Poxvirus Secreted Complement Control Proteins

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Poxvirus Secreted Complement Control Proteins 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.2000.03011.

SUMMARY Numerous members of the herpesvirus and poxvirus families express secreted proteins related to the superfamily of complement regulators as defined by the presence of multiple short consensus repeats (SCRs) and which act by binding and inhibiting key elements of the classical and alternative complement cascades. In the case of poxviruses, the two most extensively studied members are from vaccinia virus and cowpox virus. The vaccinia version, also called vaccinia control protein (VCP), and the cowpox protein are both secreted from infected cells as 35 kDa glycoproteins that exhibit anticomplement activity in vitro and in vivo.

BACKGROUND

Discovery The vaccinia complement control protein (VCP) was discovered in 1988 during characterization of proteins secreted from vaccinia virus-infected cells (Kotwal and Moss, 1988a,b). Structural relatedness to the superfamily of complement control proteins was noted through sequence analysis. The protein contains 60 amino acid short consensus repeats (SCRs)

present in proteins which control complement activation. Homologs are known in cowpox and variola (smallpox) viruses (Miller et al., 1997).

Alternative names Immunomodulatory protein (IMP) has been proposed (Kotwal et al., 1998).

Structure The VCP gene is typical of poxvirus genes, and as such there are no introns. The 263 amino acid ( 35 kDa apparent molecular weight) vaccinia protein encoded by vaccinia ORF C21L contains a 19 amino acid N-terminal signal peptide which is cleaved to generate a secreted protein of 244 amino acids. Highly conserved homologs are encoded by a variety of different orthopoxviruses, including variola and cowpox viruses (CPV). The protein contains four 60 amino acid short consensus repeats (SCRs) noted in proteins that control complement activation. Sequence analysis shows relatedness to complement control proteins, with highest similarity to human protein C4bp, a 549 amino acid protein, containing eight SCRs, which binds to the C4b fragment of the

1304 Grant McFadden and Richard Moyer fourth component of complement. The cellular protein functions to inhibit the classical pathway of complement activation. The smaller vaccinia protein exhibits 38% identity with the first half of the cellular C4b binding protein and 28% identity with the second half. Within the homologous regions, 16 of 17 cysteine residues are conserved, as are the majority of glycines and prolines (Isaacs and Moss, 1995; Howard et al., 1998).

GENE AND GENE REGULATION

Accession numbers X13166, g60690.

Chromosome location ORF C21L in vaccinia virus WR.

Main activities and pathophysiological roles

Cells and tissues that express the gene

Initially assayed by the ability to inhibit complementmediated hemolysis of sheep red blood cells, VCP also inhibits complement-mediated antibody-enhanced neutralization in vitro (Isaacs et al., 1992). The protein acts to inhibit activation of complement by both the classical and alternative complement pathways. The protein can bind to both C4b to block formation of the classical pathway generated C3 convertase and C3b to cause accelerated decay of the classical pathway C3 convertase and block conversion of C3 to C3b by both the classical and alternative pathways. Finally, the protein can cleave C3b at one of three susceptible sites in a process mediated by cofactor I. Deletion of the gene attenuates the virus and leads to smaller skin lesions in infected animals (guinea pigs). Comparison of pathology following injection of CPV and CPV deleted for VCP (CPV-VCPÿ ) into the footpad of mice showed that the animals exposed to CPV-VCPÿ resulted in greater tissue damage accompanied by more extensive hemorrhage and induration than animals injected with CPV. Swelling at the inoculation site was also markedly enhanced in CPVVCPÿ -infected animals. It is proposed that the main function of VCP in immunocompetent animals is to limit the cellular infiltration by downregulating the complement-derived chemotactic anaphylatoxins, which serves to lessen the inflammatory response and in turn leads to diminished tissue pathology (Howard et al., 1998).

Orthopoxviruses express VCP independently of the cell or tissue infected by the virus.

PROTEIN

Accession numbers g60691

Sequence See Figure 1.

