitopes might aid vaccine development. With the availability of cell culture grown hepatitis C viral particles, the development of inactivated whole virus vaccines becomes theoretically feasible. Further, complete viral life cycle cell cultures allow the study of compounds interfering with viral assembly and release, which could neither be studied in the replicon nor in the pseudo-particle system. Finally, therapeutics targeting host cell factors might be valuable alternatives and might lead to identification of broad spectrum antivirals. Even though we have been provided with a great research tool, the challenge is not only its employment to elucidate the many unresolved questions about the hepatitis C life cycle, but also the improvement of the system itself. It would be desirable to adapt cell culture grown HCVs to yield higher titers and possibly identify other host cells in order to facilitate vaccine development. Initially, full viral life cycle cell culture systems were restricted to genotype 2a. Since major virological and clinical differences between HCV genotypes exist, and therapeutic compounds will most probably show differential efficiency toward the different genotypes, it will be of great importance to eventually develop cell culture systems for all major HCV genotypes. Intergenotypic recombinants containing Core-NS2 of prototype strains of the major genotypes could be adapted to grow to titers comparable to J6/JFH1. This now allows genotype-specific studies of HCV Core, the envelope glycoproteins, the putative ion channel p7, as well as NS2 and thus HCV entry, and assembly as well as related therapeutics. Such intergenotypic recombinants might also further the development of vaccines targeting all genotypes, because they contain genotype-specific envelopes and nucleocapsid sequences. At last, they might be the first step toward full-length cell culture systems of other genotypes.

ACKNOWLEDGMENTS We are grateful to Troels K. H. Scheel for discussion and ciritical review of this manuscript.

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CHAPTER

3 Poxvirus Host Range Genes Steven J. Werden, Masmudur M. Rahman, and Grant McFadden

Contents

Abstract

I. Introduction II. Orthopoxvirus Host Range Genes A. SPI-1 B. K1L C. C7L D. CHOhr E. p28/N1R F. B5R (ps/hr) G. E3L H. K3L III. Myxoma Virus Host Range Genes A. M-T2 B. M-T4 C. M-T5 D. M11L E. M13L F. M063 IV. Molluscum Contagiosum: An Extreme Example of Host Range Restriction V. Conclusions Acknowledgments References

136 137 140 143 145 146 147 148 149 152 153 154 155 155 157 158 159 160 160 162 163

As a family of viruses, poxviruses collectively exhibit a broad host range and most of the individual members are capable of replicating in a wide array of cell types from various host species, at least

Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida 32610 Advances in Virus Research, Volume 71 ISSN 0065-3527, DOI: 10.1016/S0065-3527(08)00003-1

#

2008 Elsevier Inc. All rights reserved.

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in vitro. At the cellular level, poxvirus tropism is dependent not upon specific cell surface receptors, but rather upon: (1) the ability of the cell to provide intracellular complementing factors needed for productive virus replication, and (2) the ability of the specific virus to successfully manipulate intracellular signaling networks that regulate cellular antiviral processes downstream of virus entry. The large genomic coding capacity of poxviruses enables the virus to express a unique collection of viral proteins that function as host range factors, which specifically target and manipulate host signaling pathways to establish optimal cellular conditions for viral replication. Functionally, the known host range factors from poxviruses have been associated with manipulation of a diverse array of cellular targets, which includes cellular kinases and phosphatases, apoptosis, and various antiviral pathways. To date, only a small number of poxvirus host range genes have been identified and studied, and only a handful of these have been functionally characterized. For this reason, poxvirus host range factors represent a potential gold mine for the discovery of novel pathogen–host protein interactions. This review summarizes our current understanding of the mechanisms by which the known poxvirus host range genes, and their encoded factors, expand tropism through the manipulation of host cell intracellular signaling pathways.

I. INTRODUCTION Poxviruses are a highly successful family of pathogens, which are unique among animal viruses in several respects. Members of the poxvirus family are characterized by a complex enveloped brick-shaped virion, large genomes that range from 130 to 300 kb and a replication site exclusive to the cytoplasm of the host cell (Moss, 2007). As a family of viruses, albeit with a few exceptions like Molluscum contagiosum virus (MCV) (described later), most poxviruses have the capacity to infect a relatively wide spectrum of eukaryotic hosts in vitro, whereas individual virus members within the family are commonly restricted to either an exclusive host, or a relatively small number of potential host species (McFadden, 2005). In cultured mammalian cells, most poxviruses can bind and initiate the virus infectious cycle but whether the subsequent infection is permissive (to produce infectious progeny virions) or nonpermissive (where the infection aborts at some intracellular stage) depends on the specific poxvirus–cell pairing. In nonpermissive cells, the replicative block for a specific poxvirus infection usually occurs following virion binding and entry, and frequently results from either the inability to circumvent innate intracellular barriers crucial to completion of the virus replication cycle,

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or the failure of the cell to provide necessary complementing factors ( Johnston and McFadden, 2003; McFadden, 2005). Consequently, the ability of poxviruses to manipulate the signal transduction networks of infected cells has a major impact on the outcome of a specific viral infection. The distinct tropism of each individual poxvirus is thus strictly dependent on the unique repertoire of host range genes expressed by that virus (McFadden, 2005). Some of the proteins encoded by poxvirus host range genes, called host range factors, have been demonstrated to specifically target host intracellular pathways, which are often configured to prevent viral infections, to establish a cellular microenvironment more favorable to viral replication (Seet et al., 2003). In fact, the outcome of the dynamic struggle between viral host range factors and cellular antiviral pathways can determine whether a specific infection will be permissive or not. Before one can gain a better appreciation of the sophisticated interplay between viral and cellular proteins, the operational roles that host range factors play in the manipulation of the targeted host intracellular signaling pathways must be better understood. Generally speaking, the discovery of specific poxvirus host range genes has usually been the result of targeted gene-knockout analysis in which the mutant virus isolate is subsequently shown to be replication deficient in a subset of cultured cells for which the parental virus is permissive (Johnston and McFadden, 2004). All poxviruses are predicted to encode a unique collection of host range factors; however, their specific identification as host range genes has been largely fortuitous. Thus, our current understanding of the host target pathways with which the protein products of these poxvirus host range genes interact is incomplete. This chapter will specifically highlight host range genes that have been discovered and analyzed to date, and the majority of these are from members of only two of the eight chordopoxvirus genera; namely, the orthopoxviruses and the leporipoxviruses (Table I).

II. ORTHOPOXVIRUS HOST RANGE GENES The family of orthopoxviruses collectively exhibits the broadest host range of the chordopoxviruses, and most of the individual orthopoxvirus members are capable of replicating in a wide array of cell types from various species, at least in vitro. This unique cellular tropism can in part be explained by the large coding capacity of the canonical orthopoxviral genome, which encodes a diverse spectrum of specific viral proteins that enable the virus to cross species barriers (Alcami and Koszinowski, 2000). The interaction between host cell proteins and poxvirus host range proteins is so diverse that no single viral host range ortholog common to all poxvirus genomes has yet been identified (Seet et al., 2003). However,

TABLE I

Poxvirus host range genes

Gene

Protein type

Function

SPI-1

Serpin

K1L

Ankyrin-repeats

C7L

Cytoplasmic

May inhibit caspaseindependent pathway of apoptosis Inhibit NF-kB activation Inhibition of apoptosis

C7L/K1L

–

–

CHOhr

Ankyrin-repeats

P28/N1R

E3-ubiquitin ligase

B5R

Membrane glycoprotein

Prevent protein synthesis shutoff Degradation of proteins, inhibit apoptosis Activate Src

Cultured cells with defects in virus host range

References

PK15 cells, A549

Brooks et al. (1995)

RK13 cells

Shisler and Jin (2004)

RK13, hamster Dede cells PK1, RK13 and most human cells CHO

Najera et al. (2006)

Orthopoxvirus

Murine macrophages

Vero, CEF, PK-15 and quail (QT-6)

Perkus et al. (1990) Spehner et al. (1988) Brick et al. (2000), Nerenberg et al. (2005) Stern et al. (1997)

E3L

dsRNA-BP

Inhibit IFN responses

K3L

eIF2a homologue

Pseudosubstrate inhibitor of PKR

M-T2 M-T4 M-T5

TNF receptor ER-localized Ankyrin-repeats

M11L

Mitochondrial

Inhibit rabbit TNFa Inhibit apoptosis Cell cycle progression, phosphorylation of AKT Inhibit apoptosis

M13L

Pyrin (PYD) domain C7L like

Inhibit inflammasome Unknown

HeLa, Vero, murine dendritic cell line, CEF (MVA-E3L) BHK cells, mouse L929

Beattie et al. (1995b), Langland and Jacobs (2002) Langland and Jacobs (2002)

Rabbit T-cells Rabbit T-cells Rabbit T-cells, human tumour cells

Macen et al. (1996) Barry et al. (1997) Mossman et al. (1996), Wang et al. (2006)

Rabbit T-cells Rabbit T-cells

Everett et al. (2000), Macen et al. (1996) Johnston et al. (2005a)

Rabbit cells

Barrett et al. (2007b)

Leporipoxvirus

M063

Note: The defects in host-range are specifically exhibited by viral gene knockout constructs or viral recombinants engineered to express heterologous host ranges genes. Revised from (McFadden, 2005).

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many of the better characterized host range genes in orthopoxviruses are sometimes fairly well conserved among the family members and suggest the importance of these genes in poxvirus biology by counteracting host cell responses and manipulation of the host cellular microenvironment (Table II). Vaccinia virus (VACV) is the most extensively studied orthopoxvirus and for that reason has provided the bulk of our knowledge in regards to the discovery and functional understanding of orthopoxvirus host range genes.

A. SPI-1 Historically, the first host-range gene encoded by a poxvirus was indirectly discovered using mutants of rabbitpox virus (RPXV) nearly a halfcentury ago (Fenner, 1958). RPXV commonly produces red hemorrhagic lesions or pocks when cultured on the chorioallantoic membrane (CAM) of developing chick embryos; however, ‘‘white pock’’ mutants were observed to spontaneously arise within the wild-type population (Fenner, 1958). Pair-wise crosses between isolated RPXV mutants were performed and many pairs recombined to yield red pocks resembling wild-type RPXV. On the basis of the presence or absence of recombinants, the mutants could be arranged in a linear order, suggesting that the white pock mutants of RPXV were single-locus mutations (Gemmell and Cairns, 1959). However, certain crosses were unable to yield wild-type RPXV, which demonstrated that they comprised an overlapping set of viral genomic deletions. Ultimately, the extent of the deletions were correlated with the severity of the replication defect on nonpermissive cells through biochemical examination of the mutants (Moyer et al., 1980). Individual RPXV white pock mutants were characterized and a recombination matrix was constructed (Gemmell and Fenner, 1960), which was later amended to include an additional category of virus mutants that failed to replicate on pig kidney cells (Sambrook et al., 1966). The collection of RPXV mutants could be distinguished from each other on the basis of their distinct pock morphology and their inability to produce plaques in vitro in certain indicator mammalian cell lines (McClain, 1965; McClain and Greenland, 1965). On further examination, it was concluded that individual RPXV mutants differed in the stage at which virus replication was blocked in nonpermissive cells (Fenner and Sambrook, 1966). Genomic analysis determined that the RPXV host range mutants contained viral genomic rearrangements, including extensive terminal deletions up to 30 kb (Lake and Cooper, 1980; Moyer and Rothe, 1980). The specific genetic locus responsible for the host range phenotype of the RPXV mutants was later mapped to the SPI-1 gene, which expresses a member of the serine protease inhibitor (serpin) superfamily (Ali et al., 1994). SPI-1 is one of the three related orthopoxvirus serpin genes and is

TABLE II

Orthopoxvirus orthologs of characterized host range proteins VACV WRa SPI-1

VACV COPb

SPI-1/205 C12L/015 353 353 100/100 98/99

RPXV UTRc

VACV MVAd

005/005 357 97/98

No gene

VARV BSHe

CPXV Ger91f

B21R/185 B20R/201 372 375 93/96 95/96

ECTV MOSg

MPXV ZREh

CMLV CMSi

168/169 370 95/97

B19R/180 357 96/97

200R/257 372 94/96

K1L

K1L/032 284 100/100

K1L/036 284 98/99

024/024 284 99/99

No gene

C1L/024 66 95/97

M1L/038 284 97/99

022/022 283 96/98

C1L/027 283 95/98

30L/032 67 97/99

C7L

C7L/021 150 100/100

C7L/024 150 100/100

013/013 150 100/100

018L/006 150 100/100

D11L/011 150 99/100

C13L/027 150 100/100

015/015 150 95/98

D10L/013 150 96/98

19L/021 150 98/99

CHOhr/ Fragment No gene CP77j

No gene

No gene

D8L/008 451 89/94

C9L/023 668 100/100

No gene

D7L/010 658 92/96

No gene

p28/ N1Rk

Fragment No gene

008/008 007R/003 242 88 96/98 74/79

D6R/006 242 95/97

C7R/021 242 97/98

012/012 241 100/100

D5R/008 242 95/97

14R/016 242 93/96

B5R

B5R/187 317 100/100

B5R/232 317 99/99

167/167 317 99/99

173R/151 317 97/99

B6R/170 316 93/97

B4R/185 317 97/99

155 156 317 95/98

B6R/167 317 97/99

178R/229 317 93/97

E3L

E3L/059 190 100/100

E3L/071 190 97/98

048/048 90 99/99

050L/034 190 96/97

E3L/047 192 94/96

F3L/063 190 96/98

043/043 190 91/95

F3L/052 153 88/90

55L/063 190 95/97

K3L

K3L/034 88 100/100

K3L/040 88 99/99

026/026 88 99/99

024L/011 88 98/98

C3L/026 87 80/89

M3L/040 88 98/98

No gene

C3L/029 42 93/93

32L/035 88 85/91

a

Ortholog in Vaccina virus strain Western Reserve. Ortholog in Vaccinia strain Copenhagen. Ortholog in Rabbitpox virus strain Utrecht. d Ortholog in Vaccina virus strain modified vaccina Ankara. e Ortholog in Variola major virus strain Bangladesh 1975. f Ortholog in Cowpox virus strain GRI90. g Ortholog in Ectromelia virus strain Moscow. h Ortholog in Monkeypox virus strain Zaire. i Ortholog in Camelpox virus strain CMS. j CHOhr is fragmented in VACV-WR (WR014, WR016, WR015, WR016, WR017) but has no ortholog in VACV-COP. k p28/N1R is fragmented in VACV-WR (WR011, WR012) but has no ortholog in VACV-COP. Gene name/ORF; protein length (aa); % identity/% similarity. b c

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47% homologous to SPI-2 (also known as crmA, the gene ortholog from cowpox virus), which is the most extensively studied orthopoxvirus serpin. To date, only SPI-1 has been shown to have a specific host range function, but it is formally possible that restrictive host cells also exist for the SPI-2/crmA or SPI-3 genes, so at the time of writing neither SPI-2 nor SPI-3 are considered to be host range genes. Deletion of the SPI-1 gene from RPXV generated white-pox variants and reduced host range in vitro, as measured by the inability of the virus to replicate in pig kidney and normally permissive human lung carcinoma cells (A549) (Ali et al., 1994). No gross defects in protein or DNA synthesis was observed in nonpermissive cells infected with an RPXV mutant disrupted in the SPI-1 gene; however, few infectious mature virions were produced and signs of excessive virus-induced apoptosis were evident (Brooks et al., 1995). During RPXV infection, SPI-1 appeared to inhibit cellular proteinases with chymotrypsin-like activity and was presumed to inhibit a caspase-independent pathway of apoptosis (Brooks et al., 1995). In contrast, a comparable VACV SPI-1 deletion mutant was unable to replicate efficiently in primary human keratinocytes or A549 cells but did not induce an early apoptosis response (Shisler et al., 1999). The exact cellular protein targets of SPI-1 that mediate host range have yet to be identified, but in vitro studies demonstrated that the SPI-1 protein of RPXV functions as an inhibitor of human neutrophil cathepsin G (Moon et al., 1999) and can act synergistically with SPI-2 to block granule-mediated cell killing pathways (Macen et al., 1996). Deletion of the SPI-1 gene from either ectromelia virus (ECTV) (Wallich et al., 2001) or VACV strain Western Reserve (WR) (Kettle et al., 1995) did not effect virus virulence in animal models, suggesting that SPI-1 is not a crucial determinant for poxvirus virulence, at least in these animal model systems when injected by the intranasal route of inoculation in BALB/c mice. In contrast, VACV strain WR constructs deficient of SPI-1 were reported to be severely attenuated in CB6F1 mice, but did not compromise the effectiveness of the immune response when used as a vaccine (Legrand et al., 2004). This discrepancy may be due to differences in the degree of susceptibility of each mouse strain to VACV. Although the role SPI-1 plays in host range determination needs to be better defined, the early studies involving RPXV mutants provided the first evidence that poxvirus host range genes can profoundly regulate host cell tropism and offered a novel collection of molecular tools to study the mechanics that define poxvirus permissiveness at the cellular level.

B. K1L The first VACV host range genes were discovered during the screening of mutagenized viral stocks used to generate temperature sensitive VACV mutants. Individual VACV host range mutants were initially noted when

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they exhibited altered plaque morphology on permissive cell lines and when plated on indicator human cell lines, the mutant virus replication was restricted regardless of cell origin (Drillien et al., 1981). For the first of these VACV mutants described, an 18 kb deletion near the left terminus of the viral genome was identified in the mutant virus and used to map the exact location of the viral gene locus responsible for VACV tropism in human cells. Fragments of DNA from wild-type VACV were inserted into the thymidine kinase (tk) locus of the mutant virus and recombinants were screened for their ability to replicate in human cells (Gillard et al., 1985, 1986). The minimal VACV DNA fragment capable of rescuing host range of the mutant virus was 855 bp long and contained a single open reading frame (ORF) encoding a 32.5-kDa polypeptide (Gillard et al., 1986) named K1L, as was based on the current standard protocol for naming VACV (most commonly, strains WR or Copenhagen) genes (Rosel et al., 1986). Since that time, the K1L gene has also been assigned the nomenclature WR032 in VACV strain WR (see www.poxvirus.org). For this chapter, we will refer to this VACV gene as simply K1L. Deletion of the K1L gene from wild-type VACV strain Copenhagen unexpectedly did not restrict growth of the knockout virus in human cells; however, rabbit fibroblast (RK13) cells were rendered nonpermissive to infection by the K1L knockout VACV virus (Perkus et al., 1990). The K1L protein is expressed as an early-intermediate gene product that appears to directly or indirectly influence early delayed virus gene products, since shutoff of K1L gene product synthesis occurs prior to viral DNA replication (Gillard et al., 1989). On further investigation, a rapid shutdown of both viral and cellular protein synthesis was observed in nonpermissive RK13 cells infected with K1L-deleted VACV strain WR (Ramsey-Ewing and Moss, 1996). Hence, the host range function of K1L appeared to involve either mRNA stability or protein translation in VACV-infected cells. Ectopic expression of the VACV K1L gene in stably transfected RK13 cells rescued the virus replication of modified vaccinia Ankara (MVA), which lacks many genes found in the parental VACV strains used to generate MVA (Sutter et al., 1994). This was the first example of the rescue of a poxvirus genetic mutant by host cells that stably express a complementing viral transgene. Similarly, marker rescue of the partially deleted K1L gene with wild-type K1L sequences released the cellular block restricting MVA replication in RK13 cells, but the recombinant K1L-restored MVA remained nonpermissive in other mammalian cells tested (Meyer et al., 1991). The K1L gene from VACV is fairly well conserved within the orthopoxvirus family (see Table II) with the exception of camelpox virus (CMLV) and variola virus (VARV), in which the putative K1L ORF contains a premature stop codon encoding a nonfunctional gene product (Cowley and Greenaway, 1990; Gubser and Smith, 2002). ECTV also

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encodes a protein which shares 98% amino acid similarity to VACV K1L but the ECTV ortholog failed to support optimal virus replication of MVA in RK13 cells, suggesting that K1L is not as critical for ECTV replication in rabbit cells as it is for VACV (Chen et al., 1993). At the molecular level, K1L has more recently been demonstrated to inhibit the activation of NF-kB by preventing the degradation of IkBa (Shisler and Jin, 2004). For example, infection of HeLa cells with the highly attenuated NYVAC strain of VACV strain Copenhagen (in which the gene for K1L, and many other viral genes, are deleted) stimulated the induction of NF-kB, which could be blocked when the host cells were transfected with a plasmid expressing VACV K1L (Guerra et al., 2006). Additionally, VACV K1L has also been found to interact with a host protein called ACAP2, a GTPase activating protein, via its ankyrin (ANK) repeats (Bradley and Terajima, 2005; Meng and Xiang, 2006). Although K1L was identified as a VACV host range factor many years ago, the exact nature of the host restriction in human cells and the mechanism by which K1L overcomes the host restriction still remain elusive today. However, it is assumed that various orthopoxviruses encode orthologs of the K1L gene family to evade host antiviral responses by specifically targeting innate host factors responsible for cellular self-defense and thereby subvert the host cellular signaling environment to promote virus replication.

C. C7L Earlier studies have demonstrated that the VACV host range gene K1L mapped to a gene locus essential for virus replication in human cells; however, following targeted deletion of K1L in strain Copenhagen, the replicative ability of the knockout virus in human cells remained unchanged (Perkus et al., 1990). This result suggested the possibility of an alternative viral host range gene that mediates VACV permissiveness in cells of human origin. The search for additional VACV host range genes identified C7L, which was shown to be functionally similar to the K1L gene in terms of permitting VACV strain Copenhagen growth on human cells (Perkus et al., 1990). In previous studies, the human cell host range of VACV had been exclusively attributed to the K1L gene but it soon became clear that viral host range genes could be functionally redundant in some cells, but not in others. The C7L gene encodes a 19-kDa protein and can substitute VACV K1L providing VACV strain Copenhagen the ability to replicate in cell lines derived from humans and pigs, but not in rabbit (RK13) (Perkus et al., 1990) or hamster (Dede) cells (Oguiura et al., 1993). Like K1L, the molecular role of C7L still remains unknown but the gene is well conserved in all of the orthopoxvirus genomes that have been sequenced, thus indicating the importance of this gene in poxvirus biology (Gubser et al., 2004).

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Recent evidence suggests that the viral C7L gene may play a critical role in the inhibition of apoptosis triggered in response to viral infection (Najera et al., 2006). In HeLa cells infected with NYVAC, a translational block was observed that corresponded to a significant increase in the phosphorylation of the eukaryotic protein synthesis initiation factor 2a (eIF-2a) late in the course of infection. Both K1L and C7L are deleted from the genome of NYVAC, derived from VACV strain Copenhagen, and when the C7L gene was reinserted into the genome of NYVAC the virus was now able to block the induction of apoptosis and successfully infect HeLa cells (Najera et al., 2006). It is still unclear why VACV would encode two seemingly equivalent genes but it is reasonable to speculate that C7L and K1L perform distinctive functions in some cell types, but not in others.

D. CHOhr In tissue culture, VACV has the ability to replicate in a broad spectrum of cell types from many diverse mammalian hosts, but one notable exception is the Chinese hamster ovary (CHO) cell line. VACV infection of CHO cells results in an abortive infection in which early viral gene expression is terminated very soon after infection, followed by an extremely rapid inhibition of both virus and host protein synthesis (Drillien et al., 1978; Hruby et al., 1980). Cowpox virus (CPXV) contains one of the largest genomes known in the chordopoxvirus family and in all probability encodes more host range factors involved in cell tropism than any other mammalian poxvirus family member. In contrast to VACV, CPXV can productively replicate in CHO cells, so the genetic basis for this difference in host range has been of some interest. A 77-kDa CPXV protein, called either CP77 or the CHOhr protein, encoded by the CPXV025 gene was identified for its ability to permit CPXV replication in CHO cells (Spehner et al., 1988). One functional role of CHOhr is likely to avoid the abrupt and early shutoff of protein synthesis, which is characteristic of VACV infection in CHO cells. Interestingly, the CHOhr gene was demonstrated to provide functionally equivalent host range functions as K1L and C7L by permitting VACV strain Copenhagen gene knockout constructs of K1L or C7L to replicate on human and porcine kidney cells (Perkus et al., 1990). Similarly, the host restriction of a K1L-deleted VACV strain WR could be reversed in RK13 cells when complemented by CP77 (Chung et al., 1997; Ramsey-Ewing and Moss, 1996). The functional equivalence of CHOhr, K1L, and C7L, at least in some cells, is a remarkable phenomenon, given that the primary amino acid sequences of these gene products are not significantly related. Since CHOhr gene supports orthopoxvirus replication in the broadest range of mammalian cell types tested, it would suggest this gene would be the one most conserved among members of the orthopoxviruses.

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On the contrary, the VACV homolog of CHOhr is deleted in strain Copenhagen (Goebel et al., 1990) and relative to the CPXV CHOhr sequence the orthologous pseudogene in the VACV strain WR genome has been inactivated by multiple substitutions and frame shift mutations (Kotwal and Moss, 1988). The genomic evidence suggests that VACV evolved from an ancestral virus that contained a functional counterpart of the intact CP77 gene and its function was lost through either mutation or deletion, possibly during the 200 years of VACV ‘‘domestication’’ since its first isolation in the days of Jenner. The ECTV genome also possesses CHOhr-related DNA sequences; however, like VACV, the ECTV gene is also severely mutated and functionally inactive, indicating that CHOhr is not important for ECTV pathogenesis in the mouse. Interestingly, insertion of the CHO gene from CPXV extended the normally narrow host range of ECTV to include CHO cells in tissue culture (Chen et al., 1992). Expression of the CHOhr gene in VACV prevented the induction of apoptosis and allowed for the expression of late virus genes, such that increased time for viral replication was available prior to cell death (Bair et al., 1996; Ink et al., 1995). However, expression of the antiapoptotic factors Bcl-2 and E1B from adenovirus failed to rescue virus growth alone, suggesting that host restriction of VACV in CHO and RK13 cells is mediated by a pathway distinct from classic Bcl-2-inhibitable apoptosis (Chung et al., 1997; Ink et al., 1995). The CHOhr protein was shown to bind and promote the dissociation of HMG20A from the viral factories, providing the first cellular target regulated by viral host range CHOhr protein (Hsiao et al., 2006). The significance of this novel host protein interaction, as well as the molecular mechanism by which CHOhr functions to expand the host range of VACV to CHO cells, remains poorly understood and merits further study.

