Dissecting the Regions of Virion-Associated Kaposi's Sarcoma-Associated Herpesvirus Complement Control Protein Required for Complement Regulation and Cell Binding

Abstract

Complement, which bridges innate and adaptive immune responses as well as humoral and cell-mediated immunity, is antiviral. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a lytic cycle protein called KSHV complement control protein (KCP) that inhibits activation of the complement cascade. It does so by regulating C3 convertases, accelerating their decay, and acting as a cofactor for factor I degradation of C4b and C3b, two components of the C3 and C5 convertases. These complement regulatory activities require the short consensus repeat (SCR) motifs, of which KCP has four (SCRs 1 to 4). We found that in addition to KCP being expressed on the surfaces of experimentally infected endothelial cells, it is associated with the envelope of purified KSHV virions, potentially protecting them from complement-mediated immunity. Furthermore, recombinant KCP binds heparin, an analogue of the known KSHV cell attachment receptor heparan sulfate, facilitating infection. Treating virus with an anti-KCP monoclonal antibody (MAb), BSF8, inhibited KSHV infection of cells by 35%. Epitope mapping of MAb BSF8 revealed that it binds within SCR domains 1 and 2, also the region of the protein involved in heparin binding. This MAb strongly inhibited classical C3 convertase decay acceleration by KCP and cofactor activity for C4b cleavage but not C3b cleavage. Our data suggest similar topological requirements for cell binding by KSHV, heparin binding, and regulation of C4b-containing C3 convertases but not for factor I-mediated cleavage of C3b. Importantly, they suggest KCP confers at least two functions on the virion: cell binding with concomitant infection and immune evasion.

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

“…A similar mechanism could also be operative for the interaction of VCP with C4b and factor I. Because domain requirements for ligand binding and CFAs in viral (34,48,49,54) and human complement regulators (55)(56)(57)(58)(59)(60)(61)(62) are conserved, it could be inferred that the recognition sites for C3b/C4b and factor I are spatially conserved in both human and viral complement regulators, and that they employ a common mechanism to inactivate C3b and C4b.…”

Molecular Characterization of the Rhesus Rhadinovirus (RRV) ORF4 Gene and the RRV Complement Control Protein It Encodes

“…Thus, KCP is likely to help protect virions and infected cells against complement eradication. Moreover, KCP may be multifunctional; we recently showed that, like other human and viral RCAs, it binds to heparin and heparan sulfate (27,40). On the surface of virions, KCP may therefore aid in viral attachment to host cells, as previously described for the envelope glycoproteins K8.1 and gB (1,3,9,44).…”

“…Cells and debris were removed by centrifugation (1,900 ϫ g for 5 min at 4°C), and virions were then concentrated (13,000 ϫ g for 180 min at 4°C) before being resuspended in serum-free medium by overnight incubation at 4°C. The IEM procedure was performed essentially as described for staining KSHV virion-associated KCP (40). Briefly, RRV was applied to Parlodion-coated 200-mesh nickel grids (Agar Scientific, Stansted, United Kingdom).…”

“…Like these cellular proteins, KCP modulates complement activation by inhibiting the C3 convertases (29,39), key enzyme complexes that are formed subsequent to complement activation and that determine progression of the complement cascade. ORF4 is a lytic gene (17,41), and the KCP protein it encodes has been found on the surface of both infected cells and viruses (40,41). Thus, KCP is likely to help protect virions and infected cells against complement eradication.…”

Viral Semaphorin Inhibits Dendritic Cell Phagocytosis and Migration but Is Not Essential for Gammaherpesvirus-Induced Lymphoproliferation in Malignant Catarrhal Fever

“…expression vector (29). The pTorsten-A3Fc was produced by amplifying the A3 coding sequence by using the primers XbaI142S and A3deltastop (see Table 1 for the primer sequence) and the AlHV-1 BAC C500 as the template DNA.…”