CC Chemokine of Molluscum Contagiosum Virus

CC Chemokine of Molluscum Contagiosum Virus 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.2000.03018.

SUMMARY Virus-encoded members of the chemokine superfamily of ligands have been discovered in the genomes of certain poxviruses and herpesviruses. The MC148R gene of molluscum contagiosum virus, a human-specific poxvirus that induces benign skin lesions which can become clinically serious in immunocompromised patients, encodes for a member of the CC chemokine superfamily that can antagonize the activities of several human CC chemokines tested. The MC148R protein lacks certain amino acid motifs at the N-terminus that are believed to be required for signaling from cognate chemokine receptors, and this feature is believed to be important for its activity as a CC chemokine antagonist.

BACKGROUND Variola virus, the causative agent of smallpox, and molluscum contagiosum virus (MCV) are the only two poxviruses whose infections are limited to human hosts. Smallpox infections are typically a disseminated disease ultimately involving many organs and tissues. Ordinary smallpox, caused by variola major (of the genus Orthopoxvirus) exhibits a 30% mortality. Molluscum contagiosum, belonging to a separate genus, causes benign proliferative lesions of the skin in normal and

immunocompromised individuals. However, molluscum infections are a concern in immunocompromised individuals and in particular with individuals suffering from AIDS, where disfiguring illness can result.

Discovery The molluscum protein, designated as MC148R (based on the open reading frame designation), was first recognized as a possible chemokine homolog as part of the characterization and annotation of the complete DNA sequence of the virus (Senkevich et al., 1996, 1997). Based on restriction digestion patterns, there are two distinct MCV isolates, designated as type 1 and type 2 (Darai et al., 1986). A nearly identical protein is encoded by both isolates.

Alternative names ORF MC148R, MCV chemokine homolog (MCCH), MC148P.

Structure MC148R is a contiguous gene, typical of poxviruses, lacking introns.

1286 Grant McFadden and Richard Moyer

Main activities and pathophysiological roles One of the most remarkable features of molluscum contagiosum infections is the absence of inflammatory cell infiltrates within lesions that contain large numbers of viral particles. MCCH has been shown to block the chemotactic response of multiple subsets of leukocytes to both CC and CXC chemokines (Krathwohl et al., 1997; Damon et al., 1998) which might explain the absence of inflammation noted within MCV lesions. Like macrophage inflammatory protein 1 (MIP-1), MCCH (type 1 or 2) can inhibit colony formation of myeloid, erythroid, and multipotential progenitor (hematopoietic) cells (Krathwohl et al., 1997).

GENE AND GENE REGULATION

Accession numbers MCV MCCH type 1: U60315 MCV MCCH type 2: U96749

Chromosome location For MCV type 1, the M148R ORF is located approximately 167 kb from the left end of the 186 kb genome within the BamH1 C1 fragment. Transcription is towards the right end of the genome.

Regulatory sites and corresponding transcription factors MCV cannot be grown or studied in cell culture. However, cloning of the MC184R ORF into vaccinia

suggests that the gene is expressed early, before DNA replication (Bugert et al., 1998). A poxvirus sequence (TTTTTNT), typical of an early poxvirus transcription termination signal, is found 40 bp downstream of the 3 0 translation stop codon consistent with early transcription of the gene.

PROTEIN

Accession numbers MCV type MCV type GenPept: MCV type MCV type

1: U60315 2: U96749 1: g1492091 2: g2459795

Sequence See Figure 1.

Description of protein The MCCH protein is 104 amino acids, some 12 amino acids longer than its closest relative, human MIP-1, a CC chemokine (Schall et al., 1990; Miller and Krangel, 1992). MCCH contains the canonical cysteine residues, typical of CC chemokines (Ben-Baruch et al., 1995), which are found at amino acids 30, 31, 59, and 75 (Figure 2). In addition, and atypical of other known CC chemokines, the molluscum protein has two additional cysteine residues at positions 94 and 101. These motifs have been found to be conserved in 29 separate MCCH gene isolates derived from type I and type II MCV (Bugert et al., 1998). Based on cloning and expression

Figure 1 The amino acid sequence of MCCH from MCV type 1 (bottom) and MCV type 2 MCV (top).

CC Chemokine of Molluscum Contagiosum Virus 1287 Figure 2 A `GAP' alignment of MIP-1 (top) and MCCH-1 (bottom). Both proteins contain a C-C motif (bold type amino acids at positions 30/31 in MCCH-1), and two additional conserved cysteines (at positions 59 and 75 in MCCH-1). Both MIP-1 and MCCH-1 also contain an N-terminal secretory signal which is cleaved to generate the mature protein. MIP-1 is cleaved between amino acids S/A at position 23/24. MCCH-1 is cleaved between residues S/L at position 24/25. The proteoglycan-binding site is set in bold type in MIP-1 and just precedes the conserved cysteine at position 75.

of the MCCH type 1 protein in vaccinia, there is a functional N-terminal 24 amino acid secretory signal which is cleaved releasing the mature MCCH protein from the infected cell (Bugert et al., 1998). The receptor site of CC chemokines consists of an N-terminal 10 amino acid domain located adjacent to cleaved secretory signal peptide. One key feature of MCCH is deletion or absence of four critical residues within this domain. These missing amino acids lead to a protein truncated at the N-terminus following signal peptide cleavage. These missing amino acids comprise that portion of the receptor binding site which activates the engaged receptor (Clark-Lewis et al., 1995). It has been proposed that MCCH might bind but fail to activate the receptor, thereby acting as a chemokine antagonist (Senkevich et al., 1996). A second possibility for nonproductive signal mediation may reside within a proteoglycan-binding site found within both MCCH and CC chemokines. In the case of MIP-1, this motif consists of the sequence `KRSTQV'. Mutagenesis experiments have shown that introducing neutral or acidic amino acids into that site results in loss of chemotaxis while maintaining other chemokine activities (Graham et al., 1996). It is noteworthy that MCCH from both MCV-1 and MCV-2 contains neutral or acidic amino acids at this site.

Important homologies MCCH has some structural motifs in common with CC chemokines. A comparison with MIP-1 is shown in Figure 2.

RECEPTOR UTILIZATION MCCH can utilize a variety of CC and CXC chemokine receptors. Interaction has been specifically demonstrated with CCR1 and/or CCR5, CCR2, CCR8, CXCR1, and/or CXCR2 and CXCR4 (Damon et al., 1998).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles The proposed role of MCCH is the prevention of inflammation within MCV lesions.

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