Poxvirus IFN Receptor Homologs Grant McFadden1,* and Richard Moyer2 1
The John P. Robarts Research Institute and Department of Microbiology and Immunology, The University of Western Ontario, 1400 Western Road, London, Ontario, N6G 2V4, Canada 2 Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, PO Box 100266, Gainesville, FL 32610-0266, USA * corresponding author tel: (519) 663-3184, fax: (519) 663-3847, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.14014.
SUMMARY The first example discovered of a viroceptor (virusencoded receptor homolog) targeted against the interferon family was the M-T7 gene product of myxoma virus, a secreted glycoprotein of 37 kDa that shares sequence similarity to the external ligandbinding domain of the cellular IFN receptor. Later, M-T7 was shown to be closely related to a variety of homologous genes in many other poxviruses that share sequence homology to the same domain of the cellular receptors. Studies have shown that these poxvirus IFN receptors bind and inhibit IFN in a relatively species-specific fashion, with the highest affinity generally being to the ligand derived from the host species of the particular poxvirus.
BACKGROUND
Discovery The first example of a virus-encoded IFN receptor was uncovered when DNA sequencing studies revealed that myxoma virus encodes a 263 amino acid protein possessing considerable homology with the cellular IFN R (Upton et al., 1992). This 37 kDa protein is the major protein secreted from cells infected with myxoma virus (strain Lausanne) and is designated M-T7 because it is encoded by the seventh open reading from the viral DNA terminus (Upton
et al., 1992). Database analysis revealed that the conserved amino acids mapped to the extracellular ligand-binding domain of the mammalian IFN Rs. Particularly noteworthy was the cysteine residue spacing important for ligand interaction (Figure 1). Due to the availability of virus genome sequence information, soluble IFN R homologs have since been discovered in a variety of other poxviruses (Mossman et al., 1995a; AlcamõÂ and Smith, 1996a,b). The T7 open reading frame of a related leporipoxvirus, Shope fibroma virus (SFV), encodes a 265 residue soluble IFN R homolog, called S-T7 (Upton and McFadden, 1986; Upton et al., 1992). Vaccinia virus strains Copenhagen and Western Reserve (WR) also encode related 272 amino acid homologs from their respective B8R open reading frames (Goebel et al., 1990; Howard et al., 1991). By probing ectromelia virus DNA fragments with the vaccinia virus B8R gene, an ectromelia virus IFN R homolog was discovered, and subsequent cloning and sequencing revealed an open reading frame encoding a 266 residue protein, whose location within the ectromelia virus genome remains to be determined (Mossman et al., 1995b). Two strains of variola virus, the causative agent of smallpox (strains Bangladesh 1975 and India 1967), have been completely sequenced (Massung et al., 1993a; Shchelkunov et al., 1993) and found to encode related soluble IFN Rs, designated B8R and B9R, respectively. Both proteins are predicted to contain 266 amino acids, of which 265 are identical. Finally, swinepox virus, a member of the suipoxvirus genus, encodes a 274 amino acid
1840 Grant McFadden and Richard Moyer IFN R homolog from the C6L open reading frame (Massung et al., 1993b). Tanapox virus, a member of the yatapoxvirus genus, secretes a 38 kDa glycoprotein which binds and inhibits the effects of human IFN , IL-2, and IL-5 (Essani et al., 1994), but its sequence remains to be reported.
Alternative names vIFN R.
Structure All the poxvirus IFN R homologs are secreted proteins with sequence homology to the ligandbinding domain of the cellular IFN receptors. All contain from 6 to 8 of the conserved cysteines that define the folding domains of this family (Figure 1). The family has been extensively reviewed (Upton and McFadden, 1994; Mossman et al., 1995a, 1996a; AlcamõÂ and Smith, 1996b; Smith, 1996).
