Species Specificity of Vaccinia Virus Complement Control Protein for the Bovine Classical Pathway Is Governed Primarily by Direct Interaction of Its Acidic Residues with Factor I

Abstract

Poxviruses display species tropism-variola virus is a human-specific virus, while vaccinia virus causes repeated outbreaks in dairy cattle. Consistent with this, variola virus complement regulator SPICE (smallpox inhibitor of complement enzymes) exhibits selectivity in inhibiting the human alternative complement pathway and vaccinia virus complement regulator VCP (vaccinia virus complement control protein) displays selectivity in inhibiting the bovine alternative complement pathway. In the present study, we examined the species specificity of VCP and SPICE for the classical pathway (CP). We observed that VCP is ∼43-fold superior to SPICE in inhibiting bovine CP. Further, functional assays revealed that increased inhibitory activity of VCP for bovine CP is solely due to its enhanced cofactor activity, with no effect on decay of bovine CP C3-convertase. To probe the structural basis of this specificity, we utilized single- and multi-amino-acid substitution mutants wherein 1 or more of the 11 variant VCP residues were substituted in the SPICE template. Examination of these mutants for their ability to inhibit bovine CP revealed that E108, E120, and E144 are primarily responsible for imparting the specificity and contribute to the enhanced cofactor activity of VCP. Binding and functional assays suggested that these residues interact with bovine factor I but not with bovine C4(HO) (a moiety conformationally similar to C4b). Mapping of these residues onto the modeled structure of bovine C4b-VCP-bovine factor I supported the mutagenesis data. Taken together, our data help explain why the vaccine strain of vaccinia virus was able to gain a foothold in domesticated animals. Vaccinia virus was used for smallpox vaccination. The vaccine-derived virus is now circulating and causing outbreaks in dairy cattle in India and Brazil. However, the reason for this tropism is unknown. It is well recognized that the virus is susceptible to neutralization by the complement classical pathway (CP). Because the virus encodes a soluble complement regulator, VCP, we examined whether this protein displays selectivity in targeting bovine CP. Our data show that it does exhibit selectivity in inhibiting the bovine CP and that this is primarily determined by its amino acids E108, E120, and E144, which interact with bovine serine protease factor I to inactivate bovine C4b-one of the two subunits of CP C3-convertase. Of note, the variola complement regulator SPICE contains positively charged residues at these positions. Thus, these variant residues in VCP help enhance its potency against the bovine CP and thereby the fitness of the virus in cattle.

Orthopoxvirus K3 orthologs show virus- and host-specific inhibition of the antiviral protein kinase PKR

“…Thus, it is possible that poxviruses whose E3 orthologs inhibit PKR less efficiently might depend more on their K3 orthologs for PKR inhibition than is currently recognized. Similarly, orthologs of the complement-binding factor VCP from VACV, VARV and MPXV were shown to inhibit complement factors in a species-specific manner [ 33 – 37 ]. Another example of variations in poxviral immune antagonist activity is found in the poxvirus C7L family, which confers host range function in different poxviruses [ 38 – 40 ].…”

The imitation game: a viral strategy to subvert the complement system

“…Site-directed mutagenesis performed to probe the structural basis of this specificity indicated that charge dictates the species specificity in poxviral complement regulatorspositively charged residues (K108 and K120) impart specificity towards the human complement, and negatively charged (E108, E120 and E144) residues direct the specificity towards the bovine complement. Further, it was also revealed that these residues are directly involved in the interaction with FI [129,130].…”

“…Like most proteins, functional sites were present on the external face of the proteins. Site-directed mutagenesis studies performed in pox (VCP and SPICE) [118, [129][130][131] and herpesviral (HVS CCPH and Kaposica) [98,120,122] RCA proteins have identified multiple sites critical for DAA and CFA. These data, together with the information on functional sites in huRCA, provided a wealth of knowledge on the structure-function relationship.…”

“…Thus, if the mutations help these viruses in adaptation, smallpox regulator SPICE and vaccinia regulator VCP must function in a species-specific manner. Functional analysis indicated that the above premise is indeed true: SPICE displays selectivity in inhibiting human CP and APs [93,118,130], while VCP exhibits selectivity in inhibiting bovine CP and AP [129,130]. Site-directed mutagenesis performed to probe the structural basis of this specificity indicated that charge dictates the species specificity in poxviral complement regulatorspositively charged residues (K108 and K120) impart specificity towards the human complement, and negatively charged (E108, E120 and E144) residues direct the specificity towards the bovine complement.…”

Virus-Encoded Complement Regulators: Current Status

“…Studies on the species specificity of SPICE and VCP showed that both exhibit strong selectivity, which is consistent with the species tropism of variola and vaccinia viruses, i.e., SPICE is human complement specific and VCP bovine complement specific. Remarkably, the determinant for the switch in the selectivity of SPICE and VCP is the presence of oppositely charged residues in CCP domains 2–3 [ 125 , 126 ].…”