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.

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.

Yellow fever virus NS2B-NS3 protease: characterization of charged-to-alanine mutant and revertant viruses and analysis of polyprotein-cleavage activities.

A series of 46 charged-to-alanine mutations in the yellow fever virus NS2B-NS3 protease, previously characterized in cell-free and transient cellular expression systems, was tested for their effects on virus recovery. Four distinct plaque phenotypes were observed in cell culture: parental plaque-size (13 mutants), reduced plaque-size (17 mutants), small plaque-size (8 mutants) and no plaque-formation (8 mutants). No mutants displayed any temperature sensitivity based on recovery of virus after RNA transfection at 32 versus 37 degrees C. Most small plaque-mutants were defective in growth efficiency compared with parental virus. However not all small plaque-mutants had defective 2B/3 cleavage, with some showing selective defects at other non-structural protein cleavage sites. Revertant viruses were recovered for six mutations that caused reduced plaque sizes. Same-site and second-site mutations occurred in NS2B, and one second-site mutation occurred in the NS3 protease domain. Some reversion mutations ameliorated defects in cleavage activity and plaque size caused by the original mutation. These data indicate that certain mutations that reduce NS2B-NS3 protease cleavage activity cause growth restriction of yellow fever virus in cell culture. However, for at least two mutations, processing defects other than impaired cleavage activity at the 2B/3 site may account for the mutant phenotype. The existence of reversion mutations primarily in NS2B rather than NS3, suggests that the protease domain is less tolerant of structural perturbation compared with the NS2B protein.

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.

Identification and characterization of nonsubstrate based inhibitors of the essential dengue and West Nile virus proteases.

The 72 known members of the flavivirus genus include lethal human pathogens such as Yellow Fever, West Nile, and Dengue viruses. There is at present no known chemotherapy for any flavivirus and no effective vaccines for most. A common genomic organization and molecular mechanisms of replication in hosts are shared by flaviviruses with a viral serine protease playing a pivotal role in processing the viral polyprotein into component polypeptides, an obligatory step in viral replication. Using the structure of the dengue serine protease complexed with a protein inhibitor as a template, we have identified five compounds, which inhibit the enzyme. We also describe parallel inhibitory activity of these compounds against the West Nile virus Protease. A few of the compounds appear to provide a template for design of more potent and specific inhibitors of the dengue and West Nile virus proteases. Sequence similarities among flaviviral proteases suggests that such compounds might also possibly inhibit other flaviviral proteases.

Modulation of the nucleoside triphosphatase/RNA helicase and 5'-RNA triphosphatase activities of Dengue virus type 2 nonstructural protein 3 (NS3) by interaction with NS5, the RNA-dependent RNA polymerase.

Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase and a 5'-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5'-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.

Structure of vaccinia complement protein in complex with heparin and potential implications for complement regulation.

Vaccinia virus complement control protein (VCP), a homolog of the regulators of the complement activation family of proteins, inhibits complement activation through mechanisms similar to human fluid-phase complement regulators factor H and C4b-binding protein. VCP has a heparin-binding activity that assists vaccinia in host interactions. Interaction with cell-surface polyanions like heparin is centrally important in the functioning of fluid-phase complement regulators and is the basis of host-target discrimination by the alternative pathway. We report the structure of VCP in complex with a heparin decasaccharide, which reveals changes in VCP that might be pertinent to complement regulation. Properties that VCP shares with fluid-phase complement regulators suggest that such conformational changes may be of relevance in the functioning of other complement regulators. Additionally, comparison of VCP-heparin interactions with potentially similar interactions in factor H might enable understanding of the structural basis of familial hemolytic uremic syndrome, attributed to mutational disruption of heparin and C3b binding by factor H.