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Polly Roy

Publications and source records attributed to Polly Roy.

At least 19 recordsLinked to original sources
PubMed2004

Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system.

Virus-like particles (VLPs) produced by recombinant expression of the major viral structural proteins could be an attractive method for severe acute respiratory syndrome (SARS) control. In this study, using the baculovirus system, we generated recombinant viruses that expressed S, E, M and N structural proteins of SARS-CoV either individually or simultaneously. The expression level, size and authenticity of each recombinant SARS-CoV protein were determined. In addition, immunofluorescence and FACS analysis confirmed the cell surface expression of the S protein. Co-infections of insect cells with two recombinant viruses demonstrated that M and E could assemble readily to form smooth surfaced VLPs. On the other hand, simultaneous high level expression of S, E and M by a single recombinant virus allowed the very efficient assembly and release of VLPs. These data demonstrate that the VLPs are morphological mimics of virion particles. The high level expression of VLPs with correct S protein conformation by a single recombinant baculovirus offers a potential candidate vaccine for SARS.

Animals↗
PubMed2004

Mapping the assembly pathway of Bluetongue virus scaffolding protein VP3.

The structure of the Bluetongue virus (BTV) core and its outer layer VP7 has been solved by X-ray crystallography, but the assembly intermediates that lead to the inner scaffolding VP3 layer have not been defined. In this report, we addressed two key questions: (a) the role of VP3 amino terminus in core assembly and its interaction with the transcription complex (TC) components; and (b) the assembly intermediates involved in the construction of the VP3 shell. To do this, deletion mutants in the amino terminal and decamer-decamer interacting region of VP3 (DeltaDD) were generated, expressed in insect cells using baculovirus expression systems, and their ability to assemble into core-like particles (CLPs) and to incorporate the components of TC were investigated. Deletion of the N-terminal 5 (Delta5N) or 10 (Delta10N) amino acids did not affect the ability to assemble into CLPs in the presence of VP7 although the cores assembled using the 10 residue mutant (Delta10N) deletion were very unstable. Removal of five residues also did not effect incorporation of the internal VP1 RNA polymerase and VP4 mRNA capping enzyme proteins of the TC. Removal of the VP3-VP3 interacting domain (DeltaDD) led to failure to assemble into CLPs yet retained interaction with VP1 and VP4. In solution, purified DeltaDD mutant protein readily multimerized into dimers, pentamers, and decamers, suggesting that these oligomers are the authentic assembly intermediates of the subcore. However, unlike wild-type VP3 protein, the dimerization domain-deleted assembly intermediates were found to have lost RNA binding ability. Our study emphasizes the requirement of the N-terminus of VP3 for binding and encapsidation of the TC components, and defines the role of the dimerization domain in subcore assembly and RNA binding.

Bluetongue virus↗
PubMed2004

A capsid protein of nonenveloped Bluetongue virus exhibits membrane fusion activity.

The outer capsid layer of Bluetongue virus, a member of the nonenveloped Reoviridae family, is composed of two proteins, a receptor-binding protein, VP2, and a second protein, VP5, which shares structural features with class I fusion proteins of enveloped viruses. In the replication cycle of Bluetongue virus VP5 acts as a membrane permeabilization protein that mediates release of viral particles from endosomal compartments into the cytoplasm. Here, we show that VP5 can also act as a fusion protein and induce syncytium formation when it is fused to a transmembrane anchor and expressed on the cell surface. Fusion activity is strictly pH-dependent and is triggered by short exposure to low pH. No cell-cell fusion is observed at neutral pH. Deletion of the first 40 amino acids, which can fold into two amphipathic helices, abolishes fusion activity. Syncytium formation by VP5 is inhibited in the presence of VP2 when it is expressed in a membrane-anchored form. The data indicate an interaction between the outer capsid protein VP2 and VP5 and show that VP5 undergoes pH-dependent conformational changes that render it capable of interacting with cellular membranes. More importantly, our data show that a membrane permeabilization protein of a nonenveloped virus can evolve into a fusion protein by the addition of an appropriate transmembrane anchor. The results strongly suggest that the mechanism of membrane permeabilization by VP5 and membrane fusion by viral fusion proteins require similar structural features and conformational changes.

Amino Acid Motifs↗
PubMed2004

Role of an arbovirus nonstructural protein in cellular pathogenesis and virus release.

The insect-borne Bluetongue virus (BTV) is considered the prototypic Orbivirus, a member of the Reovirus family. One of the hallmarks of Orbivirus infection is the production of large numbers of intracellular tubular structures of unknown function. For BTV these structures are formed as the polymerization product of a single 64-kDa nonstructural protein, NS1, encoded by the viral double-stranded RNA genome segment 6. Although the NS1 protein is the most abundant viral protein synthesized in infected cells, its function has yet to be determined. One possibility is that NS1 tubules may be involved in the translocation of newly formed viral particles to the plasma membrane, and NS1-specific monoclonal antibodies have been shown to react with viral particles leaving infected cells. In the present study we generated a mammalian cell line that expresses a recombinant single-chain antibody fragment (scFv) derived from an NS1-specific monoclonal antibody (10B1) and analyzed the effect that this intracellular antibody has on BTV replication. Normally, BTV infection of mammalian cells in culture results in a severe cytopathic effect within 24 to 48 h postinfection manifested by cell rounding, apoptosis, and lytic release of virions into the culture medium. However, infection of scFv-expressing cells results in a marked reduction in the stability of NS1 and formation of NS1 tubules, a decrease in cytopathic effect, an increased release of infectious virus into the culture medium, and budding of virions from the plasma membrane. These results suggest that NS1 tubules play a direct role in the cellular pathogenesis and morphogenesis of BTV.