E. p28/N1R The p28/N1R RING zinc-finger protein is a 28-kDa cytoplasmic protein that is expressed early during infection and is highly conserved among all orthopoxviruses (see Table II), and orthologs are found within most other members of the chordopoxvirinae (Senkevich et al., 1994). Notable exceptions include a truncated version of p28/N1R present in VACV strain WR, and the p28/N1R ORF is completely absent from VACV strain Copenhagen (Senkevich et al., 1995; Upton et al., 1994). Recently, certain RING-containing proteins have been shown to act as ubiquitin ligases (RING-E3s) to mediate substrate-specific ubiquitination of selected substrate proteins ( Joazeiro and Weissman, 2000). In vitro studies demonstrated the p28/N1R protein from several orthopoxviruses possessed ubiquitin ligase activity (Huang et al., 2004; Nerenberg et al., 2005), and the ECTV version was also capable of inhibiting UV-light induced apoptosis

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(Brick et al., 2000). Both p28/N1R and ubiquitin colocalized to cytoplasmic viral factories to presumably direct the ubiquitination and degradation of substrate host proteins which block viral replication through the induction of apoptosis (Nerenberg et al., 2005; Upton et al., 1994). Disruption of the p28/N1R gene from ECTV completely abolished virus pathogenicity in a murine infection model but expression of p28/ N1R was nonessential for growth of the mutant virus in several tissue culture cell lines tested (Senkevich et al., 1994). In murine macrophages, early viral gene expression was observed when infected by p28/N1R knockout ECTV but the sequential replication of viral DNA was blocked resulting in an abortive infection (Senkevich et al., 1995). The mechanism by which p28/N1R extends the host range of ECTV is unknown, but the experimental evidence suggests that p28/N1R functionally compensates for an unknown cellular factor essential for viral DNA replication in macrophages. The significance of p28/N1R as a host range factor in other poxviruses remains to be investigated.

F. B5R (ps/hr) The B5R ORF of VACV strain WR encodes a 45-kDa type I integral membrane glycoprotein that is synthesized throughout virus infection (Engelstad et al., 1992). A component of extracellular enveloped virus (EEV), the B5R protein is necessary for trans-Golgi/endosomal membrane wrapping of intracellular mature virus (IMV) (Engelstad and Smith, 1993; Isaacs et al., 1992; Martinez-Pomares et al., 1993; Wolffe et al., 1993). B5R was initially characterized in VACV strain Lister as the gene responsible for restoring both plaque size and host range to a temperature sensitive variant of VACV named LC16m8 (Takahashi-Nishimaki et al., 1991). Unlike the parental VACV strain, LC16m8 replication in Vero cells is restricted but the virus proliferates equally well as the parental strain in RK13 cells (Sugimoto et al., 1985). Sequence analysis of LC16m8 identified a single nucleotide deletion introducing an early termination sequence that caused loss of gene function (Takahashi-Nishimaki et al., 1991). Deletion of the B5R gene of VACV strain WR considerably decreased the production of EEV, dramatically reduced plaque size in vitro and was highly attenuated in vivo compared to the parental strain (Wolffe et al., 1993). Arrested morphogenesis of the B5R deficient virus following formation of IMV particles was observed by both confocal and electron microscopy (Rottger et al., 1999; Sanderson et al., 1998). The protein sequence of B5R contains four conserved short consensus repeats (SCR) that are associated with the superfamily of complement control proteins. Several studies have reported mutagenesis of the B5R protein domains, and VACV constructs lacking one or more of the SCRs produced small plaques consistent with a reduction in actin tail formation

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(Herrera et al., 1998; Mathew et al., 1998; Rodger and Smith, 2002). More recently, the SCR4 domain of B5R has been demonstrated to bind and activate Src, which is critical for VACV-induced actin polymerization (Newsome et al., 2004). A related but distinct viral protein, named VACV complement control protein (VCP), is encoded by the C3L gene of the Copenhagen strain (Goebel et al., 1990). In contrast to B5R, VCP does not contain a transmembrane domain but rather an N-terminal signal sequence and the protein is secreted from VACV-infected cells (Kotwal and Moss, 1988; Kotwal et al., 1990). Among members of the orthopoxvirdae, the B5R protein is highly conserved and orthologs are present in other poxvirus family members as well. In the absence of a functional B5R gene, RPXV produced distinctive white pocks and failed to produce plaques on several cell lines including Vero, chicken embryo fibroblast (CEF), PK-15, and quail (QT-6) all of which are normally permissive for the parental RPXV (Martinez-Pomares et al., 1993). The RPXV B5R mutant virus was severely attenuated in both mice and rabbit but no enhanced host inflammatory response was observed in the infected animals (Stern et al., 1997).

G. E3L VACV has a relatively broad host range in cultured mammalian cells, except for CHO cells, and exhibits relative resistance to the antiviral effects of interferon (IFN) from many species. This anti-IFN evasion mechanism by VACV is mediated by two of the most well studied poxvirus-encoded host range genes, E3L and K3L. The 25-kDa gene product of E3L contains a highly conserved double stranded RNA (dsRNA) binding domain located at the C-terminus and an N-terminal domain with sequence similarity to Z-DNA binding motifs (Langland et al., 2006). Over the past 15 years, numerous studies have clearly demonstrated E3L as a host range gene, necessary for efficient VACV replication in many cell lines. E3L is also required for VACV pathogenesis, at least in mice, likely by involving multiple mechanisms. The E3L protein exhibits many properties that are undoubtedly linked to pathogenesis and tropism, such as IFN-blocking activities, Z-DNA binding, nuclear localization and higher order oligomerization, but the exact roles these activities play remains to be better defined. By binding and sequestering dsRNA in the cytoplasm, E3L can inhibit stimulation of dsRNA-dependent protein kinase (PKR) and activation of 20 –50 oligoadenylate synthase, two enzymes which are activated by dsRNA and induced by IFN (Chang and Jacobs, 1993; Chang et al., 1992; Rivas et al., 1998). PKR bound to dsRNA undergoes an autophosphorylation event and is able to phosphorylate eIF-2 on the small a subunit, initiating an inhibition of protein synthesis within the infected cell

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(Williams, 1999). A 20 –50 synthetase on the contrary, activates a latent endoribonuclease (RNase L), which then targets and cleaves cellular rRNA and mRNA, halting all protein synthesis within the cell (Iordanov et al., 2000). Thus, the activation of both host proteins by dsRNA negatively affects viral replication by cleaving cellular and viral RNAs and thus causing general inhibition of protein synthesis. E3L has also been shown to inhibit induction of IFN-a/b by blocking phosphorylation of the transcription factor IFN regulatory factor 3 (IRF-3) and IRF-7, both key inducers of IFN gene expression (Smith et al., 2001; Xiang et al., 2002). E3L has been reported to localize to the nucleus but the role of E3L in nuclear events is not clear. The N-terminus of E3L has been shown to mediate binding to the left-handed Z-form of double stranded DNA and is necessary for full inhibition of PKR activation (Kim et al., 2003, 2004; Langland and Jacobs, 2004). However, the N-terminal domain of E3L is not required for IFN resistance or host range of VACV in cell culture (Shors et al., 1997), but is critical for full pathogenesis in mice (Brandt and Jacobs, 2001). VACV strain WR expressing an N-terminal deletion of 83 amino acids of E3L (VACV-E3LD83N) is attenuated in a mouse model by at least 1000-fold compared to wild-type VACV, when administered via the intranasal route (Brandt and Jacobs, 2001). Later it was demonstrated that VACV with N-terminal deletions in E3L replicates efficiently in the nasal mucosa but fails to spread from the nasal cavity to the lungs or the brain (Brandt et al., 2005). These E3L-compromised virus constructs also exhibit decreased virulence even when injected directly into the brain, likely due to poor replication in neuronal tissue, and protected against challenge with wild-type VACV (Brandt et al., 2005). When other unrelated Z-DNA binding domains, such as from RNA editing enzyme ADAR1 (double-stranded RNA adenosine deaminase) or the tumor-related DLM1, were substituted for the similar E3L domain, the knock-in viruses retained the virulence properties of the parental VACV strain WR (Kim et al., 2003). Mutations in the chimeric E3L protein, or parental E3L protein, that disrupt Z-DNA-binding property of E3L also decrease viral pathogenesis, suggesting a direct correlation between this domain and virulence (Kim et al., 2003). Further studies have shown that the role of E3L in VACV pathogenesis involves modulating expression of host cellular genes at the transcriptional level and inhibiting apoptosis in HeLa cells in a fashion that requires the N-terminal domain of E3L (Kwon and Rich, 2005). VACV E3L is defined as a host range gene because, while the E3L deleted virus can replicate in CEF, baby hamster kidney (BHK) or RK13 cells, it cannot replicate in Vero or HeLa cells (Beattie et al., 1995b; Langland and Jacobs, 2002). Deletion of E3L in VACV strain WR resulted in the inhibition of viral and cellular protein synthesis and reduced viral replication which can be restored by transient transfection of E3L (Chang et al., 1995).

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However, the C-terminal domain of E3L that is associated with IFN resistance is important for this rescue. The N-terminal domain of E3L is dispensable for VACV infection of cells in culture, but both the domains are required for full pathogenesis in mice (Brandt and Jacobs, 2001). VACV lacking a functional E3L or a replaceable dsRNA binding protein also induces apoptosis in HeLa cells (Kibler et al., 1997). The induction of apoptosis directly correlates with the amount of dsRNA synthesized in the infected cells. Recently, it was shown that E3L deleted VACV was unable to replicate productively (because of an abort at the level of late gene expression) in a murine epidermis-derived dendritic cell line XS52 (Deng et al., 2006). In this cell line, infection with VACV lacking E3L resulted in activation of NF-kB and production of proinflammatory cytokines like TNF and IL-6. The role of E3L in suppression of proinflammatory signal transduction and gene expression has also been studied by a genome wide screening approach using VACV mutants lacking specifically the N-terminal or C-terminal domain of E3L (Langland et al., 2006). E3L has also been studied in terms of defining the mechanism of evasion of host defenses. Expression of E3L in cell lines interferes with several cellular pathways, resulting in promotion of cellular growth, resistance to apoptosis, and impairment of antiviral activity (Garcia et al., 2002). Mouse NIH3T3 cells expressing E3L undergo inhibition of eIF-2a phosphorylation and IkBa degradation in response to dsRNA (Garcia et al., 2002). E3L expression also confers resistance to dsRNA-triggered apoptosis. E3L expressing cells grow faster than control cells and showed increased expression of cyclin A and decreased levels of p27kip1. The cells also formed solid tumors when injected in nude mice (Garcia et al., 2002). The E3L protein has also been demonstrated to display a host range function in other poxviruses. Mutant MVA with an E3L deletion (MVA-D E3L) was unable to replicate in CEF cells, mainly because of inhibition of viral protein synthesis at late times (Hornemann et al., 2003). In this cell line, E3L is required for inhibition of apoptosis and/or IFN induction. In HeLa cells, E3L is also required for full MVA life cycle. MVA-DE3L virus was defective in late protein synthesis, viral late transcription, and viral DNA replication in infected HeLa cells (Ludwig et al., 2005). The absence of E3L resulted in activation of 20 –50 oligoadenylate synthetase/RNase L and significant downregulation of more than 50% of cellular transcripts expressed under normal conditions. However, in MVA-DE3L infected HeLa cells, a cluster of cellular genes including transcription factors were also upregulated, suggesting the possibility for additional cellular targets of E3L function (Ludwig et al., 2005). E3L can complement the function of other diverse dsRNA binding proteins to different extents. The ortholog of E3L from Orf virus, the prototypic parapoxvirus, could complement the deletion of E3L in

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VACV in cell culture but was 1000-fold less pathogenic than wild-type VACV following intranasal infection of mice (Vijaysri et al., 2003). The same E3L replacement virus was about 100,000-fold less pathogenic than wild-type VACV on intracranial injection. Protein synthesis and replication of VACV lacking E3L can be complemented by expression of genes encoding other dsRNA-binding proteins in trans or by insertion of genes from other viruses that also inhibit antiviral responses, such as those that encode the reovirus S4 and hepatitis C virus NS5A proteins and cytomegalovirus (Beattie et al., 1995a; Child et al., 2002; He et al., 2001). VACV E3L was able to rescue vesicular stomatitis virus (VSV) but not encephalomyocarditis virus (EMCV) from the IFN-induced antiviral state. VACV K3L, on the contrary, rescued EMCV but not VSV from the effects of IFN (Shors et al., 1998).

H. K3L Like E3L, the K3L protein is also expressed early in VACV infection. K3L has limited sequence similarity to the N-terminal region of eIF2a. Subsequently, it was shown that K3L protein inhibits phosphorylation of eIF2a by PKR both in vivo and in vitro (Davies et al., 1992, 1993), and a direct interaction between the K3L protein and C-terminal half of the PKR kinase domain was demonstrated (Carroll et al., 1993; Craig et al., 1996; Jagus and Gray, 1994). K3L protein functions as a pseudosubstrate inhibitor of PKR, as mutations in residues conserved between the K3L protein and eIF2a are required for inhibition of PKR by K3L (KawagishiKobayashi et al., 1997). PKR recognizes K3L and eIF2a by a common mechanism and in this way K3L protein inhibits autophosphorylation of both PKR and eIF2a substrate phosphorylation (Carroll et al., 1993). Both K3L and eIF2a bind to PKR at a similar or overlapping region in the catalytic domain of the enzyme. By binding to PKR, and inhibiting its enzymatic activation, K3L blocks the inhibition of protein synthesis. VACV strain Copenhagen that is deleted of K3L (VACVDK3L) has much broader host range than the comparable knockout of the E3L gene but was found to be IFN sensitive in mouse L929 cells (Beattie et al., 1995b). In contrast to E3L, the K3L gene is required for replication in BHK cells, but is dispensable for replication in HeLa cells. It has been suggested that replication of VACVDK3L in BHK cells is aborted because E3L alone cannot sequester all the dsRNA generated during VACV infection (Langland and Jacobs, 2002). These results clearly demonstrate the functional importance of both E3L and K3L in manipulating host antiviral networks to maintain an intracellular environment supportive of virus replication.

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III. MYXOMA VIRUS HOST RANGE GENES In contrast to VACV, which has a relatively broad host range, the leporipoxvirus, myxoma virus (MYXV) is a rabbit specific pathogen, which has proven to be a useful model system for the identification and characterization of poxvirus host range genes, and evaluating their roles in disease pathogenesis. MYXV coevolved with rabbits (Sylvilagus sp.) of the Americas and causes only benign lesions restricted to the peripheral site of inoculation, which slowly resolve over time (Fenner, 2000). However, in the European rabbit MYXV causes a rapid systemic and highly lethal infection called myxomatosis (Fenner, 1983). The pathogen–host relationship between MYXV and the European rabbit has been well characterized and provides an excellent model for studying poxvirus tropism and its relationship with viral virulence (Kerr and Best, 1998; Krogstad et al., 2005). Like all poxviruses, the MYXV genome includes a distinct repertoire of host range genes, whose protein products target specific intracellular pathways to establish an environment within the infected cells that favors viral replication (Barrett et al., 2001; Cameron et al., 1999; Zuniga, 2003). Several of the host range gene products of MYXV have orthologs in other poxviruses. However, MYXV is the only poxvirus for which there has been a systematic study of the comparative biological roles of viral host range gene products both in vitro and in vivo (Stanford et al., 2007). A collection of MYXV host range genes have been identified (see Table I) and examined to determine the mechanism(s) by which they mediate MYXV tropism and regulate virus virulence. In European rabbits, MYXV infection and pathogenesis is heavily dependent on the ability of the virus to disseminate from the primary site of infection and establish secondary sites of viral infection in distal tissues. Spread of MYXV is achieved by means of infected migratory leukocytes, which function as viral transporters and disseminate virus infection to the distal tissues (Best et al., 2000; Garon et al., 1978). The ability of MYXV to infect lymphocytes in particular is a critical determinant to the success of MYXV and essential to the progression of myxomatosis in susceptible European rabbits. To date, a number of MYXV-encoded proteins have been identified as tissue-specific host range factors, which function to promote MYXV replication and block the induction of antiviral responses, particularly apoptosis, in rabbit lymphocytes. Many viruses, including poxviruses, encode modulatory proteins that function to block various components of the apoptotic pathway (Taylor and Barry, 2006). These antiapoptotic properties of host range factors have been shown to manipulate cell death pathways within the infected cell in a variety of ways, including the inhibition of caspases and the disruption of key mitochondrial checkpoints to prevent apoptosis (Everett and McFadden, 2001a,b, 2002).

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The mechanism by which individual MYXV host-range genes block apoptosis in virus-infected lymphocytes will be discussed in greater detail, but it should be noted that other host cell antiviral pathways are likely to be targeted by these viral proteins as well. However, since so much more is known about regulatory controls of the cellular apoptotic cascades, it is this host pathway that has received the bulk of attention so far.

A. M-T2 Identified as the first MYXV encoded viroreceptor (i.e. viral mimic of a cellular receptor), the M-T2 gene shares sequence similarity with the N-terminal ligand-binding domain of cellular tumor necrosis factor (TNF) receptors (Upton et al., 1991). M-T2 protein is secreted as monomeric and dimeric species from MYXV-infected cells and was shown to specifically bind and neutralize the cytolytic activity of rabbit TNF with high molar affinity (Schreiber and McFadden, 1994; Schreiber et al., 1996). European rabbits infected with M-T2 knockout MYXV (vMyxT2KO) exhibited a markedly attenuated disease in contrast to rabbits infected with the parental virus strain expressing M-T2 (Upton et al., 1991). The majority of rabbits were successful at mounting an effective immune response to vMyxT2KO infection and completely recovered, demonstrating the importance of M-T2 as a virulence factor for myxomatosis (Upton et al., 1991). Although vMyxT2KO replicates normally in RK13 cells, the mutant virus was unable to productively infect rabbit T-lymphocytes (RL-5) in tissue culture (Macen et al., 1996). Following infection of rabbit T-lymphocytes with vMyxT2KO, a rapid and extensive cellular apoptosis response was triggered, suggesting that M-T2 is able to short-circuit the induction of apoptosis in these cells, otherwise resulting in an abortive infection. The addition of exogenous M-T2 protein to RL-5 cells infected with vMyxT2KO failed to rescue these cells from virus-induced apoptosis (Macen et al., 1996). This observation suggested that an intracellular version of M-T2 may function to block the induction of TNF-mediated apoptosis. Very recently, it has been shown that M-T2 forms dominant negative complexes with cellular TNFR via an oligomerization domain (called preligand assembly domain or PLAD) that is distinct from the TNF-binding site of the receptor (Sedger et al., 2006). This inhibition of TNFR signaling by M-T2, unlike the inhibition of the TNF ligand (which is rabbit specific), was not species specific and was fully operational in human cells (Sedger et al., 2006). Thus, M-T2 exhibits two distinct functions; secreted M-T2 protein binds and inhibits rabbit TNF, whereas the intracellular form of M-T2 binds cellular TNFR and blocks the induction of apoptosis in virus infected lymphocytes. The mechanisms by which M-T2 coordinates

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these dual activities remains to be better defined; however, the ability of M-T2 to expand MYXV host range to include lymphocytes is critical to the progression of myxomatosis.

B. M-T4 The MYXV encoded M-T4 gene product is an endoplasmic reticulum (ER)-retained protein with no known cellular homolog (Barry et al., 1997). M-T4 posses a C-terminal endoplasmic reticulum retention signal (RDEL) motif which can function as an ER retention signal, however removal of the motif only reduced protein stability and did not affect localization of the protein within the ER (Hnatiuk et al., 1999). In the absence of the M-T4 ORF, MYXV (vMyxT4KO) was unable to replicate efficiently in cultured rabbit T-lymphocytes resulting in the induction of apoptosis that aborted the virus replication in these cells. Pathogenesis of vMyxT4KO was severely attenuated in susceptible rabbits, demonstrating M-T4 as a critical host range factor in the development of myxomatosis. Most notably the number of secondary lesions on infected animals were significantly reduced suggesting that the virus was unable to disseminate to distant tissues (Barry et al., 1997). At the molecular level the ability of M-T4 to mediate MYXV tropism in lymphocytes and protect these cells against apoptosis from within the ER is poorly understood. It has been hypothesized that M-T4 may function to disrupt the apoptotic pathway through direct interaction with Bap31, an ER-retained integral membrane protein that is a potent inducer of apoptosis (Zuniga, 2003). Despite the absence of a direct mechanism by which M-T4 blocks apoptosis in RL-5 cells, the ability of M-T4 to extend MYXV tropism is essential to the success of MYXV as a rabbit pathogen.

C. M-T5 M-T5 possesses no extensive sequence similarity to any nonviral proteins but is related to certain other poxvirus host range genes that contain ANK-repeat sequences (Mercer et al., 2005). The protein encoded by the M-T5 ORF of MYXV is expressed rapidly following infection and remains as an abundant and stable 49-kDa cell-associated protein throughout the course of viral infection ( Johnston et al., 2005b; Mossman et al., 1996). Pathogenesis studies of rabbits infected with M-T5 deleted MYXV (vMyxT5KO) demonstrated that M-T5 is an essential virulence factor in the establishment of myxomatosis. All rabbits infected with vMyxT5KO were successful at mounting a rapid and effective inflammatory response, sufficient to clear viral infection (Mossman et al., 1996). The absence of secondary lesions in the infected animals suggested that vMyxT5KO failed to progress beyond the primary site of inoculation and disseminate to distal tissues (Mossman et al., 1996).

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In cell culture, vMyxT5KO replicated with kinetics indistinguishable from cells infected with the parental virus in RK13 cells. However, RL-5 cells infected with vMyxT5KO resulted in an abortive infection characterized by rapid inhibition of both viral and host gene synthesis accompanied by extensive cellular apoptosis (Mossman et al., 1996). Thus, it would appear that M-T5 specifically promotes MYXV replication in T-lymphocytes by preventing the nonspecific shutdown of protein synthesis, which is probably the stimulus leading to the induction of apoptosis and abortive viral infection. The observations suggest that M-T5 functions as a host range protein that acts prior to the induction of apoptosis and permits virus spread via infected T-lymphocytes that traffic to multiple distal sites (Mossman et al., 1996). Structurally, M-T5 is predicted to contain seven ANK-repeat domains and a highly conserved F-box motif located at their carboxyl-terminus of the protein (Johnston et al., 2005b). The F-box motif mediates protein– protein interactions and was initially described as a recognition subunit of the E3 ubiquitin ligase complex, known as skp1, Cullin, F-box containing or SCF (Feldman et al., 1997; Skowyra et al., 1997). M-T5 forms a complex with cullin-1, a component of the cellular SCF complex, and enhances the ubiquitination and subsequent degradation of p27/Kip1 via the proteasomal pathway ( Johnston et al., 2005b). p27/Kip1 is an inhibitory regulator of the cell cycle and in the absence of M-T5, cells infected with MYXV enter cell cycle arrest and accumulate at G0/G1. In contrast, for cells infected with wild-type MYXV, the M-T5 gene product was shown to promote cell cycle progression beyond the G0/G1 ( Johnston et al., 2005b). Progression of cells through the cell cycle is a tightly regulated process and viral infection often leads to cell cycle arrest and the induction of apoptosis. Therefore, the ability of M-T5 to strategically manipulate the host cell cycle is a key factor to promote productive viral replication in T-lymphocytes as well as at least some human cancer cells (see below). Although MYXV is a rabbit specific virus, it was recently shown that a spectrum of human cancer cells were able to support the productive replication of MYXV (Sypula et al., 2004). In the absence of M-T5, the mutant MYXV was nonpermissive in a particular subset of human cancer cells, which supported replication of wild-type MYXV (Sypula et al., 2004; Wang et al., 2006). To date, M-T5 has been the only MYXV encoded protein that influences MYXV tropism in human cancer cells to be characterized in terms of mechanism of action, but there is evidence that some of the other MYXV host range factors can also play a tropism role in certain other human cancer cells (Barrett et al., 2007a). The susceptibility of human cancer cells to be infected by MYXV is directly linked to the level of endogenous phosphorylated Akt, or its ability to be activated by MYXV infection (Wang et al., 2006). The protein Akt is a serine/threonine protein kinase and a critical regulator of many

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diverse signaling pathways which include cell survival, growth, proliferation, angiogenesis, and migration (Franke et al., 1997; Hajduch et al., 2001; Vivanco and Sawyers, 2002). At the molecular level, M-T5 binds to Akt forming a stimulatory complex, which promotes the increased phosphorylation of Akt (Wang et al., 2006). Consequently, M-T5 expands the host range of MYXV by inducing Akt phosphorylation in human cancer cells that normally express levels of phosphorylated Akt too low to support MYXV replication. The ability of M-T5 to target specific cellular proteins such as Akt and the cullin-1/SCF complex, and thus micromanipulate the signaling environment of the infected host cell, is critical to the oncolytic properties of MYXV in human cancer cell lines (Wang et al., 2006). In addition to M-T5, MYXV encodes three additional ANK-repeat containing proteins, termed M148, M149, and M150, all of which are expressed from single copy genes and share some sequence similarity to each other (Cameron et al., 1999). On the basis of the presence of multiple ANK-repeats, these genes are all likely candidate host range genes but to date only M-T5 has been directly tested and shown to possess host range properties. It is believed that each of these ANK-repeat proteins performs a unique biological function because in the absence of M-T5 the remaining ANK-repeat genes were unable to rescue virus replication in rabbit lymphocytes, or type II human cancer cells, infected with vMyxT5KO (Mossman et al., 1996). The functional interrelationship shared among the ANK-repeat proteins of MYXV remains to be examined.

