Crystal structure of a complement control protein that regulates both pathways of complement activation and binds heparan sulfate proteoglycans

Vaccinia virus complement control protein (VCP) inhibits both pathways of complement activation through binding the third and fourth components. A homolog of mammalian regulators of complement activation, its ability to bind heparin endows VCP with additional activities of significance to viral infectivity. The structure of VCP reveals a highly extended molecule with a putative heparin recognition site at its C-terminal end. A second cluster of positive charges provides a possibly overlapping binding site for both heparin and complement components. Experiments suggested by the structure indicate that VCP can bind heparin and control complement simultaneously. This, the structure of any intact regulator of complement activation, along with attendant functional insights, will stimulate the design of new therapeutic inhibitors of complement.     

Vaccinia virus complement control protein is monomeric,and retains structural and functional integrity after exposure to adverse conditions

Vaccinia virus complement control protein (VCP) possesses the ability to inhibit both classical and alternative pathways of complement activation, as well as bind to heparin or heparan sulfate proteoglycans, making it a unique multifunctional protein with therapeutic potential. Recently, the structure of the complete molecule of VCP was determined by X-ray crystallography. Two or three VCP molecules were packed within the unit cells of both crystal forms. Using gel filtration, VCP has now been shown to exist as a monomer in solution. To test the stability of this molecule, VCP was studied by nuclear magnetic resonance (NMR) over a range of temperatures and by differential scanning calorimetry (DSC). It was also subjected to adverse physical conditions, including, freeze-thawing, changes in pH, changes in temperature, and storage at room temperature. VCP melts fully reversibly, and it maintained its 3-D structure and the ability to inhibit serum-induced hemolysis of sheep red blood cells after exposure to many extreme conditions. The robustness of VCP may be rationalized in terms of its architecture.     

Vaccinia virus complement control protein is monomeric,and retains structural and functional integrity after exposure to adverse conditions

Vaccinia virus complement control protein (VCP) possesses the ability to inhibit both classical and alternative pathways of complement activation, as well as bind to heparin or heparan sulfate proteoglycans, making it a unique multifunctional protein with therapeutic potential. Recently, the structure of the complete molecule of VCP was determined by X-ray crystallography. Two or three VCP molecules were packed within the unit cells of both crystal forms. Using gel filtration, VCP has now been shown to exist as a monomer in solution. To test the stability of this molecule, VCP was studied by nuclear magnetic resonance (NMR) over a range of temperatures and by differential scanning calorimetry (DSC). It was also subjected to adverse physical conditions, including, freeze–thawing, changes in pH, changes in temperature, and storage at room temperature. VCP melts fully reversibly, and it maintained its 3-D structure and the ability to inhibit serum-induced hemolysis of sheep red blood cells after exposure to many extreme conditions. The robustness of VCP may be rationalized in terms of its architecture.     

Influence of glycosylation and oligomerization of vaccinia virus complement control protein on level and pattern of functional activity and immunogenicity

Vaccinia virus complement control protein (VCP) is one of the proteins encoded by vaccinia virus to modulate the host inflammatory response. VCP modulates the inflammatory response and protects viral habitat by inhibiting the classical and the alternative pathways of complement activation. The extended structure of VCP, mobility between its sequential domains, charge distribution and type of residues at the binding regions are factors that have been identified to influence its ability to bind to complement proteins. We report that a Lister strain of vaccinia virus encodes a VCP homolog (Lis VCP) that is functional, glycosylated, has two amino acids less than the well-characterized VCP from vaccinia virus WR strain (WR VCP), and the human smallpox inhibitor of complement enzymes (SPICE) from variola virus. The glycosylated VCP of Lister is immunogenic in contrast to the weak immunogenicity of the nonglycosylated VCP. Lis VCP is the only orthopoxviral VCP homolog found to be glycosylated, and we speculate that glycosylation influences its pattern of complement inhibition. We also correlate dimerization of VCP observed only in mammalian and baculovirus expression systems to higher levels of activity than monomers, observed in the yeast expression system.     

