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(H₂O) (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.

IMPORTANCE 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.

痘病毒显示出种属嗜性-天花病毒是一种人类特异性病毒，而牛痘病毒则导致奶牛反复发作。与此相一致的是，天花病毒补体调节剂SPICE（补体酶的天花抑制剂）在抑制人替代补体途径中表现出选择性，牛痘病毒补体调节剂VCP（牛痘病毒补体控制蛋白）在抑制牛替代补体途径中表现出选择性。在本研究中，我们检查了VCP和SPICE对经典途径（CP）的物种特异性。我们观察到，在抑制牛CP方面，VCP比SPICE高约43倍。此外，功能分析还显示，VCP对牛CP的抑制活性增强仅是由于其增强的辅因子活性，对牛CP C3转化酶的衰变没有影响。为了探究这种特异性的结构基础，我们利用了单氨基酸和多氨基酸取代突变体，其中在SPICE模板中取代了11个变体VCP残基中的1个或多个。检查这些突变体抑制牛CP的能力表明，E108，E120和E144主要负责赋予特异性并有助于增强VCP的辅因子活性。结合和功能分析表明，这些残基与牛I因子相互作用，但与牛C4（H 检查这些突变体抑制牛CP的能力表明，E108，E120和E144主要负责赋予特异性并有助于增强VCP的辅因子活性。结合和功能分析表明，这些残基与牛I因子相互作用，但与牛C4（H 检查这些突变体抑制牛CP的能力表明，E108，E120和E144主要负责赋予特异性并有助于增强VCP的辅因子活性。结合和功能分析表明，这些残基与牛I因子相互作用，但与牛C4（H₂ O）（与C4b构象相似的部分）。将这些残基映射到牛C4b-VCP-牛因子I的建模结构上，支持了诱变数据。综上所述，我们的数据有助于解释牛痘病毒疫苗株为何能够在家养动物中站稳脚跟。

重要性痘苗病毒用于天花疫苗接种。源自疫苗的病毒正在传播，并导致印度和巴西的奶牛暴发。但是，这种向性的原因尚不清楚。众所周知，该病毒易受补体经典途径（CP）的中和。由于该病毒编码可溶性补体调节因子VCP，因此我们检查了该蛋白是否在靶向牛CP上显示出选择性。我们的数据表明，它确实具有抑制牛CP的选择性，这主要取决于其氨基酸E108，E120和E144，它们与牛丝氨酸蛋白酶因子I相互作用以灭活牛C4b（CP的两个亚基之一） C3-转化酶。值得注意的是，天花补体调节剂SPICE在这些位置含有带正电的残基。因此，