D. M11L Another MYXV encoded antiapoptotic protein, M11L, is targeted to the mitochondria by a short carboxyl transmembrane motif, where it inserts into the cytoplasmic surface of the outer mitochondrial membrane (Everett et al., 2000). Once localized at the mitochondria, M11L binds to the proapoptotic peripheral benzodiazepine receptor (PBR) and forms an inhibitory complex, which functions to block apoptosis induced by ligands of PBR (Everett et al., 2002). M11L maintains mitochondrial membrane potential by inhibiting the release of cytochrome c and blocking apoptotic signals at the mitochondrial checkpoints (Everett et al., 2002). Despite lacking any significant primary sequence similarity to host proteins, M11L adopts a Bcl-2 protein fold (Douglas et al., 2007; Kvansakul et al., 2007) and has been shown to block apoptosis by sequestering the proapoptotic proteins Bak (Wang et al., 2004) and Bax (Su et al., 2006) both of which are proapoptotic Bcl-2 family members that operate at the mitochondria. Virus replication was severely attenuated in rabbits infected with MYXV deficient in the M11L gene (vMyxM11LKO), demonstrating the importance of M11L as a virulence factor necessary for pathogenesis in

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the rabbit host. Despite the reduction in virulence, lesions produced by vMyxM11LKO were larger and expressed increased proinflammatory characteristics when analyzed by histology (Opgenorth et al., 1992). In cell culture, the ability of vMyxM11LKO to replicate in rabbit T-lymphocytes and monocytes was impaired and the knockout virus was unable to inhibit the apoptotic response of these cells during virus infection (Opgenorth et al., 1992). This would suggest that M11L functions as a host range factor by preventing apoptosis of leukocytes during host infection, thus compromising the effectiveness of cellular protective mechanisms designed to limit viral propagation.

E. M13L The M13L gene product from MYXV is an early expressed viral protein that shares some sequence similarity with the p200 family of IFN-inducible proteins (Cameron et al., 1999). On further evaluation, M13L was shown to contain an N-terminal pyrin domain (PYD) and was assigned to be a virus-encoded member of the PYD superfamily of proteins ( Johnston et al., 2005a). PYD-containing proteins have been identified in a variety of other poxviruses, including Yaba-like disease virus and the swinepox virus. The PYD is a well conserved sequence motif identified in more than 20 human proteins (Reed et al., 2003), and was characterized as a putative protein–protein interaction domain thought to function in the regulation of apoptotic and inflammatory processes (Bertin and DiStefano, 2000; Gumucio et al., 2002). Induction of inflammation is tightly regulated by inflammasomes, a series of related multiprotein complexes (size >700 kDa) responsible for the activation of the caspases 1 and 5, thereby leading to the activation of the proinflammatory cytokines IL-1b, IL-18, and IL-33 (Martinon et al., 2002). Little is known about the natural stimuli that lead to the assembly and activation of the inflammasomes, but their regulation is assumed to be critical for both the induction, and later downregulation of antimicrobial inflammatory responses. M13L was shown to interact with the ASC-1 component of the inflammasome through a PYD–PYD association disrupting the ability of ASC-1 to form the bridge between caspases and various NALPs (Johnston et al., 2005a). This complex inhibits the activation of procaspase-1 and subsequent secretion of IL-1b and IL-18 in MYXV infected cells. Deletion of the M13L gene from MYXV (vMyxM13LKO) was associated with decreased ability to productively infect rabbit T-lymphocytes but viral replication in RK13 cells was comparable to wild-type MYXV ( Johnston et al., 2005a). Moreover, the M13L gene is an essential virus virulence factor, and viral pathogenesis was severely attenuated in rabbits infected with vMyxM13LKO ( Johnston et al., 2005a). The absence of secondary

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lesions in European rabbits infected with vMyxM13LKO suggest virus dissemination by infected peripheral leukocytes was impeded and provide possible explanation why vMyxM13LKO does not cause myxomatosis in susceptible rabbits. Therefore, the ability of M13L to extend MYXV tropism to rabbit T-lymphocytes and other migratory leukocytes demonstrates its role as a critical host range factor and as an essential poxvirus immunomodulatory protein.

F. M063 Following initial analysis of the MYXV genome, no obvious K1L or CHOhr homologs were recognized, which was particularly interesting since these orthopoxvirus host range factors were partly characterized in rabbit cells, and rabbits are the natural host for MYXV. However, three tandemly arranged ORFs, designated M062R, M063R, and M064R were identified in MYXV, which share some sequence similarity with the VACV host range gene C7L (Cameron et al., 1999). Pathogenesis studies of rabbits infected with MYXV deleted of the M063R gene (vMyx63KO) failed to develop any symptoms of myxomatosis and all rabbit cell lines tested were completely nonpermissive to the mutant virus (Barrett et al., 2007b). vMyx63KO successfully binds and enters nonpermissive rabbit cells but late stage viral gene expression and DNA replication was blocked in these cells (Barrett et al., 2007b). Conversely, vMyx63KO can still productively infect both human cancer cells and certain primate cell lines (such as BGMK cells), suggesting that these latter cells are either deficient in some aspect of antiviral responses operational in rabbit cells or that M063R interacts with rabbit-specific host factors that have yet not been identified (Barrett et al., 2007b). Rabbit fibroblast that stably express VACV K1L were not successful at rescuing vMyx63KO replication, demonstrating that these genes are not interchangeable and likely interacts with different cellular targets (Barrett et al., 2007b). Although M062R and M064R ORFs have not yet been examined, it is evident that they are not functionally equivalent to M063R since vMyx63KO failed to replicate in any rabbit cells tested so far. It is interesting to note the sequence similarity between M063R and C7L, but C7L is not required for VACV replication in rabbit cells, suggesting that M063R possesses unique rabbit host range function. It remains unknown why MYXV would encode three related apparent orthologs of C7L, but one possibility is that each gene product interacts with a distinct cellular factor to promote virus replication (Barrett et al., 2007b). Among all of the other reported deletion mutants of MYXV, vMyx63KO is the only gene knockout virus reported to date that fails to replicate in all rabbit cells tested in vitro and also cannot induce any detectable primary lesion formation in infected rabbits. The molecular role of M063R is still unknown but the protein exhibits sequence similarity to the glutamate-rich

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domain of human DAXX, a FAS-binding death associated protein (Chang et al., 1998, 1999). It is possible that M063R possess similar antiapoptotic functions as described by other MYXV-encoded host range proteins, but no clear link with apoptosis has yet been demonstrated (Barrett et al., 2007b). Although M063R is not a functional equivalent of C7L, it is obvious the gene provides a critical host range function for MYXV in rabbit cells and most likely intersects with distinct intracellular signal cascade(s). Therefore, understanding the function by which the M063 family extends MYXV tropism could provide valuable insight into the narrow host range of the virus.

IV. MOLLUSCUM CONTAGIOSUM: AN EXTREME EXAMPLE OF HOST RANGE RESTRICTION Both MCV and VARV have the narrowest tropism of any poxvirus and use humans as exclusive natural hosts (Frey and Belshe, 2004; Lewis-Jones, 2004). MCV replication is strictly restricted to basal keratinocytes of the human epidermis, causing a benign tumor-like lesion in children and young adults but is far more prevalent in immunodeficient individuals (Gottlieb and Myskowski, 1994). Among the MCV genes identified in the completed DNA sequence, several potential immunomodulatory and host range proteins that control host defenses were discovered (Senkevich et al., 1996). In contrast to VARV and other orthopoxviruses, the genome of MCV is much smaller (190 kbp) and is missing more than 80 genes common to most orthopoxviruses (Senkevich et al., 1996). Many of these missing genes have been identified as having a functional role in the suppression of host response to orthopoxvirus infection (Senkevich et al., 1996, 1997). It has been hypothesized that over evolutionary time the genetic material that provides broader tissue tropism for the orthopoxviruses has been lost from MCV (Senkevich et al., 1997). For example, MCV lacks a homolog to both the E3L and K3L proteins of VACV suggesting that either the virus has evolved unique strategies to manipulate PKR and other antiviral response against viral infection, or these pathways are not operational in differentiating basal keratinocytes. The strict cellular tropism of MCV provides an ideal model for the potential discovery of novel host defense mechanisms targeted by host range genes that enable replication in human epithelia.

V. CONCLUSIONS Productive virus infection in permissive cells probably only occurs following successful manipulation of signaling networks that regulate cellular antiviral processes. Host range genes are probably present in all

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viruses, and play a significant role in modulating the intracellular environment by targeting specific host proteins to establish optimal cellular conditions for viral replication (McFadden and Murphy, 2000). Poxviruses have the specific advantage that their genomes are large and complex, such that the identification and study of viral host range factors can be studied apart from essential viral replication factors. Functionally, such host range genes from poxviruses have been associated with a diverse array of cellular processes, which includes the manipulation of cellular kinases and phosphatases and the inhibition of apoptosis and antiviral pathways like those mediated by IFN. Most of the known host range proteins from poxviruses are biochemically and structurally diverse and no single host-range gene that is common to all poxviruses has been identified to date (Johnston and McFadden, 2003). Relatively little sequence similarity is shared among the family of poxvirus host range genes, however the ANK-repeat motif is found in many of them (Mercer et al., 2005) (Fig. 1). One of the most widespread motifs found in nature, the ANK-repeat is a 33 amino acid domain that is generally found as a tandem array of 2–7 repeats (Lux et al., 1990). Structurally, the ANK-repeat consist of two alpha-helices

ANK ANK

ANK

ANK

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MYXV M-T5 (483 aa)

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CPXV CHOhr (668 aa)

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VACV K1L (284 aa)

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MYXV M150 (494 aa)

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MYXV M149 (490 aa)

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MYXV M148 (675 aa)

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FIGURE 1 Characterized poxvirus host range, Ankyrin repeat-containing proteins. Predicted Ankyrin repeats and putative C-terminal F-box domains are represented by the boxes (ANK) and (FBOX), respectively (Letunic et al., 2006; Schultz et al., 1998). Numbers indicate the amino acid length of the various host range proteins from the individual poxviruses.

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separated by a beta-hairpin and has been described as having an L-shaped organization (Mosavi et al., 2002a,b). ANK-repeats appear in a diverse array of bacterial, archaeal, and eukaryotic proteins; however, they are not commonly identified in viruses, with poxviruses being the notable exception (Bork, 1993). ANK-repeats are known to mediate protein–protein interactions and cellular proteins containing the motif have a broad range of functions including: transcriptional regulators, cytoskeletal organizers, cell cycle regulatory proteins, and developmental regulators (Mosavi et al., 2004; Sedgwick and Smerdon, 1999). The functional diversity among these ANK-repeat proteins suggests that the motif is far more important structurally, as scaffolding modules, rather than as an enzymatic role (Sedgwick and Smerdon, 1999). Presumably, the capacity of viral ANK-repeat host range factors to operate as molecular scaffolds generates novel protein–protein interactions that might otherwise not occur due to spatial and temporal restrictions. The outcome of these interactions could potentially result in novel cross-communication between distinct host signaling pathways that are normally independent of one another. Modifications of the host cellular signaling networks, induced by poxvirus-encoded host range genes, would thus be critical in establishing an environment within the host crucial for successful virus replication. Presently, the ANK-repeat has been the only identified motif shared by a majority of poxvirus host range genes, however, it is likely that additional protein motifs, critical for protein–protein interactions between viral and host partners, will be recognized in the future. Currently, the mechanism by which poxviral encoded host range proteins interact with cellular proteins to establish an environment that favors virus replication is poorly understood. Poxviruses remain a potential treasure chest to discover novel pathogen–host interactions, still waiting to be identified. The potential knowledge acquired from such interactions could provide invaluable insight into the elaborate mechanism by which viral encoded proteins micromanipulate the signal transduction pathways of the infected cells to promote an intracellular environment essential for virus replication. Understanding the function of host range genes would further develop poxviruses as potential oncolytic candidates, as selectively replicating vaccine platforms, and for other diverse biotherapeutic applications.

ACKNOWLEDGMENTS G.M. is an International Scholar of the Howard Hughes Medical Institute. We thank R. Condit and N. Moussatche for their helpful comments.

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4 Receptor Interactions, Tropism, and Mechanisms Involved in Morbillivirus-Induced Immunomodulation Ju¨rgen Schneider-Schaulies and Sibylle Schneider-Schaulies

Contents

I. Introduction A. General aspects of MV- and morbillivirus-induced immunosuppression B. Relationships between tropism of the virus, spread of infection, and immunosuppression II. Leukopenia Associated with Morbillivirus Infections III. Mechanisms and Consequences of T Cell Silencing in Morbillivirus Infections IV. Receptors and Signaling Involved in Suppression of Cell Functions V. Virus Interactions with DCS A. Virus interference with DC functions in animal models B. Experimental models and consequences of DC surface interactions with viral proteins C. Consequences of infection on DC viability and function VI. Conclusions and Perspectives References

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Institute for Virology and Immunobiology, University of Wu¨rzburg, D-97078 Wu¨rzburg, Germany Advances in Virus Research, Volume 71 ISSN 0065-3527, DOI: 10.1016/S0065-3527(08)00004-3

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Abstract

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Induction of immunomodulation and -suppression is a common feature of morbilliviruses such as measles virus (MV), rinderpest virus (RPV), and canine distemper virus (CDV) in their respective hosts. As major uptake receptor, signaling lymphocytic activation molecule (SLAM, CD150) essentially determines their tropism for immune cells, which is of considerable importance with regard to immunosuppression and the systemic spread to organs including secondary lymphoid organs, the skin, the respiratory tract, and the brain. Independent of their ability to enhance virus uptake in specialized host cells, other cell surface receptors such as the substance P receptor, DC-SIGN, Toll-like receptors (TLR), Fcgamma receptor II (FcgRII), CD46, and additional uncharacterized receptors exert a variety of immunomodulatory effects as reflected by activation of or interference with viability, differentiation, trafficking, or acquisition of effector functions of specialized immune cells. In this review, we discuss receptor interactions, tropism, and mechanisms involved in the severe, transient immunosuppression induced by MV and other morbilliviruses.

I. INTRODUCTION Morbilliviruses are members of the paramyxoviridae which have a negative-stranded RNA genome encoding for six structural and two nonstructural proteins (Fig. 1). Functionally, morbilliviral structural proteins can be grouped into those essential for replication of the genome [the nucleocapsid (N) protein and the polymerase complex consisting of the large polymerase protein (L) and its cofactor, the phosphoprotein (P)] forming the ribonucleoprotein particle (RNP) complex, and those associated with the viral lipid bilayer membrane [the fusion (F) and hemagglutinin (H) glycoproteins and the basic matrix (M) protein] forming the envelope. Viral entry relies on the interaction of the H protein with receptor molecules on the host cell surface (see below) followed by a pH-independent conformational change of the F protein, which inserts its fusogenic domain into the target cell membrane thereby initiating the membrane fusion process required for uptake of the viral core complex into the host cell cytoplasm. When expressed at the cell membrane of infected cells, the glycoprotein complex also causes fusion with adjacent uninfected cells (provided these express H-protein receptors) thereby giving rise to typical syncytia in tissue culture and in vivo. Apparently, the fusion activity of the F/H glycoprotein complex is negatively regulated by the M protein, which physically and functionally interacts with the cytoplasmic domains of F and H (Cathomen et al., 1998a,b). In addition to controlling fusogenicity, the M protein is also a driving force in

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FIGURE 1 The morbillivirus particle contains the ribonucleoprotein particle (RNP) complex consisting of the genomic RNA, the nucleocapsid protein N, the phosphoprotein P, and the polymerase L, and the envelope consisting of the matrix protein M, the fusion protein F, the hemagglutinin H, and a host cell-derived membrane. The single stranded genome of negative orientation of
16,000 nucleotides contains six genes coding for N, P (C, V), M, F, H, and L proteins, flanked by a 3,0 leader and a 5,0 trailor sequence (which contain the promotors for transcription and replication, and an encapsidation site). The transcription units are linearly arranged and separated by conserved intergenic regions typically composed of a gene stop polyadenylation site, and a trinucleotide followed by a gene start sequence. The transcription units are monocistronic except for the second, which gives rise to P and two additional proteins, V and C, by RNA editing or usage of an alternative translation start codon, respectively.

promoting viral budding, but also acts as a negative regulator of morbilliviral transcription by as yet unknown mechanisms (Pohl et al., 2007; Reuter et al., 2006). In contrast to the strictly human pathogen, their type species measles virus (MV), the other morbilliviruses, rinderpest virus (RPV), canine and phocine distemper viruses (CDV and PDV), peste des petits ruminants virus (PPRV), and the cetacean dolphin and porpoise morbilliviruses (DMV and PMV) are animal viruses, which, in common with MV, are highly contagious and cause systemic infections which can result in similar devastating diseases in their respective hosts (Barrett, 1999; Barrett and Rossiter, 1999; Katz, 1995). Following entry via the respiratory tract, they commonly cause fever, cough, and conjunctivitis, but also a severe transient immunosuppression favoring acquisition and aggravation of secondary infections which may follow a lethal course. In humans, immunosuppression induced during the acute infection is actually the

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major cause of MV-associated infant mortality worldwide. Despite implementation of the efficient vaccine, more than 30 million cases of acute measles are still reported annually with
345,000 fatalities (WHO, 2007), the majority of which develops in Third World countries as a consequence of immunosuppression (Katz, 1995). Especially virulent strains of RPV, infecting large ruminants and also pigs, reach death rates of up to 100% in cattle and wildlife such as buffalos (Syncerus caffer). In contrast to MV and RPV, CDV has a broader host range infecting many carnivores including dogs, lions, seals, and ferrets. CDV strains substantially differ in virulence. Because of the considerable worldwide socioeconomical impact of their infections, the WHO thus proclaimed and started eradication programs against MV and RPV. Most of the present knowledge about morbillivirus-induced immunosuppression has been achieved from patients infected with measles or experimental approaches in tissue culture and animal models using MV. Therefore, MV will take the biggest part of this review serving as a model virus for the other morbilliviruses.

A. General aspects of MV- and morbillivirus-induced immunosuppression Measles is a well-defined clinical entity normally contracted by children and young adults. While a long lasting virus-specific immunity is efficiently induced in the course of acute measles and after vaccination, there is a generalized transient suppression of immune responses to other infections lasting for several weeks. Characteristically, a marked leukopenia and a loss of delayed-type hypersensitivity (DTH) reactions are observed. Lymphopenia affects both the B and the T cell compartment (Okada et al., 2000). It was long before its isolation and molecular characterization that MV was recognized as the first immunosuppressive pathogen. The term ‘‘’anergy’’ was coined by von Pirquet in 1908 (von Pirquet, 1908) to describe the loss of DTH reactions to tuberculin in MV-infected individuals. In addition to the marked lymphopenia, proliferative responses of lymphocytes to polyclonal and antigen-specific stimulation ex vivo are highly impaired (Borrow and Oldstone, 1995; Griffin, 1995; Schneider-Schaulies et al., 2001). This can be documented for several weeks after acute measles, and also, albeit to a moderate extent, after vaccination (Hussey et al., 1996). Infiltration of mononuclear cells into local areas of virus replication and the appearance of antiviral antibodies and virus-specific T cells in the blood mark the onset of virus-specific immune responses. Activation of virus-specific T cells is reflected by soluble CD4, CD8, IL-2R, and b2 microglobulin in serum, and a Th1 cytokine profile which switches to a Th2 type as indicated by a rise in IL-4 plasma levels (reviewed in Griffin, 1995).

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Natural MV infections may also occur in primates such as macaques, in which they are accompanied by immunosuppression, secondary infections, and reactivation of persistent viral infections (Choi et al., 1999). The diseases caused by other morbilliviruses in their specific hosts are also associated with immunosuppression (Barrett, 1999; Barrett and Rossiter, 1999). The clinical course observed after experimental infection of ferrets with a virulent CDV strain strongly resembles that seen with MV in humans: After intranasal infection with 104 TCID50, the ferrets developed a rash beginning at day 6, which was accompanied by a severe leukopenia. Signs of disease included severe dehydration caused by diarrhea, sometimes accompanied by pneumonia and conjunctivitis. One week after infection, the DTH response to tetanus toxoid was completely suppressed, the proliferative capacity of T cells in response to phytohemagglutinin (PHA) was strongly reduced, and all animals had to be euthanized between 12 and 16 days post infection (von Messling et al., 2003). When gene functions of the envelope genes (M, F, and H) and the RNP complex genes (N, P, and L) of a recombinant virulent and an attenuated strain were compared using recombinant viruses for experimental infection, virulence-relevant mutations were found distributed throughout the genome suggesting that both the activity of the core replication complex and viral functions determining attachment, cell targeting, and spread, contribute to pathogenicity (von Messling et al., 2003).

B. Relationships between tropism of the virus, spread of infection, and immunosuppression Initial infection with morbilliviruses and subsequent spread are mainly determined by the presence of specific host cell receptors. Though MV infection doubtlessly occurs via the respiratory tract, primary target cells are, however, not yet clearly identified. Epithelial cells are susceptible to infection with certain MV strains in vitro (see below), yet do not express the receptor for MV wild-type strains, CD150, which is restricted to the hematopoetic system (see below). Possibly, MV is rather acquired by tissue resident macrophages or dendritic cells (DCs) within the epithelium and transported to local lymphatic tissues (Ingrid Allen, personal communication; Fig. 2). In fact, MV-infected MHC class II positive cells with DC morphology have recently been detected in peripheral mucosal tissues and isolated from skin explants of macaques experimentally infected with a eGFP-tagged recombinant wild-type MV (de Swart et al., 2007). This study impressively confirmed the pronounced tropism of MV for the lymphatic system including lymph nodes, where the virus is believed to infect B cells, T cells, and monocytes, which subsequently mediate systemic spread by a cell-associated viremia. Thus, MV can be

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FIGURE 2 Main route of virus spread. Infection starts most likely by infection of dendritic cells (DCs) or macrophages in the respiratory epithelium followed by virus transport in infected cells to draining lymph nodes. There, other cells of the immune system, predominantly B and T cells are infected, followed by a cell-associated viremia distributing the infection to other organs. Virus reaches the respiratory tract again, where it then may infect also epithelial cells from the basolateral side, and is released from the host.

reisolated from human peripheral blood lymphocytes (which is greatly enhanced by mitogen stimulation) and MV-specific RNA and proteins are detectable in a small proportion of peripheral lymphocytes and monocytes during and for few days after the rash (Esolen et al., 1993; Forthal et al., 1992; Schneider-Schaulies et al., 1991). Though figures vary depending on the method used for detection, the overall percentage of infected peripheral blood mononuclear cells does not exceed 2% at any stage of infection, and similar frequencies were recently determined in experimentally infected macaques (de Swart et al., 2007). In CDV-infected ferrets, a high percentage of lymphocytes and monocytes was found to be infected in the thymus and secondary lymphoid organs as revealed by histology. Interestingly, in contrast to MV, CDV-infection leads to a high percentage of infection of peripheral blood lymphocytes (up to 40% of T cells and 60% of B cells) already at day 7 post infection (von Messling et al., 2004). The first MV receptor identified was CD46 (membrane cofactor protein, MCP), a member of the complement regulatory proteins which is ubiquitously expressed on human nucleated cells (Do¨rig et al., 1993; Naniche et al., 1993) (Table I). High affinity binding to CD46 is, however, confined to

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attenuated virus strains and isolates adapted to growth on Vero cells. As a consequence of surface interaction and infection with the H protein, CD46 is downregulated from the cell surface and this, in agreement with the natural function of this molecule, has been associated with an enhanced sensitivity to complement-mediated lysis (Schneider-Schaulies et al., 1995b; Schnorr et al., 1995). In contrast to this special property of attenuated MV strains, all MV strains (including lymphotropic wild-type viruses and attenuated viruses) use CD150 (signaling lymphocyte activation molecule, SLAM), a member of the CD2 subset of the immunoglobulin (Ig) superfamily, as entry receptor (Erlenhoefer et al., 2001; Hsu et al., 2001; Ono et al., 2001b; Tatsuo et al., 2000, 2001; Yanagi et al., 2006). The basis for the restriction of particularly wild-type MV H proteins to functional interaction with this molecule has recently been directly documented by resolution of the H-protein structure (Hashiguchi et al., 2007). Corroborating their similarity in cell tropism and pathogenicity, other morbilliviruses such as CDV and RPV also use the species-specific orthologues of CD150 as major uptake receptors (Baron, 2005; Tatsuo and Yanagi, 2002). CD150 is expressed on activated B cells, activated and memory T cells including activated regulatory T cells (Browning et al., 2004), and immature thymocytes (Aversa et al., 1997). In line with this expression pattern, CD150þ B cells represented the prime target cell population in human tonsillar tissue material infected with MV wild-type strains in vitro (Condack et al., 2007). Within the T cell compartment, MV infection clearly segregated with CD45ROþ memory cells that also expressed CD150. Interestingly, these, though less frequently infected than B cells, appeared to be preferentially depleted from the infected tissue. Though its basis is unclear as yet, preferential infection-mediated depletion of T cells in secondary lymphoid tissues might well contribute to peripheral T cell

TABLE I

Receptors interacting with MV, their functions, and expression patterns

Receptor

Function

Expression on

CD150 CD46 DC-SIGN TLR2 FcgRII Unknown

Virus entry memory cells Virus entry (vaccine virus) Virus binding Signaling (wild-type virus) N-protein binding, signaling Virus uptake

Unknown

Proliferation inhibition

Activated T, B, Mo, DCs Ubiquitous DC DC, Mo DC, Mo, B Epithelial, endothelial, neural cells Lymphoid cells

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lymphopenia (see below). The preferential tropism of wild-type MV for B cells (both in lymphoid tissues and in peripheral blood) was also confirmed in eGFP-MV-infected macaques (de Swart et al., 2007), where expression of the marker transgene clearly segregated with that of CD150 on the individual cell compartments analyzed. If at all and to what extent monocytes (tissue resident or peripheral) are infected is unclear. Though believed to serve as MV carriers in humans (Esolen et al., 1993), peripheral blood monocytes were found essentially uninfected in macaques. This is in agreement with the absence of detectable levels of CD150 on these cells, as also seen for freshly isolated human primary monocytes and monocytic cell lines (Minagawa et al., 2001). CD150 expression is, however, inducible on monocytes and on maturing DCs, particularly in response to inflammatory signals (Kruse et al., 2001; Minagawa et al., 2001). In addition to supporting entry of morbilliviruses into hematopoetic cells, interaction with this molecule may also contribute to immunomodulation independently of infection. As described for CD46 after interaction with attenuated MV, CD150 is downregulated from the cell surface by wild-type MV contact or infection, with functional consequences being unknown as yet (Erlenhoefer et al., 2001; Welstead et al., 2004). As revealed by ligation with specific antibodies, CD150 can favor CD95-mediated apoptosis in some B and T cell lines (Mikhalp et al., 1999), but also costimulate T cells by promoting enhanced IFN-g production and thereby a Th1 response (Cocks et al., 1995; Engel et al., 2003; Sidorenko and Clark, 2003). Strikingly, studies using T cells from CD150-deficient mice fail to support a critical role of this molecule in IFN-g production, but rather indicate that CD150 enhances TCR-stimulated IL-4 release. This study also provided evidence that CD150 may modulate Toll-like receptor (TLR) 4 but not TLR2 or TLR9 signaling in macrophages. Thus, LPS-stimulated production of IL-12, TNFa, and NO were diminished and that of IL-6 was enhanced in the absence of CD150 (Wang et al., 2004). In DCs, consequences of CD150 ligation either by antibodies or MV have not yet been addressed. The tropism of MV during natural infection predominantly, but not completely, segregates with its usage of CD150 as virus entry receptor. Given the importance of CD150 as a receptor for infection with presumably all morbilliviruses, it is unclear how these viruses access CD150 negative cells in vivo such as epithelial cells, endothelial cells, and in brain infections neurons, oligodendrocytes, and astrocytes. Moreover, in vitro infection of primary endothelial cells (Andres et al., 2003), small airway epithelial cells (Takeuchi et al., 2003), and a lung carcinoma epithelial cell line (Takeda et al., 2007) by wild-type MV clearly occurred independently of CD150. With the help of the latter cell line (H358), which is highly susceptible to wild-type MV forming large syncytia in the absence of CD150 and independently of CD46, a novel receptor-binding site on the viral H protein could be identified (Takeda et al., 2007).

