Domain Swapping Reveals Complement Control Protein Modules Critical for Imparting Cofactor and Decay-Accelerating Activities in Vaccinia Virus Complement Control Protein

Abstract

Vaccinia virus encodes a structural and functional homolog of human complement regulators named vaccinia virus complement control protein (VCP). This four-complement control protein domain containing secretory protein is known to inhibit complement activation by supporting the factor I-mediated inactivation of complement proteins, proteolytically cleaved form of C3 (C3b) and proteolytically cleaved form of C4 (C4b) (termed cofactor activity), and by accelerating the irreversible decay of the classical and to a limited extent of the alternative pathway C3 convertases (termed decay-accelerating activity [DAA]). In this study, we have mapped the VCP domains important for its cofactor activity and DAA by swapping its individual domains with those of human decay-accelerating factor (CD55) and membrane cofactor protein (MCP; CD46). Our data indicate the following: 1) swapping of VCP domain 2 or 3, but not 1, with homologous domains of decay-accelerating factor results in loss in its C3b and C4b cofactor activities; 2) swapping of VCP domain 1, but not 2, 3, or 4 with corresponding domains of MCP results in abrogation in its classical pathway DAA; and 3) swapping of VCP domain 1, 2, or 3, but not 4, with homologous MCP domains have marked effect on its alternative pathway DAA. These functional data together with binding studies with C3b and C4b suggest that in VCP, domains 2 and 3 provide binding surface for factor I interaction, whereas domain 1 mediates dissociation of C2a and Bb from the classical and alternative pathway C3 convertases, respectively.

Mutational analysis of Kaposica reveals that bridging of MG2 and CUB domains of target protein is crucial for the cofactor activity of RCA proteins

“…Our results provided here clearly point out that CCP2 and CCP3 of Kaposica are most vital for its CFA, because swapping of these with the homologous domains of DAF results in complete loss of the CFA. These data also gain support from our earlier studies with viral RCAs Kaposica and VCP (16,17). Furthermore, by swapping the intercysteine segments and by site-directed mutagenesis, we also identify the intercysteine regions and residues in CCP2 and CCP3 of Kaposica critical for its CFA.…”

“…There is, however, a caveat-swapping strategy, although superior than domain deletions, may lead to structural alteration because of disruption of the interaction between modules. Having said the above, we would like to point out that this strategy did prove successful in identifying the functional modules in another viral RCA: vaccinia virus complement control protein (VCP) (17). We, thus, swapped each of the Kaposica modules with the homologous modules of human DAF, which is also composed of four CCP modules but lacks CFA.…”

“…Thus, these viral proteins can serve as useful tools to understand the functioning of RCA proteins. Extensive mutagenesis studies in these viral regulators have led to the understanding that CCP modules 2 and 3 are critical for imparting CFA and that the factor I binding site resides in these domains (16)(17)(18)(19). However, the underlying molecular mechanisms of complement regulation by viral RCA are still enigmatic.…”

Virus–Complement Interactions: An Assiduous Struggle for Dominance

“…Investigation of the mechanisms of interaction of VCP with its target proteins showed that it follows a simple 1:1 binding model, with very fast on-and-off rates [ Attempts to identify the functional domains suggested that different domains contribute to different functional activities, but that all four modules contribute to its optimal activity [91]. Our very recent data indicate that domains 2 and 3 in VCP provide the binding surface for factor I, while domain 1 mediates the dissociation of the protease subunit from the classical and AP C3 convertases, respectively [100]. Owing to its inhibitory nature, the molecule has also been evaluated for its effectiveness in several disease conditions (reviewed in [101,102]).…”

Glycosylated and Nonglycosylated Complement Control Protein of the Lister Strain of Vaccinia Virus

“…VCP is a homolog of the human regulators of complement activation proteins and has been shown to inhibit antibody-mediated complement-enhanced neutralization of vaccinia virus in vitro (10). Mechanistically, VCP functions to facilitate viral pathogenesis by inhibiting complement activation (11,12) through enhancement of factor I-mediated inactivation of complement proteins C3b (13) and C4b and by accelerating the dissociation of the C3 convertase of the complement pathway (14,15). The C3L gene is conserved among vaccinia virus strains, and homologs of C3L encoding inhibitors of complement enzymes (ICEs) have been described in other orthopoxviruses, including variola virus (VARV), monkeypox virus (MPXV), cowpox virus (CPXV), and ectromelia virus (ECTV), where the encoded proteins are known as SPICE, MOPICE, CPV-IMP, and EMICE, respectively (16)(17)(18)(19).…”