scholarly journals Glycosylated and Nonglycosylated Complement Control Protein of the Lister Strain of Vaccinia Virus

2014 ◽  
Vol 21 (9) ◽  
pp. 1330-1338
Author(s):  
Clement A. Meseda ◽  
Jordan Kuhn ◽  
Vajini Atukorale ◽  
Joseph Campbell ◽  
Jerry P. Weir
Keyword(s):  
Biological Activity ◽  
Vaccinia Virus ◽  
Sequence Data ◽  
Smallpox Vaccine ◽  
Complement Components ◽  
Complement Control Protein ◽  
Complement Proteins ◽  
Alternative Complement ◽  
Vaccinia Viruses ◽  
Control Protein

ABSTRACTThe vaccinia virus complement control protein (VCP) is a secreted viral protein that binds the C3b and C4b complement components and inhibits the classic and alternative complement pathways. Previously, we reported that an attenuated smallpox vaccine, LC16m8, which was derived from the Lister strain of vaccinia virus (VV-Lister), expressed a glycosylated form of VCP, whereas published sequence data at that time indicated that the VV-Lister VCP has no motif for N-linked glycosylation. We were interested in determining whether the glycosylation of VCP impairs its biological activity, possibly contributing to the attenuation of LC16m8, and the likely origin of the glycosylated VCP. Expression analysis indicated that VV-Lister contains substrains expressing glycosylated VCP and substrains expressing nonglycosylated VCP. Other strains of smallpox vaccine, as well as laboratory strains of vaccinia virus, all expressed nonglycosylated VCP. Individual Lister virus clones expressing either the glycosylated VCP or the nonglycosylated species were isolated, and partially purified VCP from the isolates were found to be functional equivalents in binding human C3b and C4b complement proteins and inhibiting hemolysis and in immunogenicity. Recombinant vaccinia viruses expressing FLAG-tagged glycosylated VCP (FLAG-VCPg) and nonglycosylated VCP (FLAG-VCP) were constructed based on the Western Reserve strain. Purified FLAG-VCP and FLAG-VCPg bind human C3b and C4b and blocked complement-mediated hemolysis. Our data suggest that glycosylation did not affect the biological activity of VCP and thus may not have contributed to the attenuation of LC16m8. In addition, the LC16m8 virus likely originated from a substrain of VV-Lister that expresses glycosylated VCP.

scholarly journals Kinetic Analysis of the Interactions between Vaccinia Virus Complement Control Protein and Human Complement Proteins C3b and C4b

2004 ◽  
Vol 78 (17) ◽  
pp. 9446-9457 ◽  
Author(s):  
John Bernet ◽  
Jayati Mullick ◽  
Yogesh Panse ◽  
Pradeep B. Parab ◽  
Arvind Sahu
Keyword(s):  
Vaccinia Virus ◽  
Binding Site ◽  
Molecular Mechanisms ◽  
Accurate Determination ◽  
Factor H ◽  
Human Complement ◽  
Target Proteins ◽  
Complement Control Protein ◽  
Complement Proteins ◽  
Control Protein

ABSTRACT The vaccinia virus complement control protein (VCP) is an immune evasion protein of vaccinia virus. Previously, VCP has been shown to bind and support inactivation of host complement proteins C3b and C4b and to protect the vaccinia virions from antibody-dependent complement-enhanced neutralization. However, the molecular mechanisms involved in the interaction of VCP with its target proteins C3b and C4b have not yet been elucidated. We have utilized surface plasmon resonance technology to study the interaction of VCP with C3b and C4b. We measured the kinetics of binding of the viral protein to its target proteins and compared it with human complement regulators factor H and sCR1, assessed the influence of immobilization of ligand on the binding kinetics, examined the effect of ionic contacts on these interactions, and sublocalized the binding site on C3b and C4b. Our results indicate that (i) the orientation of the ligand is important for accurate determination of the binding constants, as well as the mechanism of binding; (ii) in contrast to factor H and sCR1, the binding of VCP to C3b and C4b follows a simple 1:1 binding model and does not involve multiple-site interactions as predicted earlier; (iii) VCP has a 4.6-fold higher affinity for C4b than that for C3b, which is also reflected in its factor I cofactor activity; (iv) ionic interactions are important for VCP-C3b and VCP-C4b complex formation; (v) VCP does not bind simultaneously to C3b and C4b; and (vi) the binding site of VCP on C3b and C4b is located in the C3dg and C4c regions, respectively.

scholarly journals 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

2017 ◽  
Vol 91 (19) ◽  
Author(s):  
Jitendra Kumar ◽  
Viveka Nand Yadav ◽  
Swastik Phulera ◽  
Ashish Kamble ◽  
Avneesh Kumar Gautam ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Classical Pathway ◽  
Variola Virus ◽  
Complement Control Protein ◽  
Factor I ◽  
Functional Assays ◽  
Alternative Complement ◽  
Cofactor Activity ◽  
Complement Regulator ◽  
Control Protein

ABSTRACTPoxviruses 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.IMPORTANCEVaccinia 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.

scholarly journals Disabling complement regulatory activities of vaccinia virus complement control protein reduces vaccinia virus pathogenicity

Vaccine ◽  
2011 ◽  
Vol 29 (43) ◽  
pp. 7435-7443 ◽  
Author(s):  
John Bernet ◽  
Muzammil Ahmad ◽  
Jayati Mullick ◽  
Yogesh Panse ◽  
Akhilesh K. Singh ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Complement Control Protein ◽  
Control Protein

scholarly journals Heavily Isotype-Dependent Protective Activities of Human Antibodies against Vaccinia Virus Extracellular Virion Antigen B5

2009 ◽  
Vol 83 (23) ◽  
pp. 12355-12367 ◽  
Author(s):  
Mohammed Rafii-El-Idrissi Benhnia ◽  
Megan M. McCausland ◽  
John Laudenslager ◽  
Steven W. Granger ◽  
Sandra Rickert ◽  
...  
Keyword(s):  
Antibody Response ◽  
Vaccinia Virus ◽  
Smallpox Vaccine ◽  
Human Antibody ◽  
Human Mabs ◽  
Complement Components ◽  
Vaccinia Virion ◽  
Virion Antigen

ABSTRACT Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro—and protection in vivo in a mouse model—by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.

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