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In addition to these known and unknown uptake receptors, a variety of cell surface receptors have been identified that interact with MV, but do not act as uptake receptors. They may support receptor-mediated virus uptake, such as the cytoskeletal protein moesin and DC-SIGN (CD209), or fusion, such as the substance P receptor (neurokinin-1), or may induce intracellular signaling, such as TLR2, and the Fc-gamma receptor II (FcgRII; see below).

II. LEUKOPENIA ASSOCIATED WITH MORBILLIVIRUS INFECTIONS Measles is associated with a pronounced leukopenia affecting B cells, monocytes, neutrophils, as well as CD4þ and CD8þ T cells, the extent of which seems to be related to the age-dependent severity of the disease (Okada et al., 2000, 2001). In contrast to B cell frequencies, which can still be below control levels for up to six weeks, numbers of T cells return to normal after 10 days and the CD4/CD8 ratio remains constant over time (Arneborn and Biberfeld, 1983; Okada et al., 2001; Ryon et al., 2002). Mechanisms accounting for B cell depletion have barely been studied as yet, do, however, certainly include infection-mediated cell death as these cells are preferentially targeted, and apoptosis induced after CD150 ligation may also contribute (Mikhalp et al., 1999) (see above). Frequencies of peripheral T cells could be affected by MV interference with thymocyte generation and viability. MV can target the development of CD34þ human hematopoetic stem cells to thymocyte precursors in vitro by infection and impairing the function of stroma cells. This relied on stromal cell infection, which occurred most likely independently of CD150 (Manchester et al., 2002). The interaction of MV with thymocytes, which express CD150 and can thus support MV entry and possibly replication, has been addressed in various experimental settings. Indeed, expression of MV proteins was detected in thymocytes (and CD4þ and CD8þ T cells) of CD150-transgenic mice after in vivo or in vitro infection (Hahm et al., 2003). This study did not address thymocyte apoptosis as a result of MV exposure, which was, however, massively seen in human thymus/liver xenografts in SCID mice, in which, interestingly, thymocytes themselves remained uninfected (Auwaerter et al., 1996; Valsamakis et al., 1999). It is thus likely that thymocyte viability and/or differentiation into functional T cells is affected secondary to infection of supporting cell types such as thymic epithelial cells, which release unidentified viral components (Valentin et al., 1999). Alternatively, thymocyte loss could result from CD150 ligation by the viral H protein, similarly as shown in studies using specific antibodies (Sidorenko and Clark, 2003). Interestingly, prolonged passage of avirulent MV vaccine strains in human

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thymic tissue enhanced their ability to cause thymocyte apoptosis, and this correlated with an amino acid exchange within the H-protein ectodomain (Valsamakis et al., 1999). Strikingly, however, ex vivo analyses in human measles patients rather suggested that thymic output was even increased as determined by T cell receptor excision circle (TREC) frequencies indicating that depletion of T cell precursors may not contribute to T cell lymphopenia (Permar et al., 2003). Rather, destruction of T cells by viral infection directly or indirectly, for instance by the induction of enhanced sensitivity to apoptosis, appears more likely. Indeed, evidence for the latter phenomenon was provided after CD3 ligation of T cells of patients ex vivo (Addae et al., 1995, 1998; Okada et al., 2000). T cell infection may cause cell loss in secondary lymphatics (see below). However, the frequency of infected cells in the periphery is low during measles at any stage of the acute disease, so that at least for peripheral T cell infection-mediated loss most likely does not substantially contribute to T cell lymphopenia (Borrow and Oldstone, 1995; Griffin, 1995). This may be different for RPV or CDV, where infected peripheral T cells were detected at high frequency in cattle or ferrets, respectively (Heaney et al., 2002; von Messling et al., 2004). As an alternative mechanism for loss of peripheral T cells, disappearance of highly activated LFA-1þ T cells possibly by disruption of recirculation and random homing has also been proposed (Nanan et al., 1999). T cell depletion could well occur in secondary lymphoid tissues by acquisition of MV or apoptotic signals from professional antigenpresenting cells (APCs). In support of this hypothesis, fusion or apoptosis was found to be induced in T cell cocultures with MV-infected DCs (Fugier-Vivier et al., 1997; Grosjean et al., 1997; Servet-Delprat et al., 2000a; Vidalain et al., 2000, 2001), and massive apoptosis in vivo is clearly seen in lymphoid tissues of RPV-infected cattle (Stolte et al., 2002). How transmission from DCs to T cells occurs and if so, whether it involves cell fusion, is unknown and questionable, since unstimulated T cells do not express CD150 (Aversa et al., 1997). MV infection of DCs may, however, not even be required for transmission, because the MV envelope glycoprotein complex was recently found to target DC-SIGN on DCs, and thus the mechanism of MV transmission to T cells could be similar to that of HIV (Geijtenbeek and van Kooyk, 2003; van Kooyk and Geijtenbeek, 2003). There, interaction with DC-SIGN results in endocytic internalization and concentration of at least a fraction of the HIV particles, which from there can be transported to the interface with a conjugating target cell forming there, what is called an infectious synapse (a process referred to as trans-infection). Successful transmission to the target cell again relies on the presence of attachment receptors, that are typically actively concentrated at the interface, which requires reorganization of the actin cytoskeleton, but not antigen recognition (Piguet and Steinman, 2007).

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As to what extent cell fusion resulting in giant cell formation in secondary lymphatics contributes to T cell loss in vivo is as yet unclear. Giant cell formation in hyperplasic lymphoid tissue was documented in a measles patient at autopsy; however, MV could not be detected in this area (Nozawa et al., 1994), and both giant cell formation and necrosis were seen in lymphoid tissues of MV-infected Rhesus macaques to a moderate extent (Kobune et al., 1996; McChesney et al., 1989; Van Binnendijk et al., 1995). In summary, in acute morbillivirus infections, various mechanisms may account for T cell depletion, which can be considered as an extreme of ‘‘silencing’’ this population. The contribution of T cell loss to the generalized immunosuppression remains, however, unclear, since the duration of the latter clearly exceeds that of lymphopenia.

III. MECHANISMS AND CONSEQUENCES OF T CELL SILENCING IN MORBILLIVIRUS INFECTIONS The ability of MV to cause cell cycle arrest in infected cells including T cells is well known. In addition, infection-dependent alterations of differentiating effector functions, but not those already established, have been intensively studied (Borrow and Oldstone, 1995; Casali et al., 1984). However, the vast majority of T cells isolated from patients or experimentally infected animals is uninfected, yet in spite of this resists mitogenic activation signals in terms of proliferation. Therefore, mechanisms other than direct infection must be involved, which have to be provided in vivo by infected cells. As farreaching effects often relate to the production of soluble inhibitory factors, these have been looked for in measles as well, however, with moderate success. Ex vivo analyses do not support deficiencies in IL-2 production or a lack of IL-2R expression (Griffin and Ward, 1993; Moss et al., 2002). Elevated levels of IL-10 were not consistently seen (Moss et al., 2002; Okada et al., 2001) and it is still unclear whether type I IFNs are produced at significant levels in vivo upon MV infection. In human peripheral blood lymphocytes (PBL) cultures, attenuated but not wild-type MV strains were potent interferon inducers (Naniche et al., 2000). More recently, the ability of attenuated strains to induce production of type I IFN also in DCs directly correlated with production of subgenomic defective interfering RNAs, which, for unknown reasons, were absent from wild-type MV strains tested in this study (Shingai et al., 2007). In contrast, wild-type MV strains caused induction of type I IFN in CD150 transgenic murine cells including DCs (Shingai et al., 2005), and this was linked to developmental alterations of DCs in CD150 transgenic mice (Hahm et al., 2004). The ability of wild-type MV strains to induce IFN-a/b in myeloid DCs is thus apparently species specific, and therefore, the role of these cytokines in immunomodulation by infected DCs in vivo remains unresolved as yet.

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Beyond any doubt, the ability of morbilliviruses to modulate the type I IFN response by blocking IFN receptor signaling is of considerable pathogenic importance. As shown for related viruses, the MV nonstructural V and/or C proteins have this activity (Ohno et al., 2004; Palosaari et al., 2003; Shaffer et al., 2003). In addition, the MV P protein also contributes, and tyrosine 110 is required to block STAT1 phosphorylation and nuclear translocation of STAT proteins in response to type I interferon exposure (Devaux et al., 2007). Similarly, RPV P and V proteins block IFN action by sequestering STAT1, while RPV C protein only weakly blocks IFNinduced STAT1 activation as revealed by in vitro transfection experiments (Nanda and Baron, 2006). In agreement with these findings, studies with recombinant RPV identified the V protein as dominant inhibitor of IFN signaling in the context of infection, since IFN sensitivity of infected cells was restored upon ablation of V-protein expression. Although STAT2 was obviously redistributed in virus-infected cells, STAT2 was not bound by any viral protein (Nanda and Baron, 2006). Studies on morbilliviral evasion from IFN action were, however, mainly performed in nonhematopoetic cells and thus, the relative contribution to immunosuppression in general and to inhibition of lymphocyte expansion or shaping of their effector functions such as cytokine release needs to be established. Production of other soluble proliferation inhibiting factor(s) from MVinfected B- or T cell cultures has been proposed, and though these were never identified. Not surprisingly, they clearly did not involve IL-10 or type I IFN as revealed by inclusion of neutralizing antibodies (Fujinami et al., 1998; Sun et al., 1998). In contrast, polyclonal MV-specific antibodies restored the ability of MV-infected T cell cultures to proliferate in response to mitogenic stimulation (Sanchez-Lanier et al., 1988; Yanagi et al., 1992) indicating that viral proteins were directly involved in inhibition. Evidence for inhibitory activity of the MV glycoprotein complex consisting of the hemagglutinin (H) and the fusion protein heterodimer (F1/2) came from a number of in vitro and in vivo experiments. In vitro, the MV glycoproteins (expressed on the surface of infected cells or on virions), but not those derived from a recombinant virus expressing the vesicular stomatitis virus (VSV) G protein instead of MV-F/H (Fig. 3), prevented expansion of T cell lines and that of mitogen- or CD3/CD28-driven primary human and rodent T cells in a dose- and contact-dependent, yet infection-independent manner (Schlender et al., 1996). The inhibitory activity of the viral glycoprotein complex was confirmed in experiments where coculture of T cells with cell lines transfected to coexpress F and H proteins, but not those expressing F or H protein alone, caused T cell arrest in vitro (Avota et al., 2001; Schlender et al., 1996; Schnorr et al., 1997a; Weidmann et al., 2000a). Importantly, transfer of these doubly transgenic cells into cotton rats significantly impaired the ability of T cells from these animals to expand in response to mitogenic stimulation ex vivo (Niewiesk et al., 1997, 1999). The activity of the

Morbillivirus-Mediated Immunomodulation

MLR

A

PHA-stimulation

B

30

185

40,000

MGV

20,000 + MV

15

LPS

MGV

Mock

Proliferation (cpm)

Stimulation index

LPS

+ MV 0 10,000 1000 100 DC number

0

5 1 2 3 4 DC-T cell coculture

C N

N

P

P

M

M

F

VSV-G

H

L

L

MV

MGV

FIGURE 3 Typical findings of morbillivirus envelope protein-mediated suppression of T cell proliferation as exemplified for measles virus (MV). Both, the mixed lymphocyte reaction (MLR, allogen, T cells, and DCs from different donors) (A), and PHA-stimulated T cell proliferation in DC-T cell cocultures (B), are suppressed in the presence of MV-infected DCs. There is no difference between vaccine and wild-type MV (open circles and closed squares in A, or lanes 3 and 4 in B, respectively) in their capacity to suppress these reactions. When a recombinant virus (MGV) (C) expressing the vesicular stomatitis virus (VSV) G protein instead of the morbilliviral envelope proteins F and H is used, the MLR and proliferation of T cells is not suppressed (A, closed triangles; B, lane 5).

viral F/H glycoprotein complex to serve as effector structure for T cell inhibition is obviously conserved within morbilliviruses since it applied as well to that of RPV, CDV, and PPRV (Heaney et al., 2002). Proteolytic processing, but not complex glycosylation of the F protein, is required for contact-mediated T cell arrest (Weidmann et al., 2000a, b), and restoration of the inhibitory activity of the F0/H complex by exogenous cleavage suggests that the actual effector domain resides within the F protein and is conformational. The H protein may strengthen the interaction of the complex with the target cell surface by binding to its receptor (s) or act as a chaperone to stabilize the conformation of the F1/2 protein. Interestingly, the F protein of the respiratory syncytial virus (RSV) can also inhibit T cell proliferation in vitro (Schlender et al., 2002). T cell inhibition induced by the glycoprotein complex does not involve

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induction of apoptosis or interfere with stimulated upregulation of surface markers (including the IL-2R a-chain), or cytokine release, but rather prevented S-phase entry of these cells (Avota et al., 2001; Engelking et al., 1999; Niewiesk et al., 1999; Schneider-Schaulies and ter Meulen, 2002; Schnorr et al., 1997a). It is likely that contact with the MV–glycoprotein complex, particularly if expressed on the surface of infected DCs, may essentially contribute to aberrant shaping of ensuing T cell responses. Coligation of CD3 and CD46 by antibodies was found to induce regulatory T cells in vitro (Kemper et al., 2003, 2005), and a similar scenario could be envisaged with DCs expressing both MHC-II and MV H protein though of CD46 interacting MV strains. Incomplete or aberrant DC maturation and production of type I IFN and IL-10 rather than IL-12 (reviewed in SchneiderSchaulies et al., 2003) has been described to occur in infected cultures and could further promote an environment favoring differentiation and/or expansion of regulatory T cells. As yet, these cells have not been demonstrated to be specifically enhanced in measles or other morbillivirus infections. It would, however, be interesting to analyze them given their established capability to control establishment and maintenance of persistent infections, but also immunopathology particularly in autoimmune conditions (Sakaguchi et al., 2006). Both disease patterns are induced by morbilliviral infections: persistent CNS infections such as the subacute sclerosing panencephalitis (SSPE) and old dog encephalitis (ODE), and acute postinfectious encephalites are generally believed to represent a virus-induced autoimmune disease (Johnson, 1987).

IV. RECEPTORS AND SIGNALING INVOLVED IN SUPPRESSION OF CELL FUNCTIONS Interestingly, transmission of the MV glycoprotein-induced inhibitory signal does not rely on CD46 or CD150, since (1) murine T cells, which do not express functional uptake receptors, are also effectively arrested, (2) CD46- or CD150-specific antibodies do not prevent silencing of human T cells (Erlenhoefer et al., 2001; Schlender et al., 1996), and (3) both receptors can modulate CD3 signals upon coligation in vitro, act, however, costimulatory rather than inhibitory (Astier et al., 2000; Sidorenko and Clark, 2003; Zaffran et al., 2001). Within the T cell compartment, CD150 expression is confined to activated cells, and this, together with the costimulatory properties of the molecule, strongly argues against a direct role in T cell silencing. Moreover, it certainly does not interact with morbilliviral F proteins, which are likely to harbor the inhibitory effector domain (as discussed above).

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Various effects of CD46 ligation by its natural ligands (complement components C3b/C4b), antibodies and, importantly, the MV H protein on antigen presenting or T cells have been described in molecular and functional terms. These include enhanced vulnerability to complementmediated lysis (due to receptor downregulation) (Schneider-Schaulies et al., 1996; Schnorr et al., 1995), modulation of inflammatory cytokine production (Marie et al., 2001), or potential interference with TLR signaling and thereby inhibition of IL-12 production (Karp et al., 1996). Since, however, firm interaction with CD46 is restricted to attenuated MV strains (Erlenhoefer et al., 2002; Hashiguchi et al., 2007; Hsu et al., 2001; Ono et al., 2001a), the relevance of these findings for immunosuppression in vivo remains to be determined. Other cell surface molecules interacting with morbilliviruses (Fig. 4) or RSV include TLR2 and TLR4 (Bieback et al., 2002; Hahm et al., 2007; KurtJones et al., 2000), DC-SIGN (de Witte et al., 2006), and the substance P receptor (neurokinin) (Harrowe et al., 1992; Makhortova et al., 2007). These are, however, either not expressed on human T cells (such as the TLRs and DC-SIGN), or not further analyzed in terms of their ligands and potential signaling. Moesin has also been shown to enhance MV uptake (Dunster et al., 1994; Schneider-Schaulies et al., 1995a) although it is not supposed to act as direct MV-binding partner. It may have a role in reorganization of the actin cytoskeleton once MV has interacted with the cell surface, and this could be of importance not only for viral uptake, but also for

FIGURE 4

Receptors interacting with measles virus (MV).

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clustering of signaling receptors or T cell plasticity (Mu¨ller et al., 2006). At present, these data support the view that an unknown receptor system mediates signals induced by the F/H-complex at the cell surface of lymphoid cells (Table I). In agreement with a largely unaffected release of cytokines, MV contact does not affect the IL-2R-dependent activation of the JAK/STAT pathway in T cells. In contrast, MV exposure efficiently prevents activation of the phosphatidyl-inositol-3-kinase (PI3K)/Akt kinase pathway after IL-2R or CD3/CD28 ligation in vivo and in vitro (Fig. 5)

FIGURE 5 Activities of the inhibitory ‘‘Receptor X’’ triggered in T cells by interaction with the viral F/H glycoprotein complex expressed by antigen-presenting cells (APCs). Contact with the glycoprotein complex does not cause T cell death or prevent early activation, but rather causes G1 arrest (Akt-kinase-dependent, CDKlow, p27Kiphigh) in response to TCR activation, prevents late activation after 24 h (ERK phosphorylation), and regulates splicing (PI3-kinase-dependent). The subunits p85 and p110 form the PI3-kinase, which activates Akt-kinase via phosphatidyl-inositol-3,4,5-phosphate (PIP3). Phosphorylation of proteins is indicated by small circles. The T cell receptor (TCR), costimulatory molecules such as CD28, and receptor X are located in detergent-resistant microdomains (DRM) also called lipid rafts.

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(Avota et al., 2001, 2004). So far, interference with activation of this particular pathway is unique to morbilliviruses, while, given its important role in conveying survival and mitogenic or possibly oncogenic signals, other viruses rather support activation of the PI3K/Akt kinase pathway (Cacciotti et al., 2005; Dawson et al., 2003; Yu and Alwine, 2002; Yuan et al., 2002). As subunits of cyclin-dependent kinases essentially involved in S-phase entry are known as downstream effectors of this pathway, it is not surprising that these were found deregulated in MV-exposed or -infected T cell cultures (Engelking et al., 1999; Naniche et al., 1999). The importance of interruption of PI3K/Akt kinase activation for MV-induced T cell silencing was directly documented as transgenic expression of a catalytically membrane targeted active Akt kinase largely abolished the inhibitory signal (Avota et al., 2001). The regulatory subunit of the PI3K, p85, which acts upstream Akt kinase activation, was tyrosinephosphorylated shortly after TCR ligation in MV exposed T cells, yet failed to redistribute to cholesterol-rich membrane microdomains (also referred to as lipid rafts) and this correlated with a lack of TCR-stimulated degradation of Cbl-b protein (Fig. 5) (Avota et al., 2004). Importantly, MV can bind and cluster lipid rafts on the T cell surface indicating that signaling by the F/H protein complex is likely initiated by interaction of MV with molecules within these membrane domains. Further lending support to the signaling activity of the glycoprotein complex in T cells, microvillar protrusions on their surfaces almost entirely collapse shortly after MV exposure. Most likely as a consequence of its ability to block PI3K activation, MV signaling interferes with TCR ligation-dependent activation of the guanosine exchange factor Vav and its downstream substrates, the small GTPases Rac and Cdc42. Thereby, cytoskeletal rearrangement in response to TCR ligation and formation of filopodia and lamellopodia is severely impaired (Mu¨ller et al., 2006), and this may have important consequences with regard to the ability of these cells to form stable conjugates with professional APCs as required for their activation (see below). Splice regulatory factors, the activity of which can also be subject to regulation by PI3K, were recently also found to be modulated upon MV contact in T cells. In these cells, a splice isoform of the SHIP-1 phosphatase, SIP110, was found to be induced by MV and to accumulate (Avota et al., 2006). Interestingly, this constitutively membrane-associated lipid phosphatase isoform was found to deplete the cellular pool of phosphatidyl-inositol-3,4,5-phosphates (PIP3) thereby raising the threshold levels required for TCR-dependent activation of these cells. In line with this observation, overexpression of SIP110 strongly impaired expansion of primary T cells driven by CD3/CD28 ligation or phorbolester stimulation (Avota et al., 2006).

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V. VIRUS INTERACTIONS WITH DCS A. Virus interference with DC functions in animal models Because of their unique role in initiating and shaping immune responses, DCs are regarded as central to the understanding of both, induction of a virus-specific immune response, and key aspects of the virus-induced immunosuppression. Association of MV with follicular DCs has been documented in macaques (McChesney et al., 1997), yet it is unknown whether these cells can be considered as long-term repositories for MV. Infection of macaques with a recombinant wild-type MV expressing enhanced green fluorescent protein (eGFP) combined a tool allowing detection with unprecedented sensitivity and the best suitable animal model (de Swart et al., 2007). At the peak of viremia, eGFP fluorescence was detected in skin, the respiratory and digestive tract, but highest percentages of infected cells (up to 30%) were found in secondary lymphoid organs. In peripheral tissues, large numbers of MV-infected myeloid DCs were detected in conjunction with infected T cells, suggesting transmission of MV between these cell types (de Swart et al., 2007). Another animal model allowing for MV infection via the respiratory tract involves cotton rats (Sigmodon hispidus). In these animals, the H-protein dominated tropism of the virus directly reflects virulence of wild-type virus in humans by enabling the spread of recombinant viruses expressing wild-type H proteins to draining lymph nodes, and immunosuppression as evidenced by impaired proliferation of lymphocytes ex vivo (Niewiesk et al., 1997; Pfeuffer et al., 2003; Wyde et al., 1992). Analysis of DC involvement in this model has, however, not yet been possible. In CD46 transgenic mice, splenic DCs were found to be strongly activated and infected by attenuated vaccine MV (strain Edmonston) only after depletion of monocyte/macrophages (Mrkic et al., 1998, 2000). Since type I IFN has been identified as an important restriction factor for MV replication in mice, these particular transgenic mice were also deficient for the type I IFN receptor and thus, the relevance of these findings remains to be established. In immunocompetent mice transgenic for CD150 (expression of which was driven by the CD11c promotor), MV antigens were detected in a very limited number of splenic CD11cþ DCs after intravenous MV wildtype infection (Hahm et al., 2004). Because of the restricted expression pattern of the transgene, DCs and a limited amount of macrophages were the only cells the virus could access, and thus potential infection of other cell types (expressing CD150 in humans) could not be evaluated. Interaction of MV with CD150 on these cells reduces their capacity to synthesize IL-12 in response to TLR4 agonists (Hahm et al., 2007). Thus, the vast majority of findings related to the role of DCs in MV pathogenesis and immunomodulation relies on in vitro findings obtained in pure DC or DC/T cell cocultures.