Description of protein The 263 amino acid ( 35 kDa apparent molecular weight) vaccinia protein encoded by vaccinia ORF C21L contains a 19 amino acid N-terminal signal peptide which is cleaved to generate a secreted protein of 244 amino acid secreted protein. The protein contains four 60 amino acid short consensus repeats (SCRs) noted in proteins that control complement activation. The repeats comprise amino acids 20±82, 85±144, 147±202, 205±262. The protein belongs to the superfamily of the regulators of complement activation (RCA).

Figure 1 Amino acid sequence for the vaccinia complement control protein. 1 61 121 181 241

MKVESVTFLT QKMGPIYAKC ESKSYCELGS SLIGNSGVLC SGSSSSTCSP

LLGIGCVLSC TGTGWTLFNQ TGSMVWNPEA SGGEWSDPPT GNTWKPELPK

CTIPSRPINM CIKRRCPSPR PICESVKCQS CQIVKCPHPT CVR

KFKNSVETDA DIDNGQLDIG PPSISNGRHN ISNGYLSSGF

NANYNIGDTI GVDFGSSITY GYEDFYTDGS KRSYSYNDNV

EYLCLPGYRK SCNSGYHLIG VVTYSCNSGY DFKCKYGYKL

Poxvirus Secreted Complement Control Proteins 1305

Important homologies Sequence analysis reveals relatedness to complement control proteins with highest similarity to human protein C4bp, a 549 amino acid protein, containing eight SCRs, which binds to the C4b fragment of the fourth component of complement. Significant homologies to other proteins of the complement cascade include: decay-accelerating factor, factor H, membrane cofactor protein, and complement receptors. Significant homologies to viral proteins include ORF 04 of human herpesvirus 8 and herpesvirus saimiri secreted complement control proteins ORF4 and ORF15.

complement-mediated inflammatory responses of the host. Deletion of the gene from the virus generates significantly more severe tissue destruction and swelling at the site of inoculation.

Species differences Nearly identical homologs of the vaccinia VCP are found in variola (smallpox), and cowpox viruses. Differences between the various proteins are concentrated in the N-terminal region of the protein. It is likely that the protein will be found in other orthopoxviruses of significance, such as ectromelia virus.

Posttranslational modifications

References

Cleavage of an N-terminal 19 amino acid signal sequence.

Howard, J., Justus, D. E., Totmenin, A. V., Shchelkunov, S., and Kotwal, G. J. (1998). Molecular mimicry of the inflammation modulatory proteins (IMPs) of poxviruses: evasion of the inflammatory response to preserve viral habitat. J. Leukoc. Biol. 64, 68±71. Isaacs, S. N., and Moss, B. (1995). In ``Viroceptors, Virokines and Related Immune Modulators Encoded by DNA Viruses'' (ed G. McFadden), Inhibition of complement activation by vaccinia virus, pp. 55±66. R.G. Landes & Co., Austin, TX. Isaacs, S. N., Kotwal, G. J., and Moss, B. (1992). Vaccinia virus complement-control protein prevents antibody-dependent complement-enhanced neutralization of infectivity and contributes to virulence. Proc. Natl Acad. Sci. USA 89, 628±632. Kotwal, G. J., and Moss, B. (1988a). Analysis of a large cluster of nonessential genes deleted from a vaccinia virus terminal transposition mutant. Virology 167, 524±537. Kotwal, G. J., and Moss, B. (1988b). Vaccinia virus encodes a secretory polypeptide structurally related to complement control proteins. Nature 335, 176±178. Kotwal, G. J., Miller, C. G., and Justus, D. E. (1998). The inflammation modulatory protein (IMP) of cowpox virus drastically diminishes the tissue damage by down-regulating cellular infiltration resulting from complement activation. Mol. Cell. Biochem. 185, 39±46. Miller, C. G., Shchelkunov, S. N., and Kotwal, G. J. (1997). The cowpox virus-encoded homolog of the vaccinia virus complement control protein is an inflammation modulatory protein. Virology 229, 126±133.

IN VITRO ACTIVITIES

In vitro findings The protein inhibits complement-mediated lysis of red blood cells, blocks formation of C3 convertase, causes accelerated decay of the classical and alternative convertase, facilitates cleavage of C3 in the presence of cofactor factor I, blocks complementmediated antibody-enhanced neutralization, and inhibits activation of complement by both the classical and alternative pathways.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles Within the context of the virus, the protein plays a significant role in the downregulation of the