Main activities and pathophysiological roles IFN -binding proteins from supernatants infected with 10 different vaccinia virus strains, as well as cowpox, rabbitpox, camelpox, buffalopox, elephantpox, and tanapox viruses have been identified by crosslinking studies (AlcamõÂ and Smith, 1995; Mossman et al., 1995b). The secreted versions of all poxvirus IFN R homologs possess apparent molecular masses of 37±43 kDa (AlcamõÂ and Smith, 1995; Mossman et al., 1995b). To date, all poxviruses tested express an IFN -binding protein, indicating the universal importance of IFN as a critical ligand in the orchestration of an effective immune response against virus infection. While all of the sequenced poxviral IFN R homologs have been found to exhibit significant homology with the mammalian IFN R ligand-binding domain, the extent of overall sequence similarity with either the human or murine IFN Rs is relatively low (20± 25% identity). While the percentage identity between IFN R homologs from different poxvirus genera is equally low, the homology between IFN R proteins within the same poxvirus genus is high. The leporipoxvirus homologs, myxoma virus M-T7 and SFV ST7, are 68% identical and among the orthopoxvirus homologs encoded by vaccinia, ectromelia, and variola viruses there is greater than 90% identity. An important feature of all poxviral IFN R
homologs is the strong conservation of the cysteine residues shown to be critical for proper folding and functioning of the corresponding mammalian receptors (Figure 1). An intriguing observation, however, is that the orthopoxvirus receptor homologs lack the corresponding first two cysteine residues which form the lone disulfide bond within domain 1 of the mammalian receptors (Figure 1). This observation is particularly unexpected since the domain 1 disulfide bond is the only bond whose removal completely abolishes activity of the mammalian receptors (Stuber et al., 1993). The significance of this important missing disulfide bond with respect to the function of the orthopoxvirus IFN R homologs remains to be determined.
GENE
Accession numbers Myxoma virus (M-T7): M81919 Shope fibroma virus (S-T7): M14899 Vaccinia virus Western Reserve (B8R): M58056 Vaccinia virus Copenhagen (B8R): M35027 Swinepox virus (C6L): L22013 Variola virus major (B8R): L22579 Variola virus minor (H9R): U18339 Cowpox virus (B7R): Y15035 Ectromelia virus: U19584
PROTEIN
Accession numbers Myxoma virus (M-T7): 332308 Vaccinia virus Western Reserve (B8R): 335312 Vaccinia virus Copenhagen (B8R): 335553 Swinepox virus (C6L): 418186 Variola virus major (B8R): 439086 Variola virus minor (H9R): 885773 Cowpox virus (B7R): 3097028 Ectromelia virus: 761722
Sequence See Figure 1.
Description of protein All the poxvirus IFN R homologs are secreted from virus-infected cells as 35±45 kDa glycoproteins that
Poxvirus IFN Receptor Homologs 1841 Figure 1 Amino acid sequence alignment of the poxviral IFN R homologs: myxoma virus M-M-T7, Shope fibroma virus S-M-T7, swinepox virus SPV C6L, vaccinia virus B8R (VV-B8R), and variola virus Bangladesh 1975 B8R (VAR-B8R), and the cellular murine (muIFNR) and human (huIFNR) IFN receptor chains. Boxes indicate amino acid identity among all proteins, while ( ) denotes amino acids which are conserved in at least four of the seven proteins. The disulfide-forming cysteine residues conserved between the mammalian and viral proteins are both boxed and numbered 1±8. The arrow indicates the location of the N-terminal residue, determined by sequencing, of the mature secreted myxoma M-T7 protein, while the predicted transmembrane domains of the mammalian IFN Rs are underlined. The full lengths of the human and murine IFN Rs are 489 and 477 amino acids, respectively.
