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

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.

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Identification of Hot Spots in the Variola Virus Complement Inhibitor (SPICE) for Human Complement Regulation

Journal of Virology ◽

10.1128/jvi.01935-07 ◽

2008 ◽

Vol 82 (7) ◽

pp. 3283-3294 ◽

Cited By ~ 30

Author(s):

Viveka Nand Yadav ◽

Kalyani Pyaram ◽

Jayati Mullick ◽

Arvind Sahu

Keyword(s):

Hot Spots ◽

Classical Pathway ◽

Variola Virus ◽

Amino Acid Residues ◽

Human Complement ◽

Binding Ability ◽

Complement Control Protein ◽

Functional Advantage ◽

Complement Regulator ◽

Control Protein

ABSTRACT Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is ∼90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement.

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Interaction of vaccinia virus complement control protein with human complement proteins: Factor I-mediated degradation of C3b to iC3b1 inactivates the alternative complement pathway

Molecular Immunology ◽

10.1016/s0161-5890(98)90695-2 ◽

1998 ◽

Vol 35 (6-7) ◽

pp. 371 ◽

Cited By ~ 14

Author(s):

Arvind Sahu ◽

Stuart N. Isaacs ◽

Athena M. Soulika ◽

John D. Lambris

Keyword(s):

Vaccinia Virus ◽

Alternative Complement Pathway ◽

Human Complement ◽

Complement Pathway ◽

Complement Control Protein ◽

Complement Proteins ◽

Factor I ◽

Alternative Complement ◽

Control Protein

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The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

Frontiers in Immunology ◽

10.3389/fimmu.2021.607641 ◽

2021 ◽

Vol 12 ◽

Author(s):

Wen Q. Qiu ◽

Shaopeiwen Luo ◽

Stefanie A. Ma ◽

Priyanka Saminathan ◽

Herman Li ◽

...

Keyword(s):

Alternative Pathway ◽

Family Members ◽

Dendritic Morphology ◽

Classical Pathway ◽

Strong Inhibitor ◽

Alternative Pathways ◽

Complement Control Protein ◽

Factor I ◽

Complement Regulator ◽

The Brain

The Sez6 family consists of Sez6, Sez6L, and Sez6L2. Its members are expressed throughout the brain and have been shown to influence synapse numbers and dendritic morphology. They are also linked to various neurological and psychiatric disorders. All Sez6 family members contain 2-3 CUB domains and 5 complement control protein (CCP) domains, suggesting that they may be involved in complement regulation. We show that Sez6 family members inhibit C3b/iC3b opsonization by the classical and alternative pathways with varying degrees of efficacy. For the classical pathway, Sez6 is a strong inhibitor, Sez6L2 is a moderate inhibitor, and Sez6L is a weak inhibitor. For the alternative pathway, the complement inhibitory activity of Sez6, Sez6L, and Sez6L2 all equaled or exceeded the activity of the known complement regulator MCP. Using Sez6L2 as the representative family member, we show that it specifically accelerates the dissociation of C3 convertases. Sez6L2 also functions as a cofactor for Factor I to facilitate the cleavage of C3b; however, Sez6L2 has no cofactor activity toward C4b. In summary, the Sez6 family are novel complement regulators that inhibit C3 convertases and promote C3b degradation.

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The Sez6 family inhibits complement at the level of the C3 convertase

10.1101/2020.09.11.292623 ◽

2020 ◽

Cited By ~ 1

Author(s):

Wen Q. Qiu ◽

Shaopeiwen Luo ◽

Stefanie A. Ma ◽

Priyanka Saminathan ◽

Herman Li ◽

...

Keyword(s):

Alternative Pathway ◽

Family Members ◽

Dendritic Morphology ◽

Classical Pathway ◽

Strong Inhibitor ◽

Complement Control Protein ◽

Factor I ◽

The Brain ◽

Representative Family ◽

Control Protein

AbstractThe Sez6 family consists of Sez6, Sez6L, and Sez6L2. Its members are expressed throughout the brain and have been shown to influence synapse numbers and dendritic morphology. They are also linked to various neurological and psychiatric disorders. All Sez6 family members contain 2-3 CUB domains and 5 complement control protein (CCP) domains, suggesting that they may be involved in complement regulation. We show that all Sez6 family members inhibit C3 deposition by the classical and alterative pathways with varying degrees of efficacy. For the classical pathway, Sez6 is a strong inhibitor, Sez6L2 is a moderate inhibitor, and Sez6L is a weak inhibitor. Using Sez6L2 as the representative family member, we show that it specifically deactivates C3 convertases by accelerating the decay or dissociation of the C3 convertase components. Sez6L2 also deactivates C3 convertases of the alternative pathway by serving as a cofactor for Factor I to facilitate the cleavage of C3b. However, Sez6L2 has no cofactor activity toward C4b. In summary, the Sez6 family are novel complement regulators that inhibit C3 convertases.

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

Clinical and Vaccine Immunology ◽

10.1128/cvi.00347-14 ◽

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.

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Identification of Complement Regulatory Domains in Vaccinia Virus Complement Control Protein

Journal of Virology ◽

10.1128/jvi.79.19.12382-12393.2005 ◽

2005 ◽

Vol 79 (19) ◽

pp. 12382-12393 ◽

Cited By ~ 36

Author(s):

Jayati Mullick ◽

John Bernet ◽

Yogesh Panse ◽

Sharanabasava Hallihosur ◽

Akhilesh K. Singh ◽

...

Keyword(s):

Vaccinia Virus ◽

Alternative Pathway ◽

Deletion Mutants ◽

Classical Pathway ◽

Target Proteins ◽

Complement Control Protein ◽

Functional Activities ◽

Regulatory Domains ◽

Optimal Activity ◽

Control Protein

ABSTRACT Vaccinia virus encodes a homolog of the human complement regulators named vaccinia virus complement control protein (VCP). It is composed of four contiguous complement control protein (CCP) domains. Previously, VCP has been shown to bind to C3b and C4b and to inactivate the classical and alternative pathway C3 convertases by accelerating the decay of the classical pathway C3 convertase and (to a limited extent) the alternative pathway C3 convertase, as well as by supporting the factor I-mediated inactivation of C3b and C4b (the subunits of C3 convertases). In this study, we have mapped the CCP domains of VCP important for its cofactor activities, decay-accelerating activities, and binding to the target proteins by utilizing a series of deletion mutants. Our data indicate the following. (i) CCPs 1 to 3 are essential for cofactor activity for C3b and C4b; however, CCP 4 also contributes to the optimal activity. (ii) CCPs 1 to 2 are enough to mediate the classical pathway decay-accelerating activity but show very minimal activity, and all the four CCPs are necessary for its efficient activity. (iii) CCPs 2 to 4 mediate the alternative pathway decay-accelerating activity. (iv) CCPs 1 to 3 are required for binding to C3b and C4b, but the presence of CCP 4 enhances the affinity for both the target proteins. These results together demonstrate that the entire length of the protein is required for VCP's various functional activities and suggests why the four-domain structure of viral CCP is conserved in poxviruses.

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