Animals↗
PubMed2004

Interactions between the inner and outer capsids of bluetongue virus.

Bluetongue virus is a large and structurally complex virus composed of three concentric capsid layers that surround 10 segments of a double-stranded RNA genome. X-ray crystallographic analysis of the particles without the outer capsid layer has provided atomic structural details of VP3 and VP7, which form the inner two layers. However, limited structural information is available on the other five proteins in the virion-two of which are important for receptor recognition, hemagglutination, and membrane interaction-are in the outer layer, and the others, important for endogenous transcriptase activity are internal. Here we report the electron cryomicroscopy (cryo-EM) reconstruction of the mature particle, which shows that the outer layer has a unique non-T = 13 icosahedral organization consisting of two distinct triskelion and globular motifs interacting extensively with the underlying T = 13 layer. Comparative cryo-EM analysis of the recombinant corelike particles has shown that VP1 (viral polymerase) and VP4 (capping enzyme) together form a flower-shaped structure attached to the underside of VP3, directly beneath the fivefold axis. The structural data have been substantiated by biochemical studies demonstrating the interactions between the individual outer and inner capsid proteins.

Bluetongue virus↗
PubMed2004

Bluetongue virus outer capsid proteins are sufficient to trigger apoptosis in mammalian cells.

Bluetongue virus (BTV) is transmitted by Culicoides sp. biting midges to livestock, causing severe hemorrhagic disease in sheep, but is asymptomatic in the insect host. Similarly, BTV causes rapid cell death in infected mammalian cells in culture, whereas infections of insect cells are long-term and unapparent, despite productive virus replication. To assess whether apoptosis plays any role in these two distinct cell responses, we have investigated apoptosis in cultured insect and mammalian cells. Three different mammalian cell lines and three different insect cell lines including Culicoides variipennis (KC) cells were infected with BTV serotype 10, and the key apoptosis indicators of cell morphology, chromosomal DNA fragmentation, and caspase-3 activation were monitored. BTV infection induced apoptosis with the activation of the transcription factor nuclear factor kappaB (NF-kappaB) in all three mammalian cell lines. In contrast, no evidence for apoptosis was detected in any of the three insect cell lines in response to BTV infection. Using inhibitors of endosomal acidification and UV-inactivated virus, we established that virus uncoating, but not productive virus replication, is necessary for BTV to trigger apoptosis in mammalian cells. Intracellular expression of the viral outer capsid proteins VP2 and VP5 or the two major nonstructural proteins NS1 and NS2 was not sufficient to trigger an apoptotic response. However, extracellular treatment with a combination of purified recombinant VP2 and VP5, but not with each protein used separately, resulted in an apoptotic response. Virus- and VP2-VP5-stimulated apoptotic responses were both inhibited by inhibitors of endosomal acidification. Thus, for BTV the viral outer capsid proteins alone are sufficient to trigger apoptosis.

Animals↗
PubMed2004

Purified recombinant bluetongue virus VP1 exhibits RNA replicase activity.

The polymerase protein of all known double-stranded RNA (dsRNA) viruses is located within a complex subviral core particle that is responsible for transcription of the viral genome. For members of the family Reoviridae, this particle allows messenger sense RNA synthesis while sequestering the viral genome away from cellular dsRNA surveillance systems during infection of eukaryotic cells. The core particle of bluetongue virus (BTV) consists of the major structural proteins VP3 and VP7 and the minor enzymatic proteins VP1 (polymerase), VP4 (capping enzyme), and VP6 (helicase). In this report we have characterized fully processive dsRNA synthesis by VP1 from a viral plus-strand RNA template in the absence of the other proteins of the BTV core. This replicase activity consists of de novo initiation of synthesis, followed by elongation of the minus strand. Purified VP1 exhibits little sequence specificity for BTV plus-strand template, suggesting that the choice of viral over nonviral RNA template comes from its association with other proteins within the viral core.

Animals↗
PubMed2003

Viral infection switches non-plasmacytoid dendritic cells into high interferon producers.

Type I interferons (IFN-I) are important cytokines linking innate and adaptive immunity. Plasmacytoid dendritic cells make high levels of IFN-I in response to viral infection and are thought to be the major source of the cytokines in vivo. Here, we show that conventional non-plasmacytoid dendritic cells taken from mice infected with a dendritic-cell-tropic strain of lymphocytic choriomeningitis virus make similarly high levels of IFN-I on subsequent culture. Similarly, non-plasmacytoid dendritic cells secrete high levels of IFN-I in response to double-stranded RNA (dsRNA), a major viral signature, when the latter is introduced into the cytoplasm to mimic direct viral infection. This response is partially dependent on the cytosolic dsRNA-binding enzyme protein kinase R and does not require signalling through toll-like receptor (TLR) 3, a surface receptor for dsRNA. Furthermore, we show that sequestration of dsRNA by viral NS1 (refs 6, 7) explains the inability of conventional dendritic cells to produce IFN-I on infection with influenza. Our results suggest that multiple dendritic cell types, not just plasmacytoid cells, can act as specialized interferon-producing cells in certain viral infections, and reveal the existence of a TLR-independent pathway for dendritic cell activation that can be the target of viral interference.

3T3 Cells↗
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↗