Domain swapping reveals complement control protein modules critical for imparting cofactor and decay-accelerating activities in vaccinia virus complement control protein

Vaccinia virus encodes a structural and functional homolog of human complement regulators named vaccinia virus complement control protein (VCP). This four-complement control protein domain containing secretory protein is known to inhibit complement activation by supporting the factor I-mediated inactivation of complement proteins, proteolytically cleaved form of C3 (C3b) and proteolytically cleaved form of C4 (C4b) (termed cofactor activity), and by accelerating the irreversible decay of the classical and to a limited extent of the alternative pathway C3 convertases (termed decay-accelerating activity [DAA]). In this study, we have mapped the VCP domains important for its cofactor activity and DAA by swapping its individual domains with those of human decay-accelerating factor (CD55) and membrane cofactor protein (MCP; CD46). Our data indicate the following: 1) swapping of VCP domain 2 or 3, but not 1, with homologous domains of decay-accelerating factor results in loss in its C3b and C4b cofactor activities; 2) swapping of VCP domain 1, but not 2, 3, or 4 with corresponding domains of MCP results in abrogation in its classical pathway DAA; and 3) swapping of VCP domain 1, 2, or 3, but not 4, with homologous MCP domains have marked effect on its alternative pathway DAA. These functional data together with binding studies with C3b and C4b suggest that in VCP, domains 2 and 3 provide binding surface for factor I interaction, whereas domain 1 mediates dissociation of C2a and Bb from the classical and alternative pathway C3 convertases, respectively.     

Kinetic analysis of the interactions between vaccinia virus complement control protein and human complement proteins C3b and C4b

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.     

Structural basis for antagonism by suramin of heparin binding to vaccinia complement protein

Suramin is a competitive inhibitor of heparin binding to many proteins, including viral envelope proteins, protein tyrosine phosphatases, and fibroblast growth factors (FGFs). It has been clinically evaluated as a potential therapeutic in treatment of cancers caused by unregulated angiogenesis, triggered by FGFs. Although it has shown clinical promise in treatment of several cancers, suramin has many undesirable side effects. There is currently no experimental structure that reveals the molecular interactions responsible for suramin inhibition of heparin binding, which could be of potential use in structure-assisted design of improved analogues of suramin. We report the structure of suramin, in complex with the heparin-binding site of vaccinia virus complement control protein (VCP), which interacts with heparin in a geometrically similar manner to many FGFs. The larger than anticipated flexibility of suramin manifested in this structure, and other details of VCP-suramin interactions, might provide useful structural information for interpreting interactions of suramin with many proteins.     

Solution structure and dynamics of the central CCP module pair of a poxvirus complement control protein

The complement control protein (CCP) module (also known as SCR, CCP or sushi domain) is prevalent amongst proteins that regulate complement activation. Functional and mutagenesis studies have shown that in most cases two or more neighbouring CCP modules form specific binding sites for other molecules. Hence the orientation in space of a CCP module with respect to its neighbours and the flexibility of the intermodular junction are likely to be critical for function. Vaccinia virus complement control protein (VCP) is a complement regulatory protein composed of four tandemly arranged CCP modules. The solution structure of the carboxy-terminal half of this protein (CCP modules 3 and 4) has been solved previously. The structure of the central portion (modules 2 and 3, VCP approximately 2,3) has now also been solved using NMR spectroscopy at 37 degrees C. In addition, the backbone dynamics of VCP approximately 2,3 have been characterised by analysis of its (15)N relaxation parameters. Module 2 has a typical CCP module structure while module 3 in the context of VCP approximately 2,3 has some modest but significant differences in structure and dynamics to module 3 within the 3,4 pair. Modules 2 and 3 do not share an extensive interface, unlike modules 3 and 4. Only two possible NOEs were identified between the bodies of the modules, but a total of 40 NOEs between the short intermodular linker of VCP approximately 2,3 and the bodies of the two modules determines a preferred, elongated, orientation of the two modules in the calculated structures. The anisotropy of rotational diffusion has been characterised from (15)N relaxation data, and this indicates that the time-averaged structure is more compact than suggested by (1)H-(1)H NOEs. The data are consistent with the presence of many intermodular orientations, some of which are kinked, undergoing interconversion on a 10(-8)-10(-6) second time-scale. A reconstructed representation of modules 2-4 allows visualisation of the spatial arrangement of the 11 substitutions that occur in the more potent complement inhibitor from Variola (small pox) virus.     

