Biomedical subjects

Polly Roy

Publications and source records attributed to Polly Roy.

28 records · Page 2Linked to original sources

PubMed2003

Sequence specificity in the interaction of Bluetongue virus non-structural protein 2 (NS2) with viral RNA.

The non-structural protein NS2 of Bluetongue virus (BTV) is synthesized abundantly in virus-infected cells and has been suggested to be involved in virus replication. The protein, with a high content of charged residues, possesses a strong affinity for single-stranded RNA species but, to date, all studies have failed to identify any specificity in the NS2-RNA interaction. In this report, we have examined, through RNA binding assays using highly purified NS2, the specificity of interaction with different single-stranded RNA (ssRNA) species in the presence of appropriate competitors. The data obtained show that NS2 indeed has a preference for BTV ssRNA over nonspecific RNA species and that NS2 recognizes a specific region within the BTV10 segment S10. The secondary structure of this region was determined and found to be a hairpin-loop with substructures within the loop. Modification-inhibition experiments highlighted two regions within this structure that were protected from ribonuclease cleavage in the presence of NS2. Overall, these data imply that a function of NS2 may be to recruit virus messenger RNAs (that also act as templates for synthesis of genomic RNAs) selectively from other RNA species within the infected cytosol of the cell during virus replication.

Animals↗

PubMed2003

Virus-like particles as immunogens.

Subunit vaccines based on recombinant proteins can suffer from poor immunogenicity owing to incorrect folding of the target protein or poor presentation to the immune system. Virus-like particles (VLPs) represent a specific class of subunit vaccine that mimic the structure of authentic virus particles. They are recognized readily by the immune system and present viral antigens in a more authentic conformation than other subunit vaccines. VLPs have therefore shown dramatic effectiveness as candidate vaccines. Here, we review the current status of VLPs as vaccines, and discuss the characteristics and problems associated with producing VLPs for different viruses.

Adjuvants, Immunologic↗

PubMed2003

Intermolecular interactions in a two-layered viral capsid that requires a complex symmetry mismatch.

The surface of the bluetongue virus core forms a T=13 quasiequivalent icosahedral protein shell with 260 trimers of a single gene product: VP7 protein. Underneath is a smooth layer, made up of VP3 protein, which appears to guide and nucleate the assembly of VP7 trimers. The contacts between the two shells are extensive but nonspecific, and construction of the T=13 icosahedral shell requires polymorphism in the association of the VP7 subunits, each of which has two domains that contribute to trimer formation. We used structural and relative sequence information to guide an investigation of how such a complex structure is achieved during virus assembly and what residues are required to form a stable capsid. Fifteen single or multiple site-specific substitution mutations were introduced into the helical domain of VP7, which is closely associated with the VP3 layer, and the effects on capsid assembly were analyzed. Our data show that both the position and the nature of single residues are critical for the attachment of VP7 to VP3 and that formation of a stable VP7 lattice is not the automatic consequence of trimer formation.

Amino Acid Sequence↗

PubMed2003

Defining the structure-function relationships of bluetongue virus helicase protein VP6.

The VP6 protein of bluetongue virus possesses a number of activities, including nucleoside triphosphatase, RNA binding, and helicase activity (N. Stauber, J. Martinez-Costas, G. Sutton, K. Monastyrskaya, and P. Roy, J. Virol. 71:7220-7226, 1997). Although the enzymatic functions of the protein have been documented, a detailed structure and function study has not been completed and the oligomeric form of the protein in solution has not been described. In this study, we have characterized VP6 activity by creating site-directed mutations in the putative functional helicase domains. Mutant proteins were expressed at high levels in an insect cell by using recombinant baculoviruses purified and analyzed for ATP binding, ATP hydrolysis, and RNA unwinding activities. UV cross-linking experiments indicated that the lysine residue in the conserved motif AXXGXGK(110)V is directly involved in ATP binding, whereas mutant R(205)Q in the arginine-rich motif ER(205)XGRXXR bound ATP at a level comparable to that of the wild-type protein. The RNA binding activity was drastically altered in the R(205)Q mutant and was also affected in the K(110)N mutant. Helicase activity was altered in both mutants. The mutation E(157)N in the DEXX sequence, presumed to act as a Walker B motif, showed an intermediate activity, implying that this motif does not play a crucial role in VP6 function. Purified protein demonstrated stable oligomers with a ring-like morphology in the presence of nucleic acids similar to those shown by other helicases. Gel filtration chromatography, native gel electrophoresis, and glycerol gradient analysis clearly indicated multiple oligomeric forms of VP6.

Adenosine Triphosphatases↗

PubMed2002

Virus-derived tubular structure displaying foreign sequences on the surface elicit CD4+ Th cell and protective humoral responses.

Particulate vector systems for the presentation of immunogenic epitopes provide an alternate and powerful approach for the delivery of immunogens of interest. In this article, we have exploited a viral protein of unknown function, bluetongue virus (BTV) nonstructural protein NS1, which forms distinct tubular aggregates in infected cells, as an immunogen delivery system. Tubules are helical assemblies of NS1 protein that present the C-terminus of the protein to the outer edge effectively displaying appended residues in a regular and repeating array akin to the coat of a filamentous phage. To assess the breadth of response induced following tubule-based immunization, two different immunodominant foreign peptides were inserted at the C-terminus of NS1 and chimeric tubules generated following expression in the baculovirus expression system. Both constructs, one carrying a peptide of foot and mouth disease virus (FMDV) (aa 135-144 of VP1) and the other, a peptide of influenza A virus (aa 186-205 of HA), effectively assembled into tubules and were easily purified. Subsequently, using in vitro assay systems, we demonstrated that each purified chimeric particle was capable of eliciting strong immune responses. Further, NS1-FMDV chimeric tubules could induce a potent immune response that could protect against disease.