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B. Experimental models and consequences of DC surface interactions with viral proteins Human Langerhans cells and DCs isolated from peripheral blood or generated in vitro from monocytes or CD34þ precursor cells were infectable with both wild-type and vaccine MV strains as documented by the accumulation of viral proteins (Dubois et al., 2001; Fugier-Vivier et al., 1997; Grosjean et al., 1997; Klagge et al., 2000; Ohgimoto et al., 2001; Schnorr et al., 1997b; Servet-Delprat et al., 2000b; Steineur et al., 1998). Since CD46 is expressed on all these populations, infection with CD46utilizing MV strains is not surprising. Interestingly, however, MV wildtype strains and recombinant MVs expressing wild-type-derived H proteins (which rely on CD150 for entry) revealed a particular tropism for DCs in vitro and probably also in vivo since they replicate preferentially in secondary lymphoid tissues (de Swart et al., 2007; Ohgimoto et al., 2001; Pfeuffer et al., 2003; von Messling et al., 2003). The importance of CD150 was strongly supported by the requirement of this molecule for MV wildtype infection in CD11cþ DCs from transgenic mice (see above), by the enhanced susceptibility of monocytes to MV infection after activation induced CD150 expression (Minagawa et al., 2001), and by the inhibitory effect of CD150 antibodies on human DC fusion (Ohgimoto et al., 2001). Thus, human DCs generated in vitro express CD150 after LPS- or CD40Ldriven maturation, which is further upregulated by IL-1b (Bleharski et al., 2001; Kruse et al., 2001). More recently, DC-SIGN, a C-type lectin receptor expressed on myeloid DCs, has been found to enhance uptake of MV by DCs (de Witte et al., 2006). Mechanistically, this is not understood, since the protein does not support MV uptake when expressed alone in CHO cells. It may thus act to concentrate CD150 upon coligation by MV, and consequently, antibodies to both molecules block entry into DCs. Since DC-SIGN-mediated enhancement of infection was determined by increase of eGFP intensity in the infected cells, which can only accumulate upon viral replication, enhancement of uptake probably does not largely involve storage of MV in endocytic compartments. More recently, the ability of MV to induce IL10 specific transcripts in DCs has been attributed to its ability to conjugate to two different pattern recognition receptors on these cells, DC-SIGN and TLR2 (Gringhuis et al., 2007). MV wild-type, but not attenuated strains were found to act as TLR2 agonists, but not, as shown for RSV, TLR4 agonists on monocytes (and most likely DCs) (Bieback et al., 2002; KurtJones et al., 2000; Minagawa et al., 2001). Interestingly, the TLR2 agonistic activity of MV strains seems to inversely correlate with their ability to interact with CD46, and a recombinant MV expressing a wild-type H protein with amino acid 481 reversed to that found in attenuated strains (N ! Y) failed to activate TLR2 signaling (Bieback et al., 2002). Although

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common with DC-SIGN, the TLR2/CD14 complex also did not support MV entry into target cells; interaction of MV wild-type strains may well contribute to MV-induced maturation of immature DCs. MV wild-typedependent activation of TLR2 presumably accounts for the induction of CD86 and cytokines such as IL-6, IL-12p40, and IL-1 (Bieback et al., 2002; Michelsen et al., 2001). Most interestingly, MV wild-type strains also induced expression of CD150 on monocytes in a TLR2-dependent manner suggesting that they can promote expression of their entry receptor on these cells (Bieback et al., 2002). Surprisingly, the MV N protein also seems to directly interact with the surface of DCs via the FcgRII, and cell surface delivery of the N protein and its subsequent release has been observed (Marie et al., 2001, 2004). As it is a cytoplasmic protein, the finding that soluble N protein can act as a ligand for FcgRII on B cells to cause inhibition of antibody production was rather surprising (Ravanel et al., 1997). However, MV-infected cells dying from apoptosis and/or secondary necrosis as thymic epithelial cells are considered as a source of extracellular N protein (Laine et al., 2003, 2005). Alternatively, N protein can gain access to late endosomal compartments in infected cells where it recruits FcgRII and this allows for surface transport (where receptor bound N protein could act in neighboring cells) and/or release into the extracellular medium (Marie et al., 2004). Injection of N protein into mice revealed that the N protein indirectly acts on T cells by modulating DC functions via interaction with FcgRII thereby preventing IL-12 production and the generation of inflammatory reactions (Marie et al., 2004). In vitro, soluble N protein causes calcium influx in thymic epithelial cells and inhibits proliferation of a variety of cell types including activated, but not resting human primary T cells (Laine et al., 2003).

C. Consequences of infection on DC viability and function As mentioned above, human DCs are infectable with both wild-type and attenuated MV strains, albeit with different kinetics (Ohgimoto et al., 2001; Schnorr et al., 1997b) suggesting that MV proteins other than the glycoproteins favor intracellular replication in DCs as well. Recently, evidence for a role of M-protein stability in this differential infection kinetic has been obtained (Ohgimoto et al., 2007). Although MV replicates in DCs, which were immature or LPS matured at the infection time, as indicated by accumulation of viral proteins, virus release is low from immature and almost absent from mature DCs (Fugier-Vivier et al., 1997; ServetDelprat et al., 2000a; Schneider-Schaulies, unpublished). Apparently, the differentiation stage of the DCs imposes particular constraints on MV release as it does in differentiated monocytes/macrophages (Helin et al., 1999). MV-protein production and virus release can be enhanced upon

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coculture with activated T cells, and this was related to CD40 ligation (Fugier-Vivier et al., 1997; Servet-Delprat et al., 2000b). In contrast, CD40 ligation had no impact on viral replication or syncytium formation in another study (Klagge et al., 2004). Because of the accumulation of the viral glycoprotein complex on the surface of infected DCs, DC–DC fusion as well as DC fusion with cocultured T cells were observed (Grosjean et al., 1997). In this and a more recent study, apoptosis did occur in DC cultures late after infection (Hahm et al., 2004). When infected DCs were stabilized with a cell fusion inhibiting peptide (FIP), DCs remained viable for at least 48 h (Dubois et al., 2001; Klagge et al., 2000; Schnorr et al., 1997b). Phenotypic maturation of DCs immature at the onset of MV infection is indicated by upregulation of MHC class I and II, CD40, and costimulatory molecules such as CD80, CD83, and CD86 (Dubois et al., 2001; Schnorr et al., 1997b; Servet-Delprat et al., 2000b) and by induction of cytokine production. Transcripts specific for IL-12p35, IL-12p40, IL-10, IL-1a/b, IL-1RA, and IL-6 were induced after infection of monocytederived DCs with attenuated MV strains (Servet-Delprat et al., 2000b). Also on the transcriptional level, induction of IL-23p19 by attenuated, but not wild-type strains was documented. Both wild-type and attenuated strains failed to induce production of IL-12p70 and IL-10 on protein level (Klagge et al., 2004). In contrast, IL-10 was detected on mRNA and protein level after infection of DCs generated from CD34þ progenitor cells with an attenuated strain (Dubois et al., 2001), and, as outlined above, at least on transcriptional level in monocyte-derived DCs after coligation of DCSIGN and TLR2 (Gringhuis et al., 2007). Modes and kinetics of MVdependent regulation of IL-10 in DCs, which has been recently assigned as key cytokine in immunosuppression in other systems, are not yet fully understood. As discussed, infection of DCs with attenuated, but probably not wildtype MV strains causes production of type I IFN, and this is of obvious importance for maturation of these cells. Thus, upregulation of CD80 and CD86, but also TRAIL and TLR3 is at least partially dependent on the presence of type I IFN (Dubois et al., 2001; Klagge et al., 2000; Tanabe et al., 2003; Vidalain et al., 2000), as is the antiviral MxA protein which does, however, not efficiently prevent MV replication in these cells (SchneiderSchaulies et al., 1994; Schnorr et al., 1993). Moreover, acquisition of a cytolytic activity in MV-infected DCs has also been ascribed to the production of IFN-a/b (Vidalain et al., 2000, 2001), but it is also most likely confined to infection with attenuated strains. As detailed above, DCs exposed to wild-type MV strains at least partially rely on signals obtained after cross-linking of surface receptors such as TLRs, DC-SIGN, and/or CD150 for maturation. The latter and those triggered by other external stimuli including proinflammatory cytokines and CD40 are important for

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full activation, mobilization, and induction of terminal maturation of DCs. The ability of MV-infected DCs to migrate in response to chemokines has not been addressed as yet in detail, may, however, be impaired in response to MIP3a (Dubois et al., 2001). Apparently MV can modulate TLR signaling on APCs. For example, LPS- or SACS-stimulated production of IL-12p70 was strongly inhibited after exposure of monocytes to MV (Karp et al., 1996). This inhibition relied on crosslinking of CD46 as it also occurred with CD46-specific antibodies and C3b/C4b complement components. Long-term suppression of IL-12 release after SACS-stimulation of peripheral blood mononuclear cells (PBMCs) isolated from measles patients supports an impaired production of this cytokine (Atabani et al., 2001). In contrast, early after infection the ability of DCs isolated from peripheral blood of healthy donors to produce bioactive IL-12 in response to LPS or SACS was not impaired by infection with MV wild-type or vaccine strains (Schnorr et al., 1997b). The mechanism of how non-CD46 interacting MV wild-type strains might inhibit TLR-stimulated IL-12 production late after infection is unknown. Possibly, lack of stimulated IL-12 production in vitro may relate to an enhanced susceptibility of DCs to apoptosis ex vivo as seen in CD150 transgenic mice (Hahm et al., 2004) rather than to interference with TLR signaling. The recently documented ability of MV to cause activation of NF-kB by TLR2/DC-SIGN coligation has already been referred to (Gringhuis et al., 2007) (see above). Moreover, MV infection was found to interfere with signaling via TLR7 and TLR9 in plasmacytoid DCs, also known as natural interferon producing cells (Schlender et al., 2005). The functional consequences of this interference have, however, not yet been directly addressed. MV-infected DCs only subtly differ from LPS-matured DCs with regard to integrin activation, acquisition of a migratory phenotype, and motility. Similarly, the organization of cell interfaces between infected DCs and T cells appears consistent with that of functional immune synapses with regard to CD3 clustering and MHC Class II surface recruitment. However, the majority of MV-DC/T cell conjugates is unstable and only promotes abortive T cell activation (Shishkova et al., 2007). MVinfected DCs retain activities required for initiating, but not sustaining T cell conjugation and activation. This is partially rescued if surface expression of the MV glycoproteins on DCs is abolished by infection with a recombinant MV encoding VSV G protein instead of the MV glycoproteins, indicating that these contribute directly to synapse destabilization and thereby act as major effectors of T cell inhibition (Shishkova et al., 2007). As to what extent destabilization relates to the breakdown of microvillar extensions on T cells (as mentioned above, Section IV) or alterations of coupling of surface receptors to the cytoskeleton in DCs is currently unknown.

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VI. CONCLUSIONS AND PERSPECTIVES Many specific aspects of the morbillivirus-induced disease and profound immunosuppression appear to be based on the tropism mediated by usage of CD150 as entry receptor on activated cells of the immune system. Selecting monocytes and DCs in the respiratory epithelium as first target cells which transport the viral load to draining lymph nodes, morbilliviruses find their way right into the center of the immune system. Considering the role of DCs to induce and shape immune responses, it is evident that the virus interaction with these cells is central for immunomodulation and transient silencing of T cells. DCs may not only serve as primary target cells of morbilliviruses, but also support virus replication and distribution. Simultaneously, they may receive maturation signals, be it by TLR signaling, interferon production, or other as yet unknown stimuli. Virus-specific antigens were found to be efficiently presented both in vitro and in vivo, which results in the efficient induction of a specific immune response in the simultaneous presence of an immunosuppression versus other pathogens. However, incomplete T cell activation or active inhibition of T cell expansion by formation of unstable immunological synapses may predominate later in morbillivirus infection of DCs. Then, apoptosis, inability to respond to external maturation signals, and, in addition, accumulation of viral proteins on the cell surface may collectively act in T cell silencing or killing via TRAIL. Apoptosis is an important factor in T cell lymphopenia, but certainly does not explain why healthy uninfected peripheral T cells cannot proliferate upon exogenous stimulation. For this, direct negative signaling of infected DCs to scanning T cells by the morbillivirus envelope glycoprotein (F/H) complex expressed on the cell surface, or the N protein, is an attractive hypothesis. Obviously, T cells do not die as a consequence of this inhibitory signal and, at least in vitro, recover and regain their mitogenic responsiveness. In agreement with this, recall responses, which are suppressed during and after the acute viral infection, return to normal also in vivo. The morbillivirus-induced pronounced immunosuppression favors complications by secondary infections with high morbidity and mortality frequencies, which justify the strong effort to eradicate MV and RPV. However, once one or the other virus will be eradicated, the plasticity and wide host range of the close relative CDV may enable this virus to invade the niches that had been occupied before by the two other viruses. Thus, eradication will probably not abolish the requirement for continuing vaccination. The fact that functional vaccines against MV and RPV are available should not prevent the development of new thermostable, safer, and cheaper vaccines. In the case of measles, a vaccine is

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required which can overcome the protection by maternal antibodies and can be applied very early in life (Schlereth et al., 2003). Recombinant multivalent vaccines f.e. against measles and hepatitis B virus (del Valle et al., 2007), or marker vaccines against CDV (Silin et al., 2007), or marker vaccines inducing a broader immune response against RPV and PPRV (Parida et al., 2007), may provide progress in vaccination. The fight against morbillivirus infections of humans and animals will remain a valuable challenge in the future.

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Wyde, P. R., Ambrosi, M. W., Voss, T. G., Meyer, H. L., and Gilbert, B. F. (1992). Measles virus replication in lungs of hispid cotton rats after intranasal inoculation. Proc. Soc. Exp. Biol. Med. 201(1):80–87. Yanagi, Y., Cubitt, B. A., and Oldstone, M. B. (1992). Measles virus inhibits mitogen-induced T cell proliferation but does not directly perturb the T cell activation process inside the cell. Virology 187(1):280–289. Yanagi, Y., Takeda, M., and Ohno, S. (2006). Measles virus: Cellular receptors, tropism and pathogenesis. J. Gen. Virol. 87:2767–2779. Yu, Y., and Alwine, J. C. (2002). Human cytomegalovirus major immediate-early proteins and simian virus 40 large T antigen can inhibit apoptosis through activation of the phosphatidylinositide 300 -OH kinase pathway and the cellular kinase Akt. J. Virol. 76 (8):3731–3738. Yuan, H., Veldman, T., Rundell, K., and Schlegel, R. (2002). Simian virus 40 small tumor antigen activates AKT and telomerase and induces anchorage-independent growth of human epithelial cells. J. Virol. 76(21):10685–10691. Zaffran, Y., Destaing, O., Roux, A., Ory, S., Nheu, T., Jurdic, P., Rabourdin-Combe, C., and Astier, A. L. (2001). CD46/CD3 costimulation induces morphological changes of human T cells and activation of Vav, Rac, and extracellular signal-regulated kinase mitogenactivated protein kinase. J. Immunol. 167(12):6780–6785.

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5 Lyssaviruses—Current Trends Susan A. Nadin-Davis and Christine Fehlner-Gardiner

Contents

I. Introduction II. Developments in Diagnostic and Surveillance Tools A. Diagnosis B. Viral typing C. Evolutionary time frames D. Modeling applications III. Fundamental Aspects of Virus–Host Interactions A. What is the basis for RABV pathogenicity? B. Role of viral proteins C. Role of host cell pathways D. Considerations for future studies on rabies pathogenesis IV. Reverse Genetics—Methodology and Applications A. RABV vaccines B. Vaccines for other diseases V. Other Strategies for Rabies Vaccine Development A. Adenovirus recombinants B. DNA vaccines VI. The Challenge of Rabies Biologics for Passive Immunity VII. Novel Applications of RABV A. Use as a neuronal tracer B. Use of RABV proteins for molecular targeting VIII. Concluding Remarks References

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Centre of Expertise for Rabies, Ottawa Laboratory (Fallowfield), Canadian Food Inspection Agency, Ottawa, ON, Canada Advances in Virus Research, Volume 71 ISSN 0065-3527, DOI: 10.1016/S0065-3527(08)00005-5

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Various technological developments have revitalized the approaches employed to study the disease of rabies. In particular, reverse genetics has facilitated the generation of novel viruses used to improve our understanding of the fundamental aspects of rabies virus (RABV) biology and pathogenicity and yielded novel constructs potentially useful as vaccines against rabies and other diseases. Other techniques such as high throughput methods to examine the impact of rabies virus infection on host cell gene expression and two hybrid systems to explore detailed protein–protein interactions also contribute substantially to our understanding of virus–host interactions. This review summarizes much of the increased knowledge about rabies that has resulted from such studies but acknowledges that this is still insufficient to allow rational attempts at curing those who present with clinical disease.

I. INTRODUCTION The disease of rabies, known to mankind since the 23rd century BC (see Baer, 2007), remains the best known and most feared of all zoonotic diseases. Most rabies cases reported world-wide are caused by rabies virus (RABV), the prototype of the Lyssavirus genus of the family Rhabdoviridae, although all lyssaviruses can elicit the disease. Control of human rabies is achieved most effectively by elimination of the virus from animal reservoirs that maintain and transmit it to other species. In addition, highly efficacious regimens for human disease prevention exist (Rupprecht et al., 2006). However, rabies still accounts for an estimated 55,000 human deaths world-wide each year (WHO, 2005). Dogs, which have long been associated with the disease, form the major rabies reservoir in the developing world where most human exposures and fatalities occur. Failure to prevent disease in many developing countries is due to several factors: poor control and limited vaccination of dogs, lack of public education, poor public health infrastructure, and the high cost and limited availability of antirabies biologicals. In contrast, developed countries have eliminated rabies from their dog populations and postexposure prophylaxis (PEP) is available to those exposed to the disease via contact with wildlife reservoirs. This situation has driven many of the recent initiatives in rabies research that are reviewed in this chapter. At the more practical level, development of new reagents such as human neutralizing antirabies glycoprotein (G) monoclonal antibodies (Mabs) are being developed to replace the rabies immune globulin preparations that are in short supply. Efforts to develop improved and cost-effective rabies vaccines continue,

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primarily to support countrywide pet vaccination programs in the developing world and oral wildlife vaccination for elimination of sylvatic rabies in much of the developed world. Control programs, which require ongoing and timely disease surveillance, are driving the need for fieldbased diagnostic methods to assist in sylvatic rabies eradication, as well as a comprehensive knowledge of rabies epidemiology and evolution now being revealed through molecular epidemiological techniques. The occasional case of human rabies in the developed world provides continued impetus for development of a therapeutic regimen. However, despite the recent advances in our understanding of virus–host interactions summarized here, successful treatment of clinical rabies requires yet deeper appreciation of the pathogenic mechanisms that result in patient death. The ability to manipulate the RABV genome through reverse genetics permits detailed analysis of the viral features responsible for disease and facilitates some novel applications, including the use of RABV as a neurological tracer to provide unique insights into neuronal pathways and development of novel vaccines directed against rabies and other diseases.

II. DEVELOPMENTS IN DIAGNOSTIC AND SURVEILLANCE TOOLS A. Diagnosis While the direct fluorescent antibody (DFA) test applied to fresh brain smears or impressions remains the gold standard method for animal rabies diagnosis, its two main drawbacks are the need for sample shipping to central laboratories and the costs involved in acquiring and maintaining a fluorescence microscope. These requirements often restrict sample submissions, particularly from remote areas, for example, arctic regions, and in many parts of the tropics where sample preservation is problematic. These limitations in turn result in rabies underreporting and underappreciation of its significance. A direct rapid immunohistochemical test (DRIT) for rabies, developed by the Rabies Section of the Centers for Disease Control and Prevention (CDC), may help to address this problem. DRIT employs a light microscope to detect RABV antigen in brain impressions using a cocktail of biotinylated antinucleocapsid Mabs with visualization using a streptavidin–peroxidase complex. In a field evaluation in Tanzania, DRIT performed comparably to the DFA (performed at the CDC) with regards to sensitivity and specificity and thus may, subject to mandated biocontainment requirements, be a viable means of testing animal specimens under field conditions (Lembo et al., 2006). This could facilitate greatly improved surveillance in tropical

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regions but it might also be useful in developed countries where increased local surveillance is needed to support wildlife oral vaccination campaigns. Another interesting possibility for rapid screening in the field is the use of lateral flow immunochromatography (Kang et al., 2007), although comprehensive validation against established methods is required. The need for antemortem human testing has driven development of alternate diagnostic tools, for example, reverse transcriptase polymerase chain reaction (RT-PCR) testing for the presence of viral RNA in saliva (reviewed in Trimarchi and Nadin-Davis, 2007). RT-PCR has also been applied to oral swab screening of wild bat populations for active lyssavirus infection in Europe where most bat species have protected status (Echevarrı´a et al., 2001). Although, real-time RT-PCR (RRT-PCR) would have additional advantages for human testing in terms of rapidity and sensitivity, only a few reports describe its application for detection of specific lyssavirus genotypes (Foord et al., 2006; Wakeley et al., 2005). The complexity of designing broadly cross-reactive reagents for a TaqManbased approach is challenging due to the extent of lyssavirus sequence divergence (Hughes et al., 2004b). A SYBR green-based RRT-PCR, that was more sensitive than conventional RT-PCR for the detection of virus in human saliva samples, may be of value providing its specificity can be assured (Nagaraj et al., 2006). Further evaluation of a nucleic-acid sequence based amplification (NASBA) method, applied to human saliva and cerebrospinal fluid samples is needed (Wacharapluesadee and Hemachudha, 2001). The issue of testing solid organ transplant tissues has been broached in response to rabies transmission via organ transplantation (Srinivasan et al., 2005), but such testing will remain impractical unless faster and more readily accessible tests become available ( Jackson, 2004).

B. Viral typing The existence of multiple RABV strains, each of which is maintained by a specific host within a geographically defined area, drives the need for strain discrimination tools that provide critical information in support of control programs. Both antigenic and molecular-based typing techniques have been increasingly applied over the last 15 years (reviewed in NadinDavis, 2007). Extensive phylogenetic analyses of RABV (genotype 1) isolates recovered world-wide have identified their detailed phylogeography and revealed the existence of a limited number of major lineages: the heterogeneous ‘‘American indigenous’’ lineage, found only in the Americas, comprised mostly of bat-associated strains and a few species of Carnivora; at least three distinct canid lineages circulating in South-East Asia; an ‘‘Arctic’’ lineage that includes viruses from northern circumpolar

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regions as well as viruses from central and southern Asia; a canid lineage in western Africa; a lineage associated with herpestid and viverrid species in southern Africa; and the cosmopolitan lineage (Nadin-Davis and Bingham, 2004), probably widely dispersed as a result of human colonial activities in the 16th–18th centuries, that includes canid-associated viruses of Europe, the Middle East, Asia, and much of Africa, mongoose, skunk and canid associated viruses of the Americas and the Caribbean, as well as most vaccine strains. Ongoing genetic characterization of RABV isolates continues to extend our knowledge of RABV diversity and identifies newly emerging virus–host associations. For example, in the USA alone, a distinct viral variant associated with western pipistrelle bats (Franka et al., 2006) and sustained intraspecific transmission of a bat variant within skunk populations in Arizona (Leslie et al., 2006) were both recently recognized. In Latin America, additional bat-associated variants continue to be identified (Kobayashi et al., 2005). Consistent with current taxonomic trends in virology, phylogenetic analysis has been used to investigate membership in the Lyssavirus genus (Kuzmin et al., 2006a) and to delineate the seven species (genotypes or GTs) currently recognized within the genus (Tordo et al., 2004). However, as new viruses emerge (Kuzmin et al., 2005, 2006b), additional species may be identified.

