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Central modules of the vaccinia virus complement control protein are not in extensive contact.

ABSTRACT: The 28.6 kDa vaccinia virus complement control protein (VCP) is an inhibitor of the complement system and has therapeutic potential. It is composed of four domains or modules and is a homologue of complement receptor 1 (CR1) and other mammalian regulators of complement activation. A key aspect to structure-function relationships in these proteins is the extent of intramolecular module-module interactions, since these dictate the overall shape and flexibility of the molecules. A protein fragment (VCP approximately 2,3) encompassing modules 2 and 3 of VCP was over-expressed in Pichia pastoris. Ultracentrifugation showed that VCP approximately 2,3 is highly asymmetric with an axial ratio of 5.3:1, which is consistent with an end-to-end arrangement of the two modules. NMR spectroscopy, differential scanning calorimetry, CD and intrinsic tryptophan fluorescence were used to monitor unfolding of VCP approximately 2,3. Experiments performed over a range of temperatures and concentrations of guanidinium chloride revealed that module 2 unfolds under milder conditions than, and independently of, module 3. Unfolding of module 2 is not associated with extensive changes in amide (15)N and (1)H chemical shifts of module 3, implying that the modules do not form an extensive intermodular interface. Results obtained in this work for VCP approximately 2,3 are compared with those obtained in a study of CR1 modules 15-17 [Kirkitadze, Krych, Uhrin, Dryden, Smith, Cooper, Wang, Hauhart, Atkinson and Barlow (1999) Biochemistry 38, 7019-7031].

SUBMITTER: Kirkitadze MD

PROVIDER: S-EPMC1220627 | biostudies-other | 1999 Nov

REPOSITORIES: biostudies-other

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Project description: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.

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Central modules of the vaccinia virus complement control protein are not in extensive contact.

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