Functional Characterization of the Complement Control Protein Homolog of Herpesvirus Saimiri

Abstract

Herpesvirus saimiri (HVS) is a lymphotropic virus that causes T-cell lymphomas in New World primates. It encodes a structural homolog of complement control proteins named complement control protein homolog (CCPH). Previously, CCPH has been shown to inhibit C3d deposition on target cells exposed to complement. Here we have studied the mechanism by which it inactivates complement. We have expressed the soluble form of CCPH in Escherichia coli, purified to homogeneity and compared its activity to vaccinia virus complement control protein (VCP) and human complement regulators factor H and soluble complement receptor 1. The expressed soluble form of CCPH bound to C3b (K D ‫؍‬ 19.2 M) as well as to C4b (K D ‫؍‬ 0.8 M) and accelerated the decay of the classical/lectin as well as alternative pathway C3-convertases. In addition, it also served as factor I cofactor and supported factor I-mediated inactivation of both C3b and C4b. Time course analysis indicated that although its rate of inactivation of C4b is comparable with VCP, it is 14-fold more potent than VCP in inactivating C3b. Site-directed mutagenesis revealed that Arg-118, which corresponds to Lys-120 of variola virus complement regulator SPICE (a residue critical for its enhanced C3b cofactor activity), contributes significantly in enhancing this activity. Thus, our data indicate that HVS encodes a potent complement inhibitor that allows HVS to evade the host complement attack.

Characterization of the Complement Inhibitory Function of Rhesus Rhadinovirus Complement Control Protein (RCP)

“…It is worth noting that RCP-1 showed the strongest binding to C4b, out of all proteins tested, likely directly related to its doubled binding sites and superior decay accelerating activity. Surprisingly, RCP-1 was also highly effective in accelerating decay of the alternative C3 convertase, which makes it fairly unique among the viral complement inhibitors; the activity of KCP and RCP-H was negligible, and previous reports have only shown very weak alternative DAA for VCP and complement control protein homolog (CCPH) encoded by herpesvirus saimiri (33). However, the lower activity observed for the CCP5-8 portion suggests that the naturally occurring shorter isoform for strain 17577 (19) would be less effective than the full-length RCP.…”

“…Comparing KCP and RCP with other viral complement inhibitors such as VCP, CCPH (33), and smallpox inhibitor of complement enzymes (38), which all consist of four CCP domains, some similarities can be found as follows: (i) all of them act as cofactors for C3b and C4b cleavage by FI; (ii) all but RCP-1 exhibit decay acceleration activity for the classical pathway that is much stronger than that for the alternative pathway (if such activity can at all be detected); and (iii) inhibitors with a single binding site have lower DAA than those with double/ multiple binding sites, as shown for CR1 and C4BP compared with smallpox inhibitor of complement enzymes and VCP (39) or CR1 and factor H compared with CCPH and VCP (33). This observation also holds true for RCP-1, which dissociated convertases more effectively than its 4-CCP domain subunits or homolog RCP-H.…”

Complement Evasion Strategies of Viruses: An Overview

“…Initial characterization of this protein showed that it has the ability to inhibit complement activation (Fodor et al, 1995). Later, its detailed functional characterization showed that like poxviral RCA proteins, it also has the ability to accelerate decay of the CP/LP C3 convertase and to a lesser extent the AP C3 convertases as well as inactivate C3b and C4b resulting in inhibition of the CP/LP as well as the AP (Singh et al, 2006). Besides, the domain requirement of this protein for complement inactivation was essentially similar to that of the poxviral RCA proteins (Singh et al, 2009;Reza et al, 2013).…”

Structure-based Mapping of DAF Active Site Residues That Accelerate the Decay of C3 Convertases

“…Armed with this information on DAF, we then analyzed the extent of conservation of residues in DAF and in other C3 convertase regulators determined by mutagenesis to be important, either for cofactor activity or for DAA (12,18,21,22,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). Whereas we found an absence of strict conservation of residues which distinguish the two functions, by focusing alignment on the sequence flanking the CCP2 and -3 junction where DAF function resides, we identified differences between those regulators that 1) mediate DAA and 2) mediate cofactor activity.…”