C. Evolutionary time frames Recently, methods to estimate nucleotide substitution rates have helped to develop time-scaled phylogenetic trees and thereby explore the time frame of lyssavirus evolution. Regardless of gene region or variant type, RABV coding regions exhibit quite similar rates of nucleotide substitution, ranging from 1.2
104 to 5.3
104 nucleotide substitutions/site/ year, and have a high synonymous to nonsynonymous mutation ratio (Badrane and Tordo, 2001; Davis et al., 2006; Holmes et al., 2002; Hughes et al., 2005; Kuzmin et al., 2007). The rate at the noncoding G–L region appears to be somewhat higher, around 1
103 nucleotide substitutions/site/year (Davis et al., 2007; Hughes et al., 2004a). Evaluation of the growth properties of RABV populations by Bayesian methods generally indicate an exponential growth profile with variable-rate relaxed molecular clocks providing for the best fit to the data. The role of the Chiroptera as hosts for most current lyssavirus genotypes has suggested that the primordial lyssavirus was bat-associated and a host-switching event, between 888 and 1459 years ago, was postulated to have resulted in the emergence of carnivoran rabies (Badrane and Tordo, 2001). A rather shorter time frame of GT 1 lyssavirus diversity of approximately 500 years has also been proposed (Holmes et al., 2002). Although all lyssavirus genes appear to be equally useful for phylogenetic

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predictions (Wu et al., 2007), Davis et al. (2006) found a lack of congruence in GT 1 evolutionary history, predicted using different genes and were unable to establish whether bat-associated variants or viruses associated with other terrestrial species comprised the most basal evolutionary group. By two different approaches, the most recent common ancestor (MRCA) of North American bat RABVs was dated to the mid-1600s (combined range of 1254–1782) (Hughes et al., 2005); this phylogeny suggested that the parental virus initially branched to yield variants currently associated with free-tailed and vampire bats and this was followed by the emergence of distinct variants associated with solitary and colonial bats of North America. If this prediction is correct, genetic heterogeneity in bat RABVs must depend more on the migratory life-style of the host rather than on the age of the viral lineage. The ‘‘Arctic’’ lineage is estimated to have emerged in Asia sometime between 1255 and 1786 and then spread northward into all circumpolar regions (Kuzmin et al., 2008), as had previously been proposed (NadinDavis et al., 2007). A study of two distinct southern African biotypes concluded that the canid and mongoose variants exhibit quite different evolutionary dynamics probably reflecting the different ecological niches of their hosts (Davis et al., 2007). Moreover, the mean age for the MRCA of the mongoose variant was 73 years, placing its emergence around 1930 and not in the 1800s as had previously been supposed (Nel et al., 2005). In general, the time frames estimated by Bayesian methods have wide ranges and predict much shorter time scales than suggested from anecdotal information. Unless larger datasets generate different conclusions, it appears that some of the viruses referred to in past outbreaks belonged to now extinct lineages (Badrane and Tordo, 2001). Coalescent methods generate more coherent time frames when applied to more recent outbreaks (Biek et al., 2007; Hughes et al., 2004a). The European bat lyssavirus type-1 (EBLV-1) group (GT 5), harbored by serotine bats of several European countries, is estimated to have emerged between 500 and 750 years ago and has since diverged into two distinct populations (Davis et al., 2005). The nucleotide substitution rate (5
10-5 substitutions/site/year) exhibited by these viruses is one of the lowest values recorded for an RNA virus (Jenkins et al., 2002). The very high constraints against nonsynonymous substitution observed in EBLV-1 were explained by supposing that the virus had reached a fitness peak so that most amino acid changes reduce viral fitness.

D. Modeling applications Combination of viral phylogenetic analysis with various mathematical models has provided a valuable toolset for furthering our understanding of the factors contributing to viral disease dynamics. The rapid and

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well-documented spread of the mid-Atlantic raccoon rabies variant along the eastern seaboard of the USA over the last 30 years has provided extensive surveillance data that permit the development and assessment of such models (Real et al., 2005b). A model that reflects the initial 4–5 year epizootic period of raccoon rabies, followed by smaller epizootics of decreasing size and periodicity, was developed (Childs et al., 2000) followed by a stochastic spatial model of rabies spread in the state of Connecticut that identified rivers as key barriers to local dispersion (Smith et al., 2002) and demonstrated the role of long-distance animal translocation in the emergence of new foci of disease (Smith et al., 2005). This spatial model was also used to predict westward expansion of raccoon rabies across Ohio, following an outbreak that breached an oral vaccination zone intended to contain this epizootic, and to assist in the design of supplementary control activities (Russell et al., 2005). More recently, other models explored how to most effectively use natural barriers such as waterways during wildlife oral vaccination campaigns to prevent rabies spread (Russell et al., 2006). Coalescent-based estimates using raccoon RABV genetic data were in good agreement with the known spatial and demographic dynamics of disease spread with time and showed the importance of the initial wave of infection in determining spatial genetic structuring of the epizootic (Biek et al., 2007). In studies of Arctic fox strain rabies, the ecogeographic patterns observed in southern Ontario (Tinline and MacInnes, 2004) were explained by a model that unified the spatial population dynamics and molecular evolution of the RABV (Real et al., 2005a). Insights into patterns of viral spread may also be gained through raccoon genotyping to identify host subpopulation structure (Cullingham et al., 2005). Further gains in our understanding of rabies dynamics and evolution will undoubtedly follow future application of such methods.

III. FUNDAMENTAL ASPECTS OF VIRUS– HOST INTERACTIONS Despite its small size (about 12 kb) and limited coding capacity (five open reading frames (ORFs), in the order 30 -N-P-M-G-L-50 ), RABV exhibits remarkable neurotropic and neuroinvasive properties that are central to its transmission cycle. Following deposition of virus in peripheral tissues, via the bite of an infected animal, the virus enters peripheral nerves and is transmitted via nerve connections into the central nervous system (CNS), where extensive viral propagation occurs prior to secretion of large amounts of virus in salivary glands in readiness for the next cycle (Jackson, 2007). While viral transcription and replication proceeds in a manner highly typical of the Rhabdoviridae (Wunner, 2007), many aspects of lyssavirus biology are unique to this genus. Detailed structural aspects

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of RABV replication have recently been reviewed (Albertini et al., 2008) so will not be discussed further, but many new insights into the molecular basis of virus–host cell interactions and RABV pathogenesis will be described.

A. What is the basis for RABV pathogenicity? Despite the fatal outcome of rabies, limited histological lesions are seen in brains from clinical human cases, suggesting that rabies infection does not cause substantial neuronal death but rather neuronal dysfunction, mediated perhaps by impairment of neurotransmitter release and function and/or ion homeostasis (Fu and Jackson, 2005; Jackson, 2007). Some degeneration of neuronal processes was noted after infection with pathogenic but not attenuated viruses (Li et al., 2005). RABV-infected mouse brain exhibits a number of ultrastructural changes in neurons including degradation of neuronal axons and dendrites, disruption of cytoskeletal integrity, vacuolation and mitochondrial swelling that might explain neuronal dysfunction (Scott et al., 2008). The attenuated lab-adapted CVS-B2c strain, derived from the challenge virus standard (CVS) strain, causes much more severe histological damage to the murine CNS than the pathogenic silver-haired bat RABV (SHBRV) field isolate, (Wang et al., 2005) and such differences need to be borne in mind when inferring mechanisms of pathogenesis from studies using lab-adapted strains.

B. Role of viral proteins 1. Glycoprotein The G protein forms trimeric transmembrane spike structures that allow the virus to attach to neurons and then enter the cell via a fusion event; G protein is thus a key determinant of RABV’s neurotropism and neuroinvasiveness (Wunner, 2007). While the inability of a G-deficient RABV to transfer between neurons clearly demonstrated the importance of G protein to viral spread (Etessami et al., 2000), establishing the nature of the receptor(s) employed has proven challenging. RABV G binds to the neuronal nicotinic acetylcholine receptor (Gastka et al., 1996) and the murine neural cell adhesion molecule (NCAM) (Thoulouze et al., 1998), but neither of these receptors is absolutely required for RABV spread in mice. The low affinity nerve growth factor receptor (P75NTR) can also confer RABV binding to a nonpermissive cell line (Tuffereau et al., 1998). P75NTR is a member of the tumor necrosis factor (TNF) receptor superfamily that binds to several neurotrophins and is targeted by several viruses of many different orders (Kinkade and Ware, 2006), although the viruses and the neurotrophins often target different

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domains. The observation that RABV G binds to mammalian P75NTR but not to the avian equivalent was proposed to explain the virus’ restriction to mammalian hosts (Langevin et al., 2002). Moreover, RABV G binding to P75NTR requires Lys330 and Arg333 of antigenic site III, residues previously linked to RABV pathogenicity (see below) while mutations at residues flanking this antigenic site interfered with P75NTR binding (Langevin and Tuffereau, 2002). However, other observations argue against a universal role of P75NTR as a lyssavirus receptor. Only lyssaviruses of GT 1 (RABV) and GT 6, EBLV-2, interact with P75NTR, so lyssaviruses of all other GTs must gain cell entry via alternate receptor(s) (Tuffereau et al., 2001). Furthermore, viral spread occurs throughout the CNS in P75NTR-deficient mice (Jackson and Park, 1999) and viral infection of primary neurons can occur in the absence of RABV G/P75NTR binding (Tuffereau et al., 2007). While another receptor yet to be identified might function as a universal lyssavirus receptor, it remains possible that these viruses can use a number of different nerve cell receptors to gain entry into the host nervous system. Further study on nerve cell entry by field isolates may help to resolve this issue. The specific role of G protein in determining viral spread within the CNS, and hence the extent of pathogenicity, was demonstrated in a study that examined the distribution over time of several viral constructs after stereotaxic inoculation into the rat hippocampus (Yan et al., 2002). Structure–function studies involving the production of large numbers of chimeric viruses, each differing at just one amino acid within the G protein, reveal an increasing number of residues important to pathogenicity. In addition to an absolute requirement for Arg or Lys at residue 333 (Takayama-Ito et al., 2006a; Tuffereau et al., 1989), amino acids Ala242, Asp255 and Ile268, especially when present together, confer the highly pathogenic phenotype of the Nishigahara strain (Takayama-Ito et al., 2006b). An Asn to Lys mutation at residue 194 restored pathogenicity to a highly attenuated RABV strain by increasing viral spread, eliciting faster cellular internalization and shifting the pH threshold for membrane fusion (Faber et al., 2005b). However, in addition to the protein’s primary structure, the level of G protein expression also has important pathogenic consequences; in general, more pathogenic strains produce lower amounts of G protein that in turn may limit the host’s apoptotic (Yan et al., 2001) and innate immune system responses (Wang et al., 2005). Interestingly, nonpathogenic G genes are dominant over pathogenic versions (Faber et al., 2007). RABV virulence is increasingly being recognized as a multigenic trait. Regions of the RABV genome, including N, P, M and L genes, as well as noncoding elements, such as the trailer sequence and the G–L intergenic region, have all been reported to contribute to virulence and/or neuroinvasiveness (Faber et al., 2004; Pulmanausahakul et al., 2008; Shimizu et al., 2007;

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Yamada et al., 2006) probably through interaction with intracellular factors necessary for efficient viral propagation.

2. Phosphoprotein The P protein, with its highly modular organization that includes both conserved and variable domains (Nadin-Davis et al., 2002), is emerging as a factor that contributes to viral virulence at many levels. In addition to full-length P protein (P1), the P gene ORF can generate up to four N-terminally truncated P products (P2–P5) (Chenik et al., 1995). P proteins are targeted by two distinct cellular kinases (Gupta et al., 2000) that phosphorylate several variably conserved sites (Nadin-Davis et al., 2002). The cellular distribution of these various forms of P is determined by multiple localization signals present at different locations along its primary sequence. An N-terminal nuclear export signal (NES), present only in P1 and P2, normally directs these products into the cytoplasm while P3–P5 are localized to the nucleus; additionally the C-terminal P sequence contains both a nuclear localization signal (NLS) and a NES, functionalities of which appear to be determined by phosphorylation of a neighboring residue (Moseley et al., 2007a; Pasdeloup et al., 2005). The variable length, phosphorylation patterns, and cellular distribution of the P protein provide it significant potential versatility in its interactions with host cell factors. a. Dynein LC8 Interaction One intriguing interaction of the lyssavirus P protein is its association with the dynein light chain LC8 ( Jacob et al., 2000; Raux et al., 2000), a 10 kDa component of both the cytoplasmic dynein motor and Myosin V, which is involved in minus enddirected movement of organelles along microtubules and in actinmediated vesicle transport in axons. Although functional studies on this interaction have been performed only on GT 1 and GT 3 representatives (Jacob et al., 2000), the genus-wide importance of this association is suggested by the absolute conservation of the LC8 binding domain (BD) motif D(K/R)XTQT at P protein residues 143–148 (Nadin-Davis et al., 2002; Poisson et al., 2001). This finding fueled the hypothesis that P/LC8 interaction facilitated microtubule-directed minus-sense axonal transport of RABV ribonucleoprotein to the cell body and thus played a pivotal role in the spread of the virus within the host’s nervous system. However, doubt on this model was cast by the fact that viruses lacking the LC8 BD, although slowed in their rate of spread, could still gain entry into the CNS from the periphery (Mebatsion, 2001; Rasalingam et al., 2005). Tan et al. (2007) showed that loss of the LC8 BD from P reduced transcription levels in neuronal cells and thus proposed that P protein/LC8 association was important for efficient viral RNA polymerase activity, a role consistent with

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the P protein’s function as an L protein cofactor. Interestingly, the LC8 BD motif can facilitate nuclear protein importation, suggesting that this motif facilitates some nuclear function of P (Moseley et al., 2007b). b. Abrogation of the Interferon Pathway An early response to RABV infection of both neuronal cells (Pre´haud et al., 2005) and the mouse brain ( Johnson et al., 2006; Prosniak et al., 2001; Saha and Rangarajan, 2003; Ubol et al., 2006; Wang et al., 2005) is an increase in many of the key components of the type-1 interferon (IFN) pathway that acts to protect the host from viral infections. Many viruses have adopted strategies to circumvent this process (reviewed by Haller et al., 2006). The P protein determines RABV sensitivity to type-1 IFN (Shimizu et al., 2006) and abrogates the host’s innate immune system by interacting with the IFN pathway at multiple levels (Chelbi-Alix et al., 2006). Brzo´zka et al. (2005) demonstrated that full-length P protein inhibits phosphorylation of cytoplasmic interferon regulatory factor 3 (IRF3) by TANK-binding kinase 1 (TBK-1) thereby curtailing IFN induction by preventing IRF3-dependent transcription of the b-IFN gene in RABVinfected cells. P protein also interferes with the IFN-effector pathway through an interaction of its C-terminus with STAT1, a critical mediator of the Janus kinase-signal transducer and activator of transcription ( JAK/STAT) signal transduction pathway responsible for the expression of many IFNeffector genes. P protein does not interfere with STAT1 phosphorylation and activation but cytoplasmic forms of P prevent its nuclear accumulation in response to IFN induction (Brzo´zka et al., 2006; Vidy et al., 2005), while the nuclear-localized P3 binds directly to the DNA binding domain of activated STAT1, thereby suppressing transcription of IFN-inducible genes (Vidy et al., 2007). The higher nuclear translocation of phosphorylated STATs in the presence of the attenuated CVS-B2c strain compared to the virulent SHBRV strain suggests the importance of this P function to virulence (Wang et al., 2005). A third target for the RABV P protein is the product of the promyelocytic leukaemia (PML) gene, now recognized as a primary target gene of the IFN pathway (Blondel et al., 2002). PML exists in the nucleus as both a diffuse nucleoplasmic form and as discrete nuclear bodies in which it associates with several other nuclear proteins; this distribution is often disrupted in virus-infected cells. Through a motif present in the 125 C-terminal residues of P protein, P1 binds to PML and retains it in the cytoplasm while P3 binds to nuclear PML, reorganizes the nuclear bodies and increases their size. High levels of RABV growth in mouse embryo fibroblasts lacking PML suggest an antiviral effect of this product that is inhibited by P protein (Blondel et al., 2002), but better understanding of PML’s normal function is needed to fully appreciate this effect.

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3. Other viral proteins The multi-functional matrix protein may also contribute to viral virulence through many different mechanisms. It inhibits translation in RABVinfected cells via an interaction with the eIF3h subunit, part of the eIF3 complex involved in ribosomal dissociation that is critical to the cellular translation machinery (Komarova et al., 2007). M protein, in concert with G protein, also regulates viral replication and facilitates cell-to-cell spread (Pulmanausahakul et al., 2008). In addition, M protein mediates at least some of the virus’ apoptotic effects. In a model of RABV-induced neuronal apoptosis, Kassis et al. (2004) found that cells transfected with M protein alone could induce caspase activation and apoptosis via a pathway involving the binding of TNF-related apoptosis-inducing ligand (TRAIL) to its receptors; M mediated release of a soluble, active form of TRAIL important to the early induction of this apoptotic pathway. The L protein almost certainly contributes to RABV pathogenicity through interactions with various host cell components needed to support its role in viral transcription and replication but the details of such interactions remain to be explored.

C. Role of host cell pathways The increasing availability of methods to explore large-scale changes in cellular expression patterns have facilitated studies to understand how experimental RABV infection changes host transcript levels. Using various methods, infection of mice with lab-adapted RABVs resulted in sequential changes in host cell transcription patterns (Johnson et al., 2006; Prosniak et al., 2001; Saha and Rangarajan, 2003; Ubol et al., 2006; Wang et al., 2005). Whereas the majority of genes were down-regulated as viral propagation progressed, select groups of genes were up-regulated. A relatively early response was the increased level of many mRNAs encoding products involved in innate immunity, including type-1 IFNs, toll-like receptors (TLRs) which play a critical role in initiating innate immunity (see McCoy and O’Neill, 2008), many IFN-effectors, as well as chemokines, cytokines and complement proteins involved in the inflammatory response. Indeed, of all genes up-regulated by RABV infection of a human neuron-derivative cell line, 24% were involved in immunity; moreover these neurons were found to express TLR3 which recognizes dsRNA, and could mount a corresponding innate immune response, a novel finding given the immunologically privileged nature of the CNS (Pre´haud et al., 2005). A comprehensive study of TLR induction in the murine CNS in response to RABV infection also identified an early upregulation of TLR3 which would permit increased virus sensing mechanisms in the local vicinity of virally infected cells (McKimmie et al., 2005).

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Rabies-infected patients also exhibited enhanced TLR3 expression in neurons and occasionally in glial cells of the cerebellar cortex, a phenomenon that appeared to require induction by soluble factors rather than direct viral infection (Jackson et al., 2006). The innate immune response to RABV infection is followed by an increase in transcript levels for several products that may be significant to clinical disease and viral spread. These include growth factors, which may facilitate viral spread by promoting new axonal connections or prolonging infected neuron survival (Prosniak et al., 2003b), certain metabolic enzymes, particularly those involved in nucleotide metabolism, selected receptors and transporters, notably dopamine transporter/receptor and serotonin receptor, consistent with the concept of disruptions in neurotransmission, as well as significant increases in ion transporters that could be responsible for reductions of intracellular sodium and calcium (Dhingra et al., 2007; Prosniak et al., 2001; Saha and Rangarajan, 2003; Ubol et al., 2006; Wang et al., 2005). Moreover, down-regulation of proteins involved in docking and fusion of synaptic vesicles at the presynaptic membrane resulted in synaptic vesicle accumulation (Dhingra et al., 2007). In later stages of disease, there are increases in yet other transcripts that encode products which may support RABV replication and spread, for example, heat shock protein 90 (Hsp-90) and CDC10; proteins involved in axonal guidance and cell repair, for example, neuroleukin (NLK) and apolipoprotein D (ApoD) important for lipid recycling. Finally, there is an up-regulation of products associated with programmed cell death (Prosniak et al., 2001; Ubol et al., 2006; Wang et al., 2005).

1. Apoptosis Apoptosis is now recognized as one critical means by which multicellular organisms try to protect themselves against pathogen invasion (Adams and Cory, 1998) and its role in experimental rabies pathogenesis was suggested when changes consistent with apoptosis, including DNA fragmentation and increased expression of the apoptotic enhancer Bax, were observed in neurons of RABV-infected mice (Jackson and Rossiter, 1997). Apoptosis was especially prominent in the pyramidal neurons of the hippocampus and cerebral cortex, prime targets for rabies infection, and suckling mice, which are more susceptible to the disease, exhibited higher levels of apoptosis than adults (Jackson and Park, 1998; Theerasurakarn and Ubol, 1998). Although elevated levels of Bax were observed in murine neuroblastoma cells within 24 h of infection, followed by up-regulation of caspase 1 (Ubol et al., 1998), bax-deficient mice still exhibited significant apoptosis thereby implicating additional apoptotic modulators (Jackson, 1999). Using CVS strains that differ in their pathogenicity, Morimoto et al. (1999) observed a good correlation between high levels of apoptosis and high RABV G accumulation in mouse primary neuronal cell cultures.

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Similar conclusions were made using recombinant viruses differing in G protein expression levels (Faber et al., 2002); high expression of RABV G was accompanied by increases in caspase 3 activity and other apoptosis markers as well as higher anti-G antibody titers in mice. The speed or extent of apoptosis may directly determine the magnitude of the antibody response as suggested by experiments employing a recombinant virus expressing the pro-apoptotic cytochrome c gene (Pulmanausahakul et al., 2001). RABV pathogenesis may be determined to a large extent by the cell type undergoing apoptosis and the mechanisms inducing cell death. In a series of experiments on apoptosis in lymphocytes, the highly attenuated vaccine strain Evelyn Rokitnicki Abelseth (ERA) could induce both caspase-dependent and caspase-independent apoptotic pathways after infection of a Jurkat T cell line in vitro, unlike the neurovirulent CVS strain (Thoulouze et al., 2003). This phenomenon was prevented by constitutive expression of the antiapoptotic Bcl-2 product. These studies also showed that ERA could up-regulate Bcl-2 levels in the Jurkat cell line, a phenomenon that resulted in long-term persistently infected cultures. This observation suggested a mechanism by which live attenuated RABVs such as ERA may, through increased Bcl-2 synthesis, persist in vivo in vaccinated animals; this effect could contribute to their efficacy as vaccines, but could also potentially result in a ‘‘carrier’’ state. In an exploration of the role of the death-promoting factor Fas Ligand (FasL) and its receptor Fas in initiating apoptosis of activated lymphocytes in vivo, the virulent CVS strain, but not the attenuated PV strain, induced early production of FasL, primarily by infected neurons, which was associated with high levels of T cell apoptosis. Thus, CVS infection of mice induced only a transient migration of lymphocytes into the CNS in contrast to PV infection that permitted sustained T cell migration into the CNS and more limited CD3+ T cell apoptosis (Baloul et al., 2004). Up-regulation of FasL by the neurovirulent CVS strain may limit CD3+ T cell mediated apoptosis of neuronal cells and thereby preserve the integrity of the neuronal network critical to virus spread. The role of apoptosis in natural RABV infections is less evident. Compared to the CVS strain, infection of mice with SHBRV resulted in a lower overall level of neuronal apoptotic cell death (Yan et al., 2001). If apoptosis is important for pathogenesis of street viruses, it may depend on the triggering of cell death by selected cell types.

2. The macrophage–monocyte lineage and inflammatory factors Nitric oxide (NO) is a free radical produced by the enzyme nitric oxide synthase (NOS) that exists in both constitutive (cNOS) and inducible (iNOS) forms. High levels of NO are cytotoxic and activation of iNOS in macrophages is regarded as an inflammatory mediator (Aktan, 2004).

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Up-regulation of iNOS in the RABV-infected rat brain was proposed to contribute to neuropathogenesis (Akaike et al., 1995) and indeed treatment of RABV-infected mice with a selective inhibitor of iNOS delayed viral replication, apoptotic cell death and death of the host significantly (Ubol et al., 2001). RABV virions can enter macrophages via endocytosis, and, despite limited virion replication, activate expression of iNOS as well as the CXC chemokine ligand 10 (CXCL10), a T helper cell type-1 chemoattractant (Nakamichi et al., 2004); thus increases in NO levels contribute to macrophage activation in response to RABV infection. The significance of these observations was further extended to microglia cells, the functional equivalents of macrophages in the CNS. RABV entry into microglia was followed, even in the absence of significant replication, by strong induction of the chemokines CXCL10 and CCL5 through activation of several signaling pathways (Nakamichi et al., 2005). Both an attenuated vaccine strain and a pathogenic dog strain induced maturation of immature dendritic cells and monocytes, apparently through the induction of IFN-a1 mRNA (Li et al., 2008). Consistent with other reports, the vaccine strain strongly up-regulated 26 genes involved in the NF- kB signaling pathway, including TLR3, TLR7 and STAT1, while the pathogenic strain elicited a much reduced response. Dendritic cells of the lymphoid tonsillar tissue, which comes into direct contact with oral vaccines, probably play an important role in eliciting immunity and attenuated vaccines may elicit protection, at least in part, by strong induction of the innate immune response at this site. The role of TNF-a, another pro-inflammatory cytokine produced by cells of the monocyte/macrophage lineage, was investigated using a recombinant RABV expressing a soluble form of TNF-a (Faber et al., 2005a). High levels of soluble TNF-a reduced RABV replication in neuroblastoma cells without inducing significant levels of apoptosis. Furthermore, unlike animals challenged with a wildtype (wt) virus, mice survived challenge with the TNF-a-expressing recombinant virus due to significant inflammatory mechanisms as well as a direct antivirus effect. Consistent with such observations, a prior study had suggested the protective effect of the p55 TNF-a receptor, a mediator of T cell protection, in RABV ocular disease (Camelo et al., 2001). In human cases, RABVinfected neurons induced expression of both TNF-a and iNOS in adjacent astrocytes or microglial cells but not in the neurons themselves (Nuovo et al., 2005).

3. Role of noncoding RNAs? Saha et al. (2006) observed that neurotropic viruses including RABV induce production of a novel noncoding (nc) RNA, designated VINC (virus inducible ncRNA). While the function of this nuclear-localized RNA remains obscure, this observation sets the precedent that RABV

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infection can alter the ncRNA complement of infected cells. Given the recent recognition of the role of microRNAs (miRNAs) in control of gene expression and the recent discovery of viral-encoded miRNAs that affect gene expression patterns in virus-infected cells (Qi et al., 2006), this suggests novel means by which RABV could control host cell expression.