1842 Grant McFadden and Richard Moyer form soluble inhibitory complexes with IFN , with variable species specifications. Thus, all the downstream signaling events triggered by IFN -dependent receptor stimulators are blocked at a stage prior to receptor engagement. Studies on the myxoma virus M-T7 protein have illustrated the significant role played by viral IFN R homologs during a productive infection. M-T7 is expressed early during infection from a strong poxviral promoter (Mossman et al., 1995c), an important property considering the requirement to counteract the potent activities of IFN . M-T7 binds rabbit IFN with an affinity comparable to that demonstrated by the mammalian soluble receptors and their cognate ligands (10ÿ9 M) (Mossman et al., 1995c). M-T7 is secreted from myxoma virus-infected cells at levels which exceed 107 molecules per cell per hour at early times of infection (Mossman et al., 1995c), which constitutes a vast excess over the 102 ±104 cellular receptors typically expressed at the surface of most mammalian cells.
Relevant homologies and species differences
bind murine IFN with only low affinity (Mossman et al., 1995b) while another study failed to observe any detectable binding (AlcamõÂ and Smith, 1995). Broad ligand specificity was also demonstrated by IFN R homologs encoded by cowpox, rabbitpox, camelpox, buffalopox and elephantpox viruses, whose receptor homologs bind human, bovine, and rat IFN with comparable affinities (AlcamõÂ and Smith, 1995). Only camelpox and elephantpox viruses express proteins that showed weak binding of murine IFN . Interestingly, ectromelia virus displayed equal binding affinities for human, rabbit, and murine IFN (Mossman et al., 1995b). The IFN R homologs of variola virus (strains major and minor) were expressed in bacterial vectors and shown to be specific inhibitors of human, but not murine, IFN (Seregin et al., 1996). Interestingly, the vaccinia B8R protein is far more promiscuous than other poxviral IFN R homologs, and can inhibit even chicken IFN (Puehler et al., 1998). Very recently, the myxoma M-T7 protein was also shown to exhibit a second unpredicted property of binding to the glycosamino-glycan domain of a broad spectrum of chemokines, a property not shared by the IFN R homologs from other poxviruses (Lalani et al., 1997), but its sequence remains to be determined.
See Figure 1.
Affinity for ligand(s) IFN interacts exclusively with receptors from the same species (Farrar and Schreiber, 1993), and since the ligand specificity of individual receptor homologs has the potential to shed light on virus evolutionary history, determining the species specificity of poxviral IFN R homologs has particular relevance. For example, myxoma virus is one of the few poxviruses whose natural host, the South American bush rabbit or tapeti (Silvilagus brasiliensis), has been well established (Fenner and Ratcliffe, 1965; Fenner and Ross, 1994). M-T7 demonstrates strict species specificity for rabbit IFN (Mossman et al., 1995e), while the IFN R homologs from members of the orthopoxvirus genus, such as vaccinia, exhibit much broader species specificity for IFN inhibition (AlcamõÂ and Smith, 1995). Unlike myxoma virus, the natural host of most orthopoxviruses, particularly vaccinia virus, remains obscure. In chemical cross-linking assays, the vaccinia virus IFN R homolog has been found capable of binding a variety of radiolabeled ligands, including human, bovine, rabbit, and rat IFN (AlcamõÂ and Smith, 1995; Mossman et al., 1995b). In one study, the vaccinia virus receptor homolog was shown to
Regulation of receptor expression All the poxvirus IFN R homologs are expressed from early viral promoters, which function independently from the infected cell type.
BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY
Phenotypes of receptor knockouts and receptor overexpression mice Myxoma virus pathogenesis is an attractive poxvirus system to study virus virulence due to the established nature of both its natural host and the disease syndrome it causes. In the European rabbit (Oryctolagus cuniculus), myxoma virus induces a rapidly lethal infection known as myxomatosis (Fenner and Myers, 1978; Fenner and Ratcliffe, 1965). The disseminating nature of the infection, coupled with the extensive immunosuppression that is associated with both cellular immune defects and
Poxvirus IFN Receptor Homologs 1843 cytokine dysregulations, leads to death of the infected rabbits within 2 weeks (Strayer, 1989; McFadden, 1994). Deletion of the M-T7 gene from the myxoma virus genome dramatically attenuated the virus and confirmed the importance of this protein during viral pathogenesis (Mossman et al., 1996b).
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