Mapping of regions within the vaccinia virus complement control protein involved in dose-dependent binding to key complement components and heparin using surface plasmon resonance

The vaccinia virus complement control protein (VCP) is involved in modulating the host inflammatory response by blocking both pathways of complement activity through its ability to bind C3b and C4b. Other activities arise from VCP's ability to strongly bind heparin. To map regions within VCP involved in binding complement and heparin experimentally, surface plasmon resonance (SPR) and recombinantly expressed VCP (rVCP) constructs were employed. Using C3b or heparin as the immobilized ligand, various rVCP constructs were tested for their ability to bind. Results suggest that VCP is the smallest functional unit able to bind C3b, thereby blocking complement activity, and only a single site, the large basic region near the C-terminus, is involved in heparin binding. Kinetic analysis was also performed to determine the relative binding affinities between rVCP and complement (C3-MA and C4b), as well as rVCP and heparin. rVCP was found to possess a significantly greater affinity for C3-MA than C4b, as indicated by the 1.50e3-fold greater association rate constant (k(a)). This study provides insights for the design of new therapeutic proteins capable of blocking complement activation.     

Identification of complement regulatory domains in vaccinia virus complement control protein

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.     

Functional characterization of the complement control protein homolog of herpesvirus saimiri: ARG-118 is critical for factor I cofactor activities

Herpesvirus saimiri (HVS) is a lymphotropic virus that causes T-cell lymphomas in New World primates. It encodes a structural homolog of complement control proteins named complement control protein homolog (CCPH). Previously, CCPH has been shown to inhibit C3d deposition on target cells exposed to complement. Here we have studied the mechanism by which it inactivates complement. We have expressed the soluble form of CCPH in Escherichia coli, purified to homogeneity and compared its activity to vaccinia virus complement control protein (VCP) and human complement regulators factor H and soluble complement receptor 1. The expressed soluble form of CCPH bound to C3b (KD = 19.2 microm) as well as to C4b (KD = 0.8 microm) and accelerated the decay of the classical/lectin as well as alternative pathway C3-convertases. In addition, it also served as factor I cofactor and supported factor I-mediated inactivation of both C3b and C4b. Time course analysis indicated that although its rate of inactivation of C4b is comparable with VCP, it is 14-fold more potent than VCP in inactivating C3b. Site-directed mutagenesis revealed that Arg-118, which corresponds to Lys-120 of variola virus complement regulator SPICE (a residue critical for its enhanced C3b cofactor activity), contributes significantly in enhancing this activity. Thus, our data indicate that HVS encodes a potent complement inhibitor that allows HVS to evade the host complement attack.     

The Vaccinia virus complement control protein modulates adaptive immune responses during infection

Complement activation is an important component of the innate immune response against viral infection and also shapes adaptive immune responses. Despite compelling evidence that complement activation enhances T cell and antibody (Ab) responses during viral infection, it is unknown whether inhibition of complement by pathogens alters these responses. Vaccinia virus (VACV) modulates complement activation by encoding a complement regulatory protein called the vaccinia virus complement control protein (VCP). Although VCP has been described as a virulence factor, the mechanisms by which VCP enhances VACV pathogenesis have not been fully defined. Since complement is necessary for optimal adaptive immune responses to several viruses, we hypothesized that VCP contributes to pathogenesis by modulating anti-VACV T cell and Ab responses. In this study, we used an intradermal model of VACV infection to compare pathogenesis of wild-type virus (vv-VCPwt) and a virus lacking VCP (vv-VCPko). vv-VCPko formed smaller lesions in wild-type mice but not in complement-deficient mice. Attenuation of vv-VCPko correlated with increased accumulation of T cells at the site of infection, enhanced neutralizing antibody responses, and reduced viral titers. Importantly, depleting CD8(+) T cells together with CD4(+) T cells, which also eliminated T helper cell-dependent Ab responses, restored vv-VCPko to wild-type levels of virulence. These results suggest that VCP contributes to virulence by dampening both antibody and T cell responses. This work provides insight into how modulation of complement by poxviruses contributes to virulence and demonstrates that a pathogen-encoded complement regulatory protein can modulate adaptive immunity.     