D. Considerations for future studies on rabies pathogenesis While various experimental models of rabies, including the extensive use of laboratory strains of mice, provide important insights into the disease, many of the host’s transcriptional changes observed upon RABV infection likely represent common host pathways for countering infection by neurotrophic viruses (Saha and Rangarajan, 2003). Dissecting out those changes that are especially relevant to RABV pathogenesis will prove difficult although, based on the information now being collected, transgenic mice might yet play an important role in this regard. Furthermore, there are significant differences in the extent of gene activation and the cell types affected in mice depending on the RABV strain employed (Prosniak et al., 2003b). Street strains limit the response of type-1 IFN and inflammatory pathways far more effectively than avirulent strains, perhaps due in part to a lower level of G expression that limits TLR3associated activation of the innate immune system (Wang et al., 2005). An accurate appreciation of the pathogenic mechanisms operating in natural infections will require further studies employing street isolates inoculated into their normal reservoir hosts. Presently the lack of tools and reagents to study many of the processes described here in natural rabies hosts (e.g., dog, fox, skunk, raccoon, and bat species) impedes such work. Furthermore, it is evident that a full appreciation of RABV propagation and virulence requires not only an understanding of which host genes play pivotal roles but in which cell types these genes function. Studies that do not consider the heterogeneous cell population of the CNS may miss effects that are restricted to a particular cell type. Ultimately, a combination of immunohistochemical methods and in situ RT-PCR (see Nuovo et al., 2005) may prove invaluable for identifying the cell types and molecular mechanisms most critical to RABV pathogenicity in natural infections.

IV. REVERSE GENETICS—METHODOLOGY AND APPLICATIONS RABV was the first member of the Mononegavirales order to be successfully manipulated by reverse genetics to generate infectious, replicationcompetent virus (Schnell et al., 1994). Subsequent observations, that a

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foreign gene could be inserted within an intergenic region of the RABV genome without significant effect on in vitro replication of the virus, that this insert was translated into functional protein and that the recombinant RABV (rRABV) was genetically stable (Mebatsion et al., 1996), established the broad utility of this technology. Indeed the fact that the RABV genome can accommodate large increases in size (at least 55% more than wt RABV) (McGettigan et al., 2003b) and that reporter gene expression levels are dependent on insert orientation (Finke and Conzelmann, 1997, 1999), genomic position (Wu and Rupprecht, 2008) or the nature of the intergenic sequences (Finke et al., 2000) provides substantial flexibility in engineering novel and useful recombinants. Reverse genetics is now used for various applications, particularly to investigate RABV pathogenesis (see previous section) and to generate novel RV vaccines (Schnell et al., 2005). The RABV reverse genetics system was initially developed based on the SAD B19 genome sequence, but systems for other lab-adapted RABV strains, including RC-HL (Ito et al., 2001, 2003), HEP-Flury (Inoue et al., 2003), and ERA (Wu and Rupprecht, 2008), as well as the SHBRV-18 wildlife isolate (Faber et al., 2004), have now been described. As the technology has become more widely used, many methodological improvements have been developed. The original system employed host cells infected with a recombinant vaccinia virus expressing T7 polymerase to drive the transcription of the helper plasmids and the genomic cDNA. Use of host cell lines stably expressing T7 polymerase eliminated the need for the vaccinia construct and circumvented vaccinia virusinduced cytotoxicity and the requirement for removal of contaminating virus from the rRABV cultures (Buchholz et al., 1999; Ito et al., 2003). One such cell line facilitated the recovery of an RC-HL-derived rRABV containing two copies of the G gene (Hosokawa-Muto et al., 2006). Other improvements that increase rRABV/minigenome RNA yields have included construction of new helper plasmids that render the T7 RNA polymerase-transcripts cap-independent for translation (Ito et al., 2003) and the inclusion of ribozyme sequences at the termini of the cloned viral genome to permit generation of RNA with precise 50 and 30 genomic ends (Inoue et al., 2003; Le Mercier et al., 2002). A recovery system using expression plasmids bearing the cytomegalovirus (CMV) immediate early promoter in place of the T7 promoter, and which is transcribed by the host cell RNA polymerase II, yielded higher virus recoveries from multiple cell types compared to a comparable T7 promoter-driven system (Inoue et al., 2003). Use of both CMV and T7 promoters enhanced transcription efficiency and, using a T7 polymerase engineered to localize to the nucleus (Wu and Rupprecht, 2008), facilitated recovery of a recombinant ERA virus in which the order of the M and G genes was reversed; such a construct could not be recovered previously.

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A. RABV vaccines One application of this technology is to generate novel rabies vaccines. While the use of inactivated rabies vaccines that are safe and efficacious following parenteral administration in humans and animals is well established, logistical considerations dictate that oral vaccination will remain the primary means of controlling sylvatic rabies (Rupprecht et al., 2006). Only live attenuated vaccines have to date been proven effective via the oral route. There is continued interest in developing live attenuated RABV vaccines that are safer and more efficacious than the ERA/SAD B19 strains currently used for oral vaccination of foxes in Europe and North America and, in particular, vaccines that are efficacious in other wildlife reservoirs. A common approach is to engineer viruses that express higher levels of G protein that in turn elicits production of protective virus neutralizing antibody (VNA). A recombinant ERA, in which the order of the M and G genes was rearranged so as to increase G protein expression, was less virulent in mice than parental ERA (Wu and Rupprecht, 2008). A rRABV derived from the RC-HL strain carrying two copies of the wt G gene had increased levels of G protein in virions and in infected cells; this double G virus remained pathogenic in mice but after inactivation it was significantly more immunogenic than the parent virus and may be useful as an inactivated rabies vaccine (Hosokawa-Muto et al., 2006). SAD B19-derived constructs, in which the G gene was mutated to encode either a Glu or Gln at position 333 instead of Arg, had greatly reduced pathogenicity in mice and elicited VNA upon intramuscular (i.m.) and oral inoculation that afforded protection from lethal challenge (Faber et al., 2002; Morimoto et al., 2001). A rRABV containing two copies of the Glu333-mutated G gene (SPBNGA-GA) resulted in significantly increased G protein expression and hence elicited significantly higher VNA titers in mice and improved survivorship after challenge over the construct (SPBN-GA) with one G gene copy (Faber et al., 2002). Pulmanausahakul et al. (2001) found that a rRABV encoding the proapoptotic protein cytochrome c (SPBN-Cyto c(+)) had markedly reduced pathogenicity and increased immunogenicity in mice following intranasal administration when compared to a rRABV carrying an inactive cytochrome c gene. It was subsequently shown that SPBNGA, SPBNGA-GA, and SPBN-Cyto c(+) viruses could be grown to high titer and were genetically stable at position 333, important considerations for candidate vaccines (Dietzschold et al., 2004). However, serial passage through neonatal mice resulted in a secondary mutation, Asn to Lys at position 194, in some GA genes which led to an increased pathogenicity. Interestingly, only one GA gene copy of SPBNGA-GA acquired this mutation and this was insufficient to increase pathogenicity (Dietzschold et al., 2004).

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The utility of these constructs as oral vaccines has been examined in species other than laboratory rodents. SPBN-GA, SPBNGA-GA, and SPBN-Cyto c(+) administered per os caused no adverse effects in beagles; the majority of vaccinates seroconverted within 1–2 weeks postvaccination and all vaccinates survived i.m. challenge with street virus of canine origin (Rupprecht et al., 2005). The safety and efficacy of rRABV with one or two copies of the G gene carrying the Glu333 mutation and an additional Asn to Ser mutation at position 194 (SPBN-GAS and SPBNGASGAS) following oral vaccination in African mongooses and raccoons was explored. No adverse effects were observed in either species. SPBNGAS induced VNA and protected African mongooses from lethal challenge (Blanton et al., 2006). Only two out of five raccoons vaccinated with SPBN-GAS developed VNA and survived challenge; all five raccoons immunized with SPBN-GASGAS survived challenge despite detection of VNA in only two vaccinates (Blanton et al., 2007), consistent with the observation that rRABV overexpressing G protein had increased immunogenicity in mice (Faber et al., 2002). In a different approach to vaccine development, rRABV completely apathogenic for neonatal mice following intracranial inoculation were produced by deletion of either the P gene (Shoji et al., 2004) or the M gene (Ito et al., 2005). Progeny virions could be recovered from cells providing the missing proteins in trans and were able to infect cells normally susceptible to RABV; however, infection did not spread beyond the initially infected cell. When given to mice by either intraperitoneal (i.p.) or i.m. inoculation, the P-deficient rRABV elicited VNA in a dosedependent manner and protected against lethal intracranial challenge with CVS (Shoji et al., 2004). Similarly, i.m. and intranasal inoculation with the M-deficient rRABV elicited VNA production, but protection against challenge was not determined (Ito et al., 2005). While these preliminary studies in various animal models have examined rRABV immunogenicity and, in some cases, efficacy, evaluation of the optimal dose, duration of immunity, safety and efficacy of these candidate vaccines administered in a bait format appropriate for wildlife vaccination is needed before field application can be considered.

B. Vaccines for other diseases The use of rRABV vectors for development of vaccines against other diseases, especially for acquired immunodeficiency syndrome (AIDS), has also been explored. A chimeric protein comprising the envelope (Env) protein’s extracellular domain of human immunodeficiency virus type-1 (HIV-1) and the RABV G protein’s transmembrane and cytoplasmic domains was expressed by a rRABV construct, incorporated into

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virions, and exhibited many of the biological properties of wt Env (Foley et al., 2002; Schnell et al., 2000). The resulting viruses exhibited a cellular tropism similar to that of HIV-1. Similarly, replication-competent rRABVs containing the HIV-1 gag gene within the G/L intergenic region (McGettigan et al., 2001b) or the N/P intergenic region (McGettigan et al., 2003a) were recovered and expressed functional Gag product following infection of human cells. These results suggested that rRABV might be a suitable vaccine vector for HIV-1. A strong neutralizing antibody response to the Env-derived gp120 subunit was elicited if mice received an i.m. immunization with rRABV expressing HIV-1 Env protein and were subsequently boosted with recombinant gp41 and gp120, indicating that the HIV-1 Env-pseudotyped rRABV could prime the immune system for production of Env-specific neutralizing antibody (Schnell et al., 2000). After a single i.p. inoculation with the same rRABV, a long-lasting CD8+ cell mediated cytotoxic T lymphocyte (CTL) response directed against multiple HIV-1 Env epitopes was induced (McGettigan et al., 2001a). Similarly, rRABV expressing HIV1 Gag could elicit CD8+ cell-dependent Gag-specific CTL responses (McGettigan et al., 2001b, 2003a). A rRABV expressing both HIV-1 GagPol and an HIV-1 Env/RABV G chimera was generated (McGettigan et al., 2003b) and produced correctly-processed functional proteins. The demonstration, that development of an immune response to the vesicular stomatitis virus (VSV) glycoprotein ectodomain was not impaired by prior immunization with an identical RABV vector expressing a wt RABV glycoprotein, suggested that a heterologous prime/boost approach might be a useful strategy for foreign antigens presented in rRABV vectors (Foley et al., 2000). Priming with an RABV/HIV-1 Env recombinant followed by boosting with a heterologous rhabdovirus vector (VSV/HIV-1 Env) resulted in robust humoral and CTL responses in mice (Tan et al., 2005). A robust cellular response against the HIV antigen was achieved in macaques given a primary immunization with RABV/HIV-1 Env and secondary immunization with a rRABV expressing the HIV-1 Env as well as a chimeric VSV G with the RABV G cytoplasmic domain in place of the wt G protein (McKenna et al., 2007). Furthermore, this prime/boost approach was able to protect macaques against challenge with a pathogenic simian–HIV strain. In a different approach to modifying immune responses to HIV-1 antigens, McGettigan et al. (2006) produced RABV vectors co-expressing HIV proteins and murine cytokines. Compared to the parental RABV vector expressing HIV-1 Gag or Env, co-expression of IL-4 decreased the cellular immune response and abrogated the serological response to Gag and Env, whereas co-expression of IL-2 induced strong cellular responses and induced seroconversion against Env after two inoculations (McGettigan et al., 2006).

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Inactivated RABV particles can serve as immunostimulatory carrier molecules for display of B cell antigens. Green fluorescent protein (GFP) presented as a fusion protein with the RABV nucleoprotein induced a strong antibody response against GFP in mice, whereas recombinant GFP or a combination of wt RNP with recombinant GFP did not (Koser et al., 2004). Mice inoculated i.m. with either killed or live rRABV expressing a fusion protein of RABV G protein and the PA domain-4 of Bacillus anthracis developed anti-PA antibodies after a single inoculation and robust anamnestic responses to boosts with the same rRABV (Smith et al., 2006). Immunization of mice with live rRABV expressing the hepatitis C virus E2 envelope protein as a fusion with the RABV G cytoplasmic domain did not induce specific antibody; however, animals boosted with killed recombinant virus as a source of recombinant E2 did mount a potent humoral immune response (Siler et al., 2002). A single injection with RABV-E1E2, induced a long-lasting antigen-specific cellular immune response. Faber et al. (2005c) showed that a single i.m. inoculation of mice with rRABV expressing the SARS-CoV virus envelope spike protein (S) gene induced a strong SARS-CoV-neutralizing antibody response while the same RABV vector expressing the SARS-CoV nucleocapsid protein (N) did not elicit production of any N-specific antibodies, suggesting that the nature of the immune response to antigen introduced via RABV-derived vectors is antigen-dependent. While these preliminary studies show that antigen presented in the context of RABV can stimulate both humoral and cellular immune responses, further studies will be required to determine the quality of these responses vis-a`-vis protection from virulent challenge.

V. OTHER STRATEGIES FOR RABIES VACCINE DEVELOPMENT Further vaccine development is currently targeting two main rabies reservoir groups: wildlife species not readily immunized using current oral vaccines and dogs in the developing world, which require stable, safe, and inexpensive products given either by the oral or parenteral route. Two prominent strategies which have sought to address these deficiencies are described.

A. Adenovirus recombinants While the vaccinia-rabies glycoprotein recombinant V-RG was, until recently, the only heterologous recombinant vaccine employed for rabies control in the field (Brochier et al., 1991), the potential utility of various adenovirus recombinants has been explored. Constructs which have been generated include those based on human adenovirus type-5 (HAd5),

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using either E3-deleted (Yarosh et al., 1996) or E1-deleted vector backbones (Vos et al., 2001; Xiang et al., 1996), chimpanzee adenovirus serotype 68 (AdC68) (Xiang et al., 2002) or canine adenovirus type-2 (CAV-2) (Hu et al., 2006; Li et al., 2006). The immunogenicity of many of these constructs was established in the murine model as well as in a number of other target species. An AdRG1.3 construct, originally described by Yarosh et al. (1996), and shown to be highly efficacious in skunks via the oral route (Knowles et al., manuscript in preparation) is now being assessed in a bait formulation in limited field trials for rabies control in skunks in Ontario, Canada (see http://www.mnr.gov.on.ca/MNR/csb/news/ 2006/aug18nr2_06.html). CAV-2 recombinant vaccines, based upon a live vaccine already employed in dogs, were immunogenic in mice (Li et al., 2006) and in dogs after subcutaneous inoculation (Hu et al., 2006). Oral administration in dogs elicited strong long-term serological responses and protected against challenge (Zhang et al., 2008) while protective serological responses in cats required i.m. administration (Hu et al., 2007). Further evaluation of some of these constructs for licensing purposes appears likely. One concern with use of adenoviral-based vaccines is that prior adenovirus infection might generate immunity to the vector and thereby limit the vaccine’s efficacy. Indeed, the anti-RABV G antibody response of mice given a HAd5RABG recombinant can be compromised by pre-exposure to the homologous adenovirus (Xiang et al., 2002). The use of vaccines based on several different adenovirus vectors that elicit limited serological cross-reactivity may be one means of overcoming such a limitation (Xiang et al., 2002).

B. DNA vaccines The stability and low cost of DNA vaccines makes them ideal candidates for use in the developing world. Proof of principle for this approach, which involves inoculation of the animal with DNA of a plasmid that directs expression of the RABV G protein under a strong viral promoter, was demonstrated in the mouse model some years ago (Xiang et al., 1994). Difficulties in transferring this technology to larger mammals were encountered, though its feasibility for eliciting strong immune responses in cats and dogs was reported by Osorio et al. (1999) and protection of beagles from challenge using a two-dose immunization schedule administered i.m. was reported shortly thereafter (Perrin et al., 2000). Evaluation of different methods of immunizing dogs using a single DNA vaccine dose suggested that intradermal (i.d.) application into the ear pinnae was the method of choice (Lodmell et al., 2003) and could protect dogs challenged 1 year later (Lodmell et al., 2006). Experimental and field trials undertaken in Tunisia, suggested that DNA immunization, administered by jet injector into the inner ear of local dogs, was more effective in

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eliciting long-lasting VNA and protection than a cell culture-derived vaccine (Bahloul et al., 2006). Intranasal application of a two-dose regimen of DNA vaccine has also been reported to induce a good immune response in dogs (Tesoro Cruz et al., 2006). An evaluation of DNA vaccines to immunize horses against rabies reported that formulation of the DNA with a cationic lipid, given as a two-dose regimen, elicited a good serological response (Fischer et al., 2003); the application of this technology in equines could potentially be very important in Latin America where transmission of vampire bat rabies to livestock continues to cause significant losses. Bahloul et al. (2003) also proposed a potential role for DNA immunization in PEP based on experiments in the mouse model where a single dose of DNA vaccine given the same day as a RABV challenge was more effective than five doses of cell culture-derived vaccine given over 28 days, though it must be pointed out that overall protection rates (53% and 40%, respectively) were low. The potential application of DNA immunization to humans was suggested by a study showing that a DNA vaccine appeared to be comparable to cell culture-derived vaccine in eliciting VNA and protection from challenge in macaque monkeys, although multiple applications of vaccine were necessary (Lodmell et al., 1998). A later study showed some protection from challenge in macaques 1 year after administration of a single DNA vaccine dose given either via gene gun or i.m., though protection was not complete and did not appear to correlate well with presence of VNA (Lodmell et al., 2002a). Another study suggested the superiority of i.m. over i.d. administration of DNA vaccines in nonhuman primates and demonstrated the need for a booster dose 6 months after the initial immunization to maintain adequate VNA levels (Biswas et al., 2001). Use of DNA immunization for PEP in monkeys was less effective, with a 50% protection rate compared to 75% using the human diploid cell vaccine, despite attempts to accelerate the VNA induction using gene gun vaccination into specific tissues (Lodmell et al., 2002b). Based on these data, the further development of DNA vaccines for human disease prevention, especially for PEP, is not currently advocated (Ertl, 2003). DNA vaccines have attempted to address the issue of vaccine coverage. Present vaccines, which are based on selected strains of GT 1, do not elicit production of antibodies that effectively neutralize members of the more divergent lyssaviruses, especially those of GTs 2 and 3. However, Jallet et al. (1999) described various DNA vaccines, comprising chimeric glycoprotein constructs generated from the G genes of the PV (GT 1), Mokola (GT 3), and EBLV-1 (GT 5) lyssaviruses, which broaden the spectrum of protection compared to the homologous sequences. Moreover, attempts to generate a DNA vaccine against Mokola virus using the Mokola G gene inserted into several different plasmid vectors showed some promise in mouse models (Nel et al., 2003). Since very few reported

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rabies cases are due to lyssaviruses other than those of GT 1, a significant rise in the number of such cases would be needed to drive further commercial development of such vaccines; the recent emergence of several divergent lyssaviruses (see Section II.B) may result in greater attention to this issue in the future. Although there are still some safety concerns with DNA vaccines, for example, genome integration, overall these vaccines are considered generally safe and free of adverse reactions. New approaches for vaccine delivery (e.g. electroporation) appear to be overcoming the limited potency observed for first generation products, especially in larger mammals and humans (Ulmer et al., 2006). Thus, future licensing of some of these products for veterinary applications may proceed, especially if efficiency gains through the use of multi-cistronic DNA vaccines (see Patial et al., 2007) can be realized.

VI. THE CHALLENGE OF RABIES BIOLOGICS FOR PASSIVE IMMUNITY Effective rabies PEP of humans requires not only an efficacious vaccine but passive immunity applied to the wound area that can limit virus propagation and spread until an adaptive immune response develops. Passive immunity is normally supplied as human (HRIG) or equine rabies immunoglobulin (ERIG) preparations (Rupprecht et al., 2005) but the limited supply of these reagents often results in incomplete and ineffective PEP. Consequently, the development of alternative preparations comprising human Mabs has become a priority. Since a recent review has summarized most activities in this area (Nagarajan et al., 2008), only a few salient points are presented here. The anti-G Mabs chosen for this application should be virus neutralizing and should bind to well conserved G protein epitopes to ensure as broad a range of cross-reactivity as possible (Champion et al., 2000; Sloan et al., 2007). The divergent nature of many of the nonGT 1 lyssaviruses should be taken into consideration in this regard (Hanlon et al., 2005). Moreover, to prevent inadvertent selection of RABV escape mutants by such preparations, more than one G protein epitope should be targeted and hence, a Mab cocktail containing two or more Mabs is desirable (Bakker et al., 2005; Marissen et al., 2005; Prosniak et al., 2003a) and safety and efficacy similar to that achieved with HRIG should be demonstrated (Goudsmit et al., 2006). While such reagents can be produced in cell culture systems, production of functional Mabs in plant based systems has been proposed as a cost-effective alternative (Ko et al., 2003).

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VII. NOVEL APPLICATIONS OF RABV A. Use as a neuronal tracer The mammalian brain is composed of a series of neural networks and pathways, and knowledge of the interconnectedness of these networks is essential to an understanding of how the brain functions. Fine mapping of CNS neuronal pathways at the cellular level requires the use of neuronal tracers and although a variety of chemical compounds have been employed for such purposes (reviewed in Ko¨bbert et al., 2000), these are limited due to nonspecific cell uptake and dilution effects that restrict their usefulness to cells immediately adjacent to those of the injection site. To overcome such limitations, neurotropic viruses such as herpes simplex virus-1 (HSV-1), pseudorabies virus (reviewed in Loewy, 1998), and more recently, RABV were investigated for their utility in mapping neuronal pathways. Viral replication in infected cells naturally amplifies its signal and allows mapping of higher-order neural connections. Despite their advantages, tracer studies using a-herpesviruses can be confounded due to virus-induced cytotoxicity and the generation, under certain conditions, of spurious labeling by local and nonspecific spread between cells (reviewed in Norgren and Lehman, 1998). RABV is particularly useful for studying motor networks as it exclusively infects motoneurons, rather than sensory or sympathetic neurons (Graf et al., 2002; Kelly and Strick, 2000; Tang et al., 1999; Ugolini, 1995) and it propagates by transneuronal transfer (Astic et al., 1993; Coulon et al., 1989; Kucera et al., 1985; Lafay et al., 1991). Following stereotactic injection of virus into specific nerves, muscles or brain areas of the test subject and a defined incubation time, the animal tissue is perfused with fixative, sectioned, and finally stained to determine the distribution of infected cells. In theory, by adjusting the survival time following RABV inoculation, an unlimited number of serially connected neurons can be visualized and precise mapping of neuronal networks and connections is possible. Model systems thus employed include primates, rats, guinea pigs, and adult and neonatal mice. Since the rate of RABV transport can vary depending on the virus preparation—variant, passage history, size of inoculum—as well as on the animal species (Kelly and Strick, 2000; Ugolini, 1995), the time course of labeling must be established for each model system to allow determination of the sequential order of neurons in a pathway. While the CVS variants (CVS-11, -26, -N2c and -B2c) employed as neuronal tracers are fixed viruses, with various levels of pathogenicity, tracing studies are usually sufficiently short such that the test subjects do not develop clinical rabies. The potential use of RABV as a neuronal tracer was established using a well-characterized motoneuron network, the hypoglossal system, which

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showed that RABV could label all main groups of second-order neurons and higher order hypoglossal-related neurons without cytotoxicity (Ugolini, 1995). There was no lateral spread of RABV at the site of inoculation, either in muscle (Morcuende et al., 2002) or in the brain (Astic et al., 1993; Ugolini, 1995) and spread was strictly transneuronal occurring exclusively at synaptic connections (Kelly and Strick, 2003). RABV was transported solely in the retrograde direction following intracortical and i.m. injection in primates (Kelly and Strick, 2000) and inoculation into the hypoglossal nerve (Ugolini, 1995) or the bulbospongiosus muscle (Tang et al., 1999) of rats. RABV has been used in two main types of investigation. First, inoculation of RABV into muscle cells has been used to identify the motoneurons that innervate the tissue, as well as second- and higherorder neurons that relay signals to the CNS; this includes studies of pathways controlling digit movements (Rathelot and Strick, 2006), the oculomotor system (Graf et al., 2002; Morcuende et al., 2002), the olfactory system (Astic et al., 1993), and respiratory networks (Gaytan et al., 2002; Viemari et al., 2004). Secondly, RABV has been injected into specific function-related areas of the brain to identify cells that are pre-synaptic to those initially infected, thereby revealing the neurons that project into these sites. A number of such studies have examined the organization of neuronal pathways and the connections between the primary motor cortex and the cerebellum or the basal ganglia (Clower et al., 2005; Kelly and Strick, 2004; Lu et al., 2007), and within the visual cortex (Nassi and Callaway, 2006). Use of RABV as a neuronal tracer in these systems has delineated connections to a level that was not previously possible using conventional tracers. These studies commonly indicate a higher level of complexity than anticipated. For example, in a guinea pig model considerable cross-talk was demonstrated between neuronal systems controlling horizontal and vertical eye movement with an unexpectedly large number of labeled interneurons in and around the oculomotor and trochlear nuclei proper and surprisingly, labeling of structures known to be involved in directional heading during navigation (Graf et al., 2002). Morcuende et al. (2002) mapped the neuronal premotor networks involved in eyelid responses in a rat model and developed a comprehensive picture of the pre-motor networks mediating reflex, voluntary, and limbic-related eyelid responses. This work also highlighted the potential sites for motor learning involved in classical eyelid conditioning. Using combined studies with a retrograde tracer (RABV) and an anterograde tracer (HSV-1) Kelly and Strick (2003) demonstrated that multiple closed loop circuits characterize cerebral–cerebellar interactions in the primate brain in contradiction to the traditional view that connections between these regions comprise a massive open loop system;

