Poxvirus Secreted

Complement Control
Proteins
Grant McFadden
1,
*
and Richard Moyer
2
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: mcfadden@rri.on.ca
DOI: 10.1006/rwcy.2000.03011.
SUMMARY
Numerous members of the herpesvirus and poxvirus
families express secreted proteins related to the super-
family 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 con-
trol 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 con-
tains 60 amino acid short consensus repeats (SCRs)
present in proteins which control complement acti-
vation. Homologs are known in cowpox and variola
(smallpox) viruses (Miller et al., 1997).
Alternative names
Immunomodulatory protein (IMP) has been pro-
posed (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
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).
Main activities and
pathophysiological roles
Initially assayed by the ability to inhibit complement-
mediated hemolysis of sheep red blood cells, VCP also
inhibits complement-mediated antibody-enhanced
neutralization in vitro (Isaacs et al., 1992). The pro-
tein 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 path-
ways. 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-VCP

) into the
footpad of mice showed that the animals exposed to
CPV-VCP

resulted in greater tissue damage accom-

panied by more extensive hemorrhage and induration
than animals injected with CPV. Swelling at the
inoculation site was also markedly enhanced in CPV-
VCP

-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).
GENE AND GENE REGULATION
Accession numbers
X13166, g60690.
Chromosome location
ORF C21L in vaccinia virus WR.
Cells and tissues that express
the gene
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 con-
tains four 60 amino acid short consensus repeats
(SCRs) noted in proteins that control complement
activation. The repeats comprise amino acids 2082,
85144, 147202, 205262. 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 MKVESVTFLT LLGIGCVLSC CTIPSRPINM KFKNSVETDA NANYNIGDTI EYLCLPGYRK
61 QKMGPIYAKC TGTGWTLFNQ CIKRRCPSPR DIDNGQLDIG GVDFGSSITY SCNSGYHLIG
121 ESKSYCELGS TGSMVWNPEA PICESVKCQS PPSISNGRHN GYEDFYTDGS VVTYSCNSGY
181 SLIGNSGVLC SGGEWSDPPT CQIVKCPHPT ISNGYLSSGF KRSYSYNDNV DFKCKYGYKL
241 SGSSSSTCSP GNTWKPELPK CVR
1304 Grant McFadden and Richard Moyer
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 homo-
logies to other proteins of the complement cascade
include: decay-accelerating factor, factor H, mem-
brane 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.
Posttranslational modifications
Cleavage of an N-terminal 19 amino acid signal
sequence.
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 alter-
native convertase, facilitates cleavage of C3 in the
presence of cofactor factor I, blocks complement-
mediated antibody-enhanced neutralization, and
inhibits activation of complement by both the clas-
sical 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
complement-mediated inflammatory responses of
the host. Deletion of the gene from the virus gener-
ates 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 concen-
trated in the N-terminal region of the protein. It is
likely that the protein will be found in other ortho-
poxviruses of significance, such as ectromelia virus.
References
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, 6871.
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. 5566. R.G. Landes & Co., Austin, TX.
Isaacs, S. N., Kotwal, G. J., and Moss, B. (1992). Vaccinia virus
complement-control protein prevents antibody-dependent com-
plement-enhanced neutralization of infectivity and contributes
to virulence. Proc. Natl Acad. Sci. USA 89, 628632.
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, 524537.
Kotwal, G. J., and Moss, B. (1988b). Vaccinia virus encodes a
secretory polypeptide structurally related to complement con-
trol proteins. Nature 335, 176178.
Kotwal, G. J., Miller, C. G., and Justus, D. E. (1998). The inflam-
mation modulatory protein (IMP) of cowpox virus drastically
diminishes the tissue damage by down-regulating cellular infil-
tration resulting from complement activation. Mol. Cell.
Biochem. 185, 3946.
Miller, C. G., Shchelkunov, S. N., and Kotwal, G. J. (1997). The
cowpox virus-encoded homolog of the vaccinia virus comple-
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Poxvirus Secreted Complement Control Proteins 1305