Immunophysical properties and prediction of activities for vaccinia virus complement control protein and smallpox inhibitor of complement enzymes using molecular dynamics and electrostatics

We present immunophysical modeling for VCP, SPICE, and three mutants using MD simulations and Poisson-Boltzmann-type electrostatic calculations. VCP and SPICE are homologous viral proteins that control the complement system by imitating, structurally and functionally, natural regulators of complement activation. VCP and SPICE consist of four CCP modules connected with short flexible loops. MD simulations demonstrate that the rather complex modules of VCP/SPICE and their mutants exhibit a high degree of intermodular spatial mobility, which is affected by surface mutations. Electrostatic calculations using snapshots from the MD trajectories demonstrate variable spatial distribution of the electrostatic potentials, which suggests dynamic binding properties. We use covariance analysis to identify correlated modular oscillations. We also use electrostatic similarity indices to cluster proteins with common electrostatic properties. Our results are compared with experimental data to form correlations between the overall positive electrostatic potential of VCP/SPICE with binding and activity. We show how these correlations can be used to predict binding and activity properties. This work is expected to be useful for understanding the function of native CCP-containing regulators of complement activation and receptors and for the design of antiviral therapeutics and complement inhibitors.     

Identification of hot spots in the variola virus complement inhibitor (SPICE) for human complement regulation

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 approximately 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.     

Restoration of complement-enhanced neutralization of vaccinia virus virions by novel monoclonal antibodies raised against the vaccinia virus complement control protein

The vaccinia virus complement control protein (VCP) is secreted by infected cells and has been shown to inhibit complement activation through interactions with C3b/C4b. It contains four short consensus repeat (SCR) domains. It has been suggested that all four SCRs are required for VCP's activity. To elucidate which SCR domains are involved in abolishing complement-enhanced neutralization of vaccinia virus virions, we generated and characterized a panel of mouse monoclonal antibodies (MAbs) raised against VCP. Ten MAbs were isolated and all recognized VCP on Western blots under reducing conditions as well as native-bound VCP in a sandwich enzyme-linked immunosorbent assay. Three of the 10 MAbs (2E5, 3D1, and 3F11) inhibited VCP's abolition of complement-enhanced neutralization of vaccinia virus virions. These MAbs blocked the interaction of VCP with C3b/C4b. The seven remaining MAbs did not alter VCP function in the complement neutralization assay and recognized VCP bound to C3b/C4b. To understand MAb specificity and mode of interaction with VCP, we mapped the MAb binding regions on VCP. The seven nonblocking MAbs all bound to the first SCR of VCP. One of the blocking MAbs recognized SCR 2 while the other two recognized either SCR 4 or the junction between SCRs 3 and 4, indicating that structural elements involved in the interaction of VCP with C3b/C4b are located within SCR domains 2 and 3 and 4. These anti-VCP MAbs may have clinical significance as therapeutic inhibitors of VCP's complement control activity and may also offer a novel approach to managing vaccinia virus vaccine complications that occur from smallpox vaccination.     

Species selectivity in poxviral complement regulators is dictated by the charge reversal in the central complement control protein modules

Variola and vaccinia viruses, the two most important members of the family Poxviridae, are known to encode homologs of the human complement regulators named smallpox inhibitor of complement enzymes (SPICE) and vaccinia virus complement control protein (VCP), respectively, to subvert the host complement system. Intriguingly, consistent with the host tropism of these viruses, SPICE has been shown to be more human complement-specific than VCP, and in this study we show that VCP is more bovine complement-specific than SPICE. Based on mutagenesis and mechanistic studies, we suggest that the major determinant for the switch in species selectivity of SPICE and VCP is the presence of oppositely charged residues in the central complement control modules, which help enhance their interaction with factor I and C3b, the proteolytically cleaved form of C3. Thus, our results provide a molecular basis for the species selectivity in poxviral complement regulators.     