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rather the data suggested the existence of separate circuits for cognitive and motor operations. Another study challenged the traditional view of connections between cortex, basal ganglia, and cerebellum. By exploring outputs to the anterior intraparietal area (AIP), which responds to sight of objects as well as to the act of grasping them, from both the cerebellum and basal ganglia, Clower et al. (2005) were the first to demonstrate an anatomical pathway linking basal ganglia output with the parietal lobe in primates. Basal ganglia input into the AIP may provide an anatomical explanation for poorly understood nonmotor (visuomotor and visuospatial) deficits observed in Parkinson’s disease patients. Distinct, closed-loop circuits between cortex and sub-cortical areas were also demonstrated in RABV tracing experiments that examined the basal ganglia and cerebellar inputs to the supplementary motor area (SMA) and the pre-supplementary motor area (pre-SMA) (Akkal et al., 2007). Together with previous studies demonstrating that the SMA and pre-SMA neurons project to separate regions of the basal ganglia (Inase et al., 1999), these observations suggest that the SMA and the pre-SMA are nodes in distinct neural systems that form separated closed-loop circuits with the basal ganglia. The observation that the basal ganglia, rather than the cerebellum, provided the dominant input to these two nodes may provide an anatomical explanation for why cerebellar hyperactivity in Parkinson’s disease does not normalize SMA activity. Hoshi et al. (2005) demonstrated an anatomical pathway that directly links the output stage of cerebellar processing to the input stage of basal ganglia processing. This novel observation implies that the cerebellum may be able to adjust basal ganglia activity and suggests a role for the cerebellum in motor and cognitive functions normally associated with basal ganglia dysfunction. In many cases, the maps generated by RABV retrograde tracing have provided an anatomical explanation to support pathways or functional models established using other methodologies. Gayta´n et al. (2002) used RABV tracing to characterize the neuronal networks projecting to the main respiratory motoneurons in adult mice, work which provided a basis for the neural network that integrates respiratory-related activities in complex behavioral responses. RABV tracing confirmed the involvement of A6 neurons in development of a normal respiratory rhythm in mice (Viemari et al., 2004) and also resolved an ongoing controversy by showing that cortico-motoneuronal (CM) cells affecting digit muscles are found in only one area of the primary motor cortex but those for each muscle have a widespread distribution and overlap with the area known to contain shoulder representation, a feature that may allow a wide range of muscle synergies to occur (Rathelot and Strick, 2006). Traditionally, immunohistochemical or immunofluorescence methods were used to trace RABV progression through neuronal networks while

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infected cells were identified to type either by morphology or by doublestaining for cell-specific markers (Tang et al., 1999). Reverse genetic techniques have permitted the development of novel RABV tracers labeled with endogenously-expressed fluorescent proteins. RABV particles expressing a P protein/enhanced GFP (eGFP) fusion product were generated and used to track binding of virus to cells and virion internalization in vitro (Finke et al., 2004). More recently, double-labeled virions bearing two distinct fluorescent protein tags fused to P and G proteins were generated (Klingen et al., 2008) thereby permitting distinction of naked RNP from intact, enveloped viruses. Using neuronal cell cultures, these viruses demonstrated a role for RABV glycoprotein in virus particle transport within the neuron. While their utility for in vivo studies has yet to be tested, such viruses may allow real-time tracking of RABV infection. Virus particles, in which the G gene was replaced by that encoding eGFP, could be generated by complementation with a G proteinexpressing plasmid; these virions infected neurons and replicated their core components, but could not spread beyond the initially infected cells (Wickersham et al., 2007a). Such infected neurons expressed high levels of eGFP thus permitting a detailed study of the morphology and physiology of neurons at injection sites in the brain. RABV-eGFP was found to be superior to another monosynaptic retrograde tracer, HIV-1 pseudotyped with RABV G protein. Further refinements have been made to this system, in which RABV-eGFP was pseudotyped with the envelope protein EnvA from the subgroup A avian sarcoma and leucosis virus. RABVeGFP/EnvA can infect only those cells expressing the EnvA receptor, the gene for which is introduced by electroporation, and can spread monosynaptically to projecting cells if the RABV G is also provided in trans (Wickersham et al., 2007b). In this way, neurons directly pre-synaptic to the EnvA receptor-expressing cells can be unambiguously identified. This system is novel and powerful in that for the first time, it provides a tool for identifying neurons that are monosynaptically connected to a cell group, or even a single cell, and can distinguish weak, direct connections from strong, indirect ones, features which sometimes can confound interpretation of results using conventional RABV trans-synaptic tracers. While this system presently remains at the proof of principle stage, its implementation in vivo appears possible. While the expression of fluorescent proteins by RABVs will clearly extend their utility in physical mapping of neuronal pathways, expression of markers other than eGFP, such as sensors of neural activity or photosensitive ion channels, presents the possibility of using these recombinant viruses for physiological studies of neuronal function, thereby opening up new avenues of investigation.

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B. Use of RABV proteins for molecular targeting RABV proteins and modular functional domains derived from them have been used to target the delivery of molecules to specific cells or sites within a cell. Replacement of endogenous envelope proteins of retrovirus-derived vectors with RABV glycoprotein (‘‘pseudotyping’’) alters their cellular tropism, permitting the infection of neuronal cells in vitro and in vivo (Azzouz et al., 2004; Mazarakis et al., 2001; Mochizuki et al., 1998; Parveen et al., 2003; Teng et al., 2005). The utility of these pseudotyped virus vectors for transgene delivery to neurons in vivo has been investigated by incorporating genes encoding fluorescent proteins or b-galactosidase into the vectors, and determining the number and distribution of labeled cells (Mentis et al., 2006; Parveen et al., 2003). Transduction of neurons was observed following injection of the vectors into the CNS of rats and mice, as well as into muscles, indicating that the RABV glycoprotein facilitated retrograde transneuronal transport of the pseudotyped viruses. In addition, neurotrophic factors such as insulin-like growth factor I and the survival motor neuron gene product were efficiently delivered to and expressed in motor neurons in vitro (Teng et al., 2005) and in vivo (Azzouz et al., 2004). Targeting of genes to specific cell types will be critical to the development of gene therapies for treatment of degenerative diseases of the CNS and other neurological disorders. While full-length RABV glycoprotein targets pseudotyped viruses to neurons, delivery of small interfering RNA (siRNA) to neurons could be achieved using a synthetic peptide corresponding to the acetylcholine receptor binding site (ARBS) of the RABV glycoprotein extracellular domain (Kumar et al., 2007). Delivery of siRNA was achieved following both intracranial and intravenous administration of the ARBS-siRNA complexes, indicating that the complexes could be transported across the blood brain barrier. Furthermore, the siRNA was functional; expression of GFP in GFP transgenic mice, and endogenously expressed Cu–Zn superoxide dismutase, were both silenced by appropriate siRNAs and intravenous administration of an ARBSsiRNA targeting Japanese encephalitis virus was protective against fatal encephalitis in a mouse model of infection. While the mechanisms of ARBS peptide transport across the brain endothelium and detachment of the siRNA from the peptide in the cytoplasm remain to be delineated, use of the ARBS peptide for delivery of siRNA, and other small molecular weight therapeutics, to the CNS in a minimally invasive fashion appears most promising. Other functional domains of RABV proteins have been used to change the cellular distribution of recombinant proteins. Expression cassettes, in which cDNA for a protein of interest is fused in-frame between cDNA

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encoding the signal sequence and the transmembrane and cytoplasmic domains of the RABV G protein, direct production of recombinant proteins that are targeted to and remain anchored within the cellular membrane (Gupta et al., 1998; Klingen et al., 2008). This approach produced a membrane-anchored form of the b subunit of human chorionic gonadotropin (b (h)CG) that retained the antigenic epitopes of its secreted form (Gupta et al., 1998). Similarly, a red fluorescent protein (RFP) was efficiently targeted to the plasma membrane in cells transfected with an RFPglycoprotein construct (Klingen et al., 2008). Nuclear accumulation of various chimeric proteins, mediated by a NLS from RABV or other viruses, was enhanced by the presence of the RABV P protein LC8 BD (Moseley et al., 2007b) a finding that may provide for improved nuclear targeting of therapeutics.

VIII. CONCLUDING REMARKS Despite the many advances in our understanding of rabies–host interactions, and the ability to engineer RABV to produce novel constructs useful for many applications, clinical rabies in humans remains virtually 100% fatal. Even if current approaches to eliminate rabies from known carnivore reservoirs were eventually successful, the occasional cryptic infection of humans from chiropteran reservoirs (Messenger et al., 2002), which are generally not targeted by any comprehensive control strategies at present, will continue to drive the need for effective therapy. Indeed, much excitement surrounded the recovery of a patient from Wisconsin who survived clinical rabies without ever receiving antirabies biologics. The therapy applied in this case involved induction of coma and provision of antiviral drugs and agents to combat neuronal damage while the patient developed an adaptive immune response (Willoughby et al., 2005). However, several attempts to replicate this success failed (Christenson et al., 2007; Hemachudha et al., 2006; Schmiedel et al., 2007), perhaps either due to differences in pathogenicity of the RABV strains involved or variations in patient care, particularly prior to the diagnosis of rabies. To establish the merit of this approach, further experimentation using various animal and nonhuman primate models may be required. In a mouse model opening of the blood–brain barrier to allow infiltration of immune effectors appears to be a critical feature for preventing a lethal rabies outcome (Roy and Hooper, 2007; Roy et al., 2007). As well, future development of novel antiviral therapies (Real et al., 2004) may greatly improve the odds of surviving this most lethal of diseases.

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INDEX

A Acetylcholine receptor binding site (ARBS), RABV glycoprotein, 235 AIDS vaccine, rRABV vectors, 225–226 Alternate reading frame proteins (ARFPs), 61–62 ANK-repeats and poxvirus host genes, 161–162 sequences and M-T5 gene, 156 Antigen presenting cells (APCs), MV infection, 182, 194 Anti-IFN evasion, E3L gene, 151 Apolipoprotein D (ApoD), lipid recycling, 219 Apoptosis, polioviral proteins, 15 B Bahloul, C., 229 Bartenschlager, R., 101 Barth, H., 63 Bartosch, B., 63 Bodian, D., 3, 6–8 B5R gene, VACV strain, 148–149 Brzo´zka, K., 217 C Cai, Z., 87 Camelpox virus (CMLV), 144–145 Canine distemper viruses (CDV), 175 DTH response and leukopenia, 177 tropism of, 179 CD150 and CD46, MV receptor, 178–180 cDNA clone development and HCV, 71–73 Cell culture derived HCV (HCVcc), JFH1 cell culture system adaptive mutations, 83 applicability of, 92–94 cytopathic effect of, 83–84 growth characteristics, 91–92 Huh7 cells in, 82–83 lipoproteins role in, 89–91 pJ6CF clone and, 87–89

Central nervous system (CNS) and blood-brain barrier, 10 lansing virus role in, 22 paralytic poliomyelitis, 13–14 pathological lesions, distribution of, 14 poliomyelitis provocation, 7–10 poliovirus spread in, 3–4 and viremia, 8 Challenge virus standard (CVS) strain, rabies, 214 Chinese hamster ovary (CHO) cell line, 146 Chiroptera, lyssavirus genotypes, 211 CHOhr gene CPXV protein replication and, 146 VACV, apoptosis and, 147 Claudin-1, 62 Clower, D.M., 233 Cocquerel, L., 63 Combination therapy, HCV, 53, 56 Cosset, F.L., 63 Cowpox virus (CPXV), 146–147 Crotty, S., 25 Cullin-1/SCF complex, M-T5 gene, 156–157 CXC chemokine ligand 10 (CXCL10), RABV infection, 221 D Davis, P.L., 212 DC-SIGN lectins and L-SIGN, 80 MV receptor, 179, 181–182, 191 Delayed-type hypersensitivity (DTH), MV infections, 176–177 Dendritic cells (DCs), MV infection, 182 DC-SIGN and TLR2 in, 191–192 MV-protein production and virus release, 192–193 phenotypic maturation of, 193 TLR signaling modulation on, 194 viral interference in, 190 Diedrich, G., 63 DNA vaccines, RABV infection, 228–230 Dolphin morbilliviruses (DMV), 175 Dynein LC8 Interaction, lyssavirus, 216–217

251

252

Index

E Ectromelia virus (ECTV), SPI-1 gene, 143 E1/E2 envelope glycoprotein, 62, 79–80 E3L gene, VACV MVA life cycle, 151 protein synthesis inhibition and, 149–150 Z-DNA binding domains in, 150–151 Encephalomyocarditis virus (EMCV), 74, 76, 152 Endoplasmic reticulum retention signal, M-T4 gene, 155 Enhanced green fluorescent protein (eGFP), dendritic cells, 180, 190–191 Equine rabies immunoglobulin (ERIG), RABV infection, 230 ER membrane proteins, 64–66 European bat lyssavirus type-1 (EBLV-1), RABV, 212 European HCV database (euHCVdb), 55 Evelyn Rokitnicki Abelseth (ERA) vaccine strain, RABV pathogenesis, 220 F Faber, M., 227 Fas Ligand (FasL), T cell apoptosis, 220 F-box motif, M-T5 gene, 156 Fibroblast (NIH3T3) cell lines, HCV, 101 Flaccid paralysis lansing strain, 22 wild polioviruses, 23 Flaviviridae family, 53, 57 Foy, E.M., 79 Fusion protein heterodimer (F) protein, MV glycoprotein complex, 184 G Gale, M. Jr., 79 Gayta´n, S.P., 233 Glycoprotein, RABV, 214–216 Gromeier, M., 8 H HCV envelope proteins (HCVpp), pseudo particles, 79–82 Hemagglutinin (H) protein, MV glycoprotein complex, 184 Hepatitis C virus (HCV) buoyant density analysis, 89–91 cDNA clone development of, 71–73 cell culture systems

HeLa cells and, 101 H77-S genome and, 99 Huh7 and derived, 100 JFH1-based recombinants, 95–98 life cycle, 85–87 tissue tropism and, 102 viral titers in, 95 core proteins, 61 E1/E2 envelope glycoprotein, 62–63 genetic heterogeneity combination therapy sensitivity of, 56–57 genotypes of, 55–56 intra/intergenotypic recombination in, 57–59 genomic organization of, 58–60 host cell factors infectivity and, 68–70 replication and, 67 infection of, 53–54 kissing loop interaction of, 60 NS2/NS3 autoprotease, 64–65 NS4/NS5 proteins, 65–66 p7 protein, 63–64 pseudo-particle systems, 79–82 replicon system cellular protein modification in, 76 effects of, 74 host cell permissiveness and, 78–79 Huh7 cells and, 74–75 infectious particle formation, 77 replication regulation and, 77–78 rRABV vectors, 227 sequence databases, 55 therapeutic targeting studies, 103–104 High-density lipoprotein (HDL) and HCV, 62–63, 80 Hoshi, E., 233 Hsu, M., 80 Human cervix carcinoma (HeLa) cells and HCV, 83, 101 Human hepatoma cell line (Huh7), 78–79 Human rabies immunoglobulin (HRIG), 230 Hypervariable region 1 (HVR 1), HCV, 62–63 I IL-12 production, MV modulation, 194 Immune response and poliovirus, 15–17 Inducible nitric oxide synthase (iNOS), RABV infection, 220–221

Index

Inflammasomes, M13L gene, 158 Innate immunity, RABV infection, 218–219, 222 attenuated vaccines, 221 G protein, 215 P protein, 217 Interferon sensitivity determining region, HCV, 65 Internal ribosomal entry site (IRES), HCV, 60 J Jallet, C., 229 Janus kinase-signal transducer and activator of transcription (JAK/STAT), IFN expression, 217 Japanese hepatitis virus database (jpHCVdb), 55 J6CF genome, HCV, 92 JFH1 cell culture system, HCVcc adaptive mutations, 83 applicability of, 92–94 cytopathic effect of, 83–84 growth characteristics, 91–92 Huh7 cells in, 82–83 lipoproteins role in, 89–91 pJ6CF clone and, 87–89 J6/JFH1 life cycle, 68 Jubelt, B., 14 K Kassis, R., 218 Kelly, R.M., 232 K1L, VACV host gene expression of, 144 interferon activation and, 145 L Landsteiner, K., 3 LC16m8, VACV variant, 148 Leukopenia, MV infections thymocyte loss, 181 TREC and APCs in, 182 Lindenbach, B.D., 87, 89, 94 Lyssavirus, 208 DNA vaccines, 229–230 dynein LC8 interaction, 216 genotype evolution, 211–212 phylogenetic analysis of, 211 P75NTR interaction, 215 RRT-PCR detection of, 210

253

M M and L protein, RABV infection, 218 McGettigan, J.P., 226 Measles virus (MV), 175 dendritic cells interaction with, 190–194 F/H glycoprotein complex, 188 infection of, 177–178 leukopenia thymocyte loss, 181 TREC and APCs in, 182 leukopenia and DTH response, 176 PI3K/Akt kinase pathway in, 188–189 receptors interacting with, 179, 187 T cell silencing in, 183–186 tropism of, 177–180 Modified vaccinia Ankara (MVA) virus E3L deletion and life cycle of, 151 VACV K1L gene and, 144–145 Molluscum contagiosum virus (MCV), host restriction, 160 Mononegavirales, 222 Morbilliviruses, 174–176 genome organisation, 175 human and animal viruses of, 175–176 immunosuppression in, 177 leukopenia and, 181–183 spread of, 177–178 structural proteins and viral entry, 174 T cell silencing, 183–186 Morcuende, S., 232 Morimoto, K., 219 Most recent common ancestor (MRCA), North American bat RABVs, 212 Mouse hepatocyte (MMH, AML12) cell lines, 101 mRNA-associated protein ELAVL1 (HuR), 67 Mueller, S., 11 MV-glycoprotein complex, 184–186 Myxomatosis and MYXV, 153–155 Myxoma virus (MYXV), host genes M063, 159–160 M11L, 157–158 M13L, 158–159 M-T2, 154–155 M-T4, 155 M-T5, 155–157 N Nathanson, N., 8 Nerve growth factor receptor (P75NTR), 214–215

254

Index

Neural cell adhesion molecule (NCAM), 214 Neuroleukin (NLK), lipid recycling, 219 NS2/NS3 autoprotease and protein cleavage, 60, 64 N-Terminal nuclear export signal (NES), lyssavirus, 216 Nucleic-acid sequence based amplification (NASBA) method, rabies, 210 O Old dog encephalitis (ODE), MV infections, 186 Open reading frame (ORF) HCV replication, 60 and RBV, 213 Oral poliovirus vaccine (OPV), immunization, 16 and poliovirus fecal shedding of, 20 wild poliovirus and, 30 Orthopoxvirus, host genes B5R, 148–149 CHOhr, 146–147 C7L, 145–146 E3L, 149–152 K1L, 143–145 K3L, 152 p28/N1R, 147–148 SPI-1, 140–143 Osorio, J.E., 228 P Passive immunity, RABV infection, 230 Peripheral benzodiazepine receptor (PBR), M11L, 157 Peste des petits ruminants virus (PPRV) and Phocine distemper viruses (PDV), 175 Phosphatidyl-inositol-3-kinase (PI3K)/Akt kinase pathway, 188–189 Phytohemagglutinin (PHA), 177 Pietschmann, T., 96, 98 pJ6CF clone, HCV, 54, 87, 92 p28/N1R RING proteins murine macrophages and, 148 ubiquitin ligase activity in, 147–148 zinc-finger protein, 147 P75NTR binding, RABV infection, 214–215 Poliomyelitis acute flaccid paralysis, 21–22 animal model study of, 21–26 capsid protein VP1 role in, 23

pantropic and neurotropic attributes, 27–28 passive antibody and, 19 pathogenesis of, 3 provocation axonal transport, 10–11 CNS, impact on, 7–10 replication in glial and ependymal cells, 23–25 spinal cord transfection, 22–23 tropism role in, 26 Poliovirus CD155-mediated endocytosis, 11 eradication of, 37–38 infection immunization and circulating antibodies, 16–17 primary and secondary types, 17–18 sequential events in, 3–4 intramuscular replication and spread, 9 localization mechanism and, 7 necrosis and, 15 and neural pathways, 23–25 replication sites, 4–6 retrograde axonal transport, molecular mechanism for, 12 serotypes of, 20–21 species-specific expression of PVR and, 11 tropism of, 5, 26 virulence, 26, 29 epidemiological properties of, 33–34 neurovirulence mechanism in, 32 viremia and, 32–33 wild poliovirus vs. OPV, 30 Poliovirus receptor (PVR) apoptosis role and, 15 CNS localization and, 13–15 and mRNA expression, 11–13 pantropic polioviruses behavior in, 25 transgenic mouse model, 23–25 Polypyrimidine tract binding protein 1 (PTBP1), 67 Popper, E., 3 Porpoise morbilliviruses (PMV), 175 Postexposure prophylaxis (PEP), rabies, 208 Post-polio syndrome, 35–36 P protein and HCV, 60 RABV infection and, 217 Preligand assembly domain (PLAD), M-T2 gene, 154

Index

Promyelocytic leukemia (PML) gene, RABV infection, 217 Pseudo-particles expression, HCVpp, 79–82 Pulmanausahakul, R., 224 Pyrin domain (PYD) proteins, M13L gene, 158 R Rabbitpox virus (RPXV), SPI-1 gene, 140 Rabies Section of the Centers for Disease Control and Prevention (CDC), 209 Rabies virus (RABV) genotype evolution, 211–212 glycoprotein, molecular targeting, 235–236 infection, 208 adenovirus recombinant vaccine, 227–228 and apoptosis, 219–220 diagnosis of, 209–210 DNA vaccine, 228–230 dynein LC8 interaction in, 216–217 G protein in, 215–216 and interferon pathway, 217 M and L protein in, 218 passive immunity, 230 pathogenesis of, 214 P75NTR binding of, 214–215 reverse genetics, 222–223 rRABV vectors, 225–227 spatial model of, 213 TNF-a in, 221 VINC, 221–222 viral typing, 210–211 neuronal tracer, 231–234 nucleoprotein, immune stimulatory carrier, 227 phylogenetic analysis, 210–211 postexposure prophylaxis, 208 vaccines adenovirus recombinants, 227–228 DNA immunization, 228–230 live attenuated, 224–225 reverse genetics system in, 223 Racaniello, V.R., 14, 25 Randall, G., 67, 71, 93 Receptor binding domain (RBD), E2 envelope glycoprotein, 62 Ren, R., 14, 25 Replicon system, HCV cellular protein modification in, 76 characterization of, 74–76

255

effects of, 74 host cell permissiveness and, 78–79 Huh7 cells and, 74–75 infectious particle formation, 77 replication regulation and, 77–78 Respiratory syncytial virus (RSV), 185, 187, 191 Retinoic acid-inducible gene-I (RIG-I), 78 Rhabdoviridae family, 208, 213 Ribonucleoprotein particle (RNP) complex, 174–175 Rinderpest virus (RPV), tropism of, 179 RING-containing proteins, 147–148 RNA-dependent RNA polymerase (NS5B) and HCV genetic heterogeneity, 57 RNAinduced silencing complex (RISC), 68 rRABV vectors AIDS vaccine, 225–226 hepatitis C and SARS-CoV virus, 227 S Sabin, A.B., 3, 6, 29 Saha, S., 221 SARS-CoV virus, rRABV vectors, 227 Short consensus repeats (SCR), B5R, 148–149 Silver-haired bat RABV (SHBRV) strain, RABV, 214, 217, 220, 223 Small molecule antivirals, 103 Sparacio, S., 101 SPI-1, RPXV mutants genomic analysis of, 140–143 infection, 143 Splice regulatory factors, phosphatidylinositol-3-kinase (PI3K), 189 Strick, P.L., 232 Subacute sclerosing panencephalitis (SSPE), MV infections, 186 Sylvatic rabies, 209 T Tan, G.S., 216 TANK-binding kinase 1 (TBK-1), RABV, 216–217 T cell receptor excision circle (TREC), MV infections, 182 T cell silencing, morbillivirus infections glyco protein-mediated suppression of, 184–186 IFN inhibition in, 184 mechanisms for, 183 Tetraspanin CD81, 62

256

Index

TNF-related apoptosis-inducing ligand (TRAIL), 218, 221 Toll-like receptor (TLR) morbillivirus infection, 180, 187 RABV infection, 218 Tropism, morbilliviruses, 177–181 Trueta, J., 7 Tumor necrosis factor (TNF) receptors M-T2 gene, 154 RABV infection, 218, 221 U Untranslated regions (UTR), HCV, 53 V Vaccine-derived polioviruses (VDPV), 38–39 OPV immunization, 39–40 termination and, 40–41 Vaccines AIDS and, 225–226 DNA immunization, RABV, 228–230 hepatitis C and SARS-CoV virus, 227 mucosal and cellular immunity, 18–20 poliovirus serotypes, 20–21 primary and secondary infections, 17–18 RABV infection, 224–225 vaccinia-rabies recombinant, 227–228 Vaccinia virus (VACV). See Orthopoxvirus, host genes

VACV complement control protein (VCP), 149 Variola virus (VARV), 144–145 Vesicle-associated membrane protein-associated protein A (VAP-A), 68 Vesicular stomatitis virus (VSV), 184–185 rRABV vectors, 226 Viremia and poliomyelitis, 5 Virus inducible ncRNA (VINC), RABV infection, 221–222 Virus neutralizing antibody (VNA), RABV infection, 224 von Pirquet, C., 176 W Wakita, T., 83 Wild poliovirus vs. OPV, 30 Wimmer, E., 8 Y Yanagi, M., 71 Yang, W.X., 10 Yarosh, O.K., 228 Yi, M., 97, 99 Z Zhang, S., 25 Zhong, W., 83