Animals↗

PubMed2002

The membrane trafficking protein calpactin forms a complex with bluetongue virus protein NS3 and mediates virus release.

Bluetongue virus, an arbovirus of the Orbivirus genus, infects and replicates in both insect and mammalian cells. However, the cytopathic effect (cpe) on each host is very different. Mammalian cells show substantial cpe, most likely a result of the mechanism of virus release, whereas insect cells show little cpe and appear to release virus without cell lysis. Expression analysis of each infected cell type shows one protein, the nonstructural (NS) protein NS3, to be differentially expressed in the different cell types, suggesting it may act in the virus egress pathway. The molecular basis of such an interaction, however, has never been clear. Here, by using yeast two-hybrid analysis, we show that NS3 interacts with a cellular protein p11 (calpactin light chain), part of the annexin II complex that is involved in exocytosis. We map the NS3 region of interaction with p11 to a 13-residue peptide found at the N terminus of the protein and show it effectively competes with p36 (annexin II heavy chain) for p11 ligand binding. Further, we show that the C-terminal domain of NS3 interacts with VP2, the outermost protein of the fully assembled virus particle, suggesting that NS3 forms a bridging molecule that draws assembled virus into contact with the cellular export machinery. Our data describe the first host protein involvement in orbivirus egress and provide new insights into understanding arbovirus interactions with their hosts.

Amino Acid Motifs↗

PubMed2002

Comparative analysis of the roles of simian immunodeficiency and bovine leukemia virus matrix proteins in Gag assembly in insect cells.

The role of the matrix (MA) domain of simian immunodeficiency virus (SIV) and bovine leukaemia virus (BLV) Gag in the assembly of virus-like particles (VLP) in insect cells has been investigated. Wild-type SIV and BLV Gag assembled to form discrete VLP structures typical of many retroviruses analysed by similar systems. When amino acids predicated by the three-dimensional structure to be at the interface of SIV MA monomers were deleted, VLP assembly was abolished consistent with a role for MA multimerization in assembly. When amino acids predicted to be in the analogous positions in BLV MA were mutated, however, VLP assembly was not affected. These data indicate that the models of assembly derived from one model retrovirus may not necessarily apply to more distantly related viruses despite the structural similarity present in equivalent Gag domains.

Animals↗

PubMed2002

Induction of HLA-A2-restricted CTL responses by a tubular structure carrying human melanoma epitopes.

Epitope-based vaccination strategies designed to induce strong tumor-specific CD8(+) T cell responses are being widely considered for cancer immunotherapy. Here, two recombinant tubular structures, NS1-Mela 1 and NS1-Mela 2, carrying, respectively two HLA-A2 epitopes derived from human melanoma antigens were constructed and their capability to induce CTL responses in vivo were studied in HLA-A2 transgenic mice. Strong CTL responses specific for GnT-V/NA 17-A and gp100 (154-162) epitopes were generated in HLA-A2 transgenic mice immunized by the construct NS1-Mela l carrying these two epitopes. The second construct NS1-Mela 2 carrying both Tyrosinase (369-377Da) and Melan-A/Mart-1 (27-35) epitopes induced a weak Tyrosinase-specific CTL response in mice but failed to induce specific CTL responses against the Melan-A/Mart-1 (27-35) epitope in the tested mice. Thus, recombinant tubular structures containing multiple tumoral epitopes may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.

Amino Acid Sequence↗

PubMed2002

Induction of protective antiviral cytotoxic T cells by a tubular structure capable of carrying large foreign sequences.

Bluetongue virus (BTV) produces large numbers of tubules during infection which are formed by a single virus coded non-structural protein, NS1. The NS1 protein has been fused with full length green fluorescent protein (GFP) and was shown to retain the capacity to form tubules when expressed in heterologous expression systems. Moreover, recombinant purified chimeric tubules were demonstrated to be internalized by macrophages and dendritic cells. The ability of such chimeric tubules to induce protective cytotoxic T lymphocytes (CTL) responses has been assessed by generating chimeric tubules carrying a single CD8(+) T cell epitope from the lymphocytic choriomeningitis virus (LCMV) nucleoprotein. These chimeric tubules were recognized by MHC class I restricted T cell hybridoma in vitro and induced in vivo strong CD8(+) class I-restricted CTL responses in immunized mice. Further, the immunized mice were protected when challenged with a lethal dose of LCMV. This is the first study that demonstrates that the virus derived tubules synthesized by a recombinant non-structural protein carrying a single viral CTL epitope could induce protective immunity against a lethal viral challenge. Since recombinant tubules carrying large inserts can be purified at a large quantity from insect cells, they have potential to develop as safe multi-CTL vaccine delivery systems.

Animals↗

PubMed2000

NTP binding and phosphohydrolase activity associated with purified bluetongue virus non-structural protein NS2.

The bluetongue virus ssRNA-binding protein, NS2, is a phosphoprotein that forms viral inclusion bodies in infected cells. Recombinant NS2 was expressed in the baculovirus expression system and purified to homogeneity from insect cells. Purified NS2 bound nucleosides. Further investigation revealed that the protein bound ATP and GTP and could hydrolyse both nucleosides to their corresponding NMPs, with a higher efficiency for the hydrolysis of ATP. The increased efficiency of hydrolysis of ATP correlated with a higher binding affinity of NS2 for ATP than GTP. Ca(2+), Mg(2+) and Mn(2+) were able to function as the required divalent cation in the reactions. The phosphohydrolase activity was not sensitive to ouabain, an inhibitor of cellular ATPases, suggesting that this activity was not the result of a cellular contaminant.

Adenosine Triphosphate↗