Cell surface expression of the vaccinia virus complement control protein is mediated by interaction with the viral A56 protein and protects infected cells from complement attack

The vaccinia virus (VACV) complement control protein (VCP) is the major protein secreted from VACV-infected cells. It has been reported that VCP binds to the surfaces of uninfected cells by interacting with heparan sulfate proteoglycans (HSPGs). In this study, we show that VCP is also expressed on the surfaces of infected cells and demonstrate that surface localization occurs independently of HSPGs. Since VCP does not contain a transmembrane domain, we hypothesized that VCP interacts with a membrane protein that localizes to the infected-cell surface. We show that the VACV A56 membrane protein is necessary for the cell surface expression of VCP and demonstrate that VCP and A56 interact in VACV-infected cells. Since the surface expression of VCP was abrogated by reducing agents, we examined the contribution of an unpaired cysteine residue on VCP to VCP surface expression and VCP's interaction with A56. To do this, we mutated the unpaired cysteine in VCP and generated a recombinant virus expressing the altered form of VCP. Following the infection of cells with the mutant virus, VCP was neither expressed on the cell surface nor able to interact with A56. Importantly, the cell surface expression of VCP was found to protect infected cells from complement-mediated lysis. Our findings suggest a new function for VCP that may be important for poxvirus pathogenesis and impact immune responses to VACV-based vaccines.     

Structural stability and heat-induced conformational change of two complement inhibitors: C4b-binding protein and factor H

The complement inhibitors C4b-binding protein (C4BP) and factor H (FH) both consist of complement control protein (CCP) domains. Here we examined the secondary structure of both proteins by circular dichroism and Fourier-transform infrared technique at temperatures ranging from 30 degrees C-90 degrees C. We found that predominantly beta-sheet structure of both proteins was stable up to 70 degrees C, and that a reversible conformational change toward alpha-helix was apparent at temperatures ranging from 70 degrees C to 90 degrees C. The ability of both proteins to inhibit complement was not impaired after incubation at 95 degrees C, exposure to extreme pH conditions, and storage at room temperature for several months. Similar remarkable stability was previously observed for vaccinia virus control protein (VCP), which is also composed of CCP domains; it therefore seems to be a general property of CCP-containing proteins. A typical CCP domain has a hydrophobic core, which is wrapped in beta-sheets and stabilized by two disulphide bridges. How the CCP domains tolerate harsh conditions is unclear, but it could be due to a combination of high content of prolines, hydrophobic residues, and the presence of two disulphide bridges within each domain. These findings are of interest because CCP-containing complement inhibitors have been proposed as clinical agents to be used to control unwanted complement activation that contributes to many diseases.     

Structure and regulatory profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and variola and evidence for dimer formation

The outbreak of monkeypox in the Unites States in the summer of 2003 was the first occurrence of this smallpox-like disease outside of Africa. This limited human epidemic resulted from cross-infection of prairie dogs by imported African rodents. Although there were no human fatalities, this outbreak illustrates that monkeypox is an emerging natural infection and a potential biological weapon. We characterized a virulence factor expressed by monkeypox (monkeypox inhibitor of complement enzymes or MOPICE). We also compared its structure and regulatory function to homologous complement regulatory proteins of variola (SPICE) and vaccinia (VCP). In multiple expression systems, 5-30% of MOPICE, SPICE, and VCP consisted of function-enhancing disulfide-linked homodimers. Mammalian cells infected with vaccinia virus also expressed VCP dimers. MOPICE bound human C3b/C4b intermediate to that of SPICE and VCP. Cofactor activity of MOPICE was similar to VCP, but both were approximately 100-fold less efficient than SPICE. SPICE and VCP, but not MOPICE, possessed decay-accelerating activity for the C3 and C5 convertases of the classical pathway. Additionally, all three regulators possessed heparin-binding capability. These studies demonstrate that MOPICE regulates human complement and suggest that dimerization is a prominent feature of these virulence factors. Thus, our data add novel information relative to the functional repertoire of these poxviral virulence factors. Furthermore, targeting and neutralizing these complement regulatory active sites via mAbs is a therapeutic approach that may enhance protection against smallpox.