Polly Roy

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.

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.

Bluetongue virus proteins.

Introduction. Bluetongue virus (BTV) is the prototype virus of the Orbivirus genus in the Reoviridae family. Orbiviruses that infect and are transmitted by arthropod vectors (e.g. gnats, ticks, mosquitoes, etc.) include viruses that may cause disease in their vertebrate hosts with serious economic consequences in some regions of the world. The BTV group, which consists of at least 24 different serotypes (BTV-1, -2, etc.), infects field and domestic animals (e.g. sheep and cattle), occasionally with high morbidity and mortality but often with almost no apparent clinical symptoms. Other orbiviruses that may cause disease in animals include African horse sickness virus (AHSV; nine serotypes, AHSV-1, -2, etc.) and epizootic haemorrhagic disease virus (EHDV; seven serotypes, EHDV-1, -2, etc.) of deer. Although BTV and AHSV were isolated in 1900, and initial morphological and biochemical characterization were reported as early as 1969 (see review article, Gorman, 1990), much of the current knowledge on the molecular biology, genome structure and encoded products has been obtained only recently. The data are predominantly based on research on BTV.

Virus-like particles as a vaccine delivery system: myths and facts.

Vaccines against viral disease have traditionally relied on attenuated virus strains or inactivation of infectious virus. Subunit vaccines based on viral proteins expressed in heterologous systems have been effective for some pathogens, but have often suffered from poor immunogenicity due to incorrect protein folding or modification. In this review we focus on a specific class of viral subunit vaccine that mimics the overall structure of virus particles and thus preserves the native antigenic conformation of the immunogenic proteins. These virus‑like particles (VLPs) have been produced for a wide range of taxonomically and structurally distinct viruses, and have unique advantages in terms of safety and immunogenicity over previous approaches. With new VLP vaccines for papillomavirus beginning to reach the market place we argue that this technology has now ‘come‑of‑age’ and must be considered a viable vaccine strategy.

Development of Reverse Genetics Systems for Bluetongue Virus: Recovery of Infectious Virus from Synthetic RNA Transcripts

ABSTRACT Bluetongue virus (BTV), an insect-vectored emerging pathogen of both wild ruminants and livestock, has had a severe economic impact in agriculture in many parts of the world. The investigation of BTV replication and pathogenesis has been hampered by the lack of a reverse genetics system. Recovery of infectious BTV is possible by the transfection of permissive cells with the complete set of 10 purified viral mRNAs derived in vitro from transcribing cores (M. Boyce and P. Roy, J. Virol. 81:2179-2186, 2007). Here, we report that in vitro synthesized T7 transcripts, derived from cDNA clones, can be introduced into the genome of BTV using a mixture of T7 transcripts and core-derived mRNAs. The replacement of genome segment 10 and the simultaneous replacement of segments 2 and 5 encoding the two immunologically important outer capsid proteins, VP2 and VP5, are described. Further, we demonstrate the recovery of infectious BTV entirely from T7 transcripts, proving that synthetic transcripts synthesized in the presence of cap analogue can functionally substitute for viral transcripts at all stages of the BTV replication cycle. The generation of BTV with a fully defined genome permits the recovery of mutations in a defined genetic background. The ability to generate specific mutants provides a new tool to investigate the BTV replication cycle as well as permitting the generation of designer vaccine strains, which are greatly needed in many countries.

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.

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.

Nonstructural Protein 3 of Bluetongue Virus Assists Virus Release by Recruiting ESCRT-I Protein Tsg101

ABSTRACT The release of Bluetongue virus (BTV) and other members of the Orbivirus genus from infected host cells occurs predominantly by cell lysis, and in some cases, by budding from the plasma membrane. Two nonstructural proteins, NS3 and NS3A, have been implicated in this process. Here we show that both proteins bind to human Tsg101 and its ortholog from Drosophila melanogaster with similar strengths in vitro. This interaction is mediated by a conserved PSAP motif in NS3 and appears to play a role in virus release. The depletion of Tsg101 with small interfering RNA inhibits the release of BTV and African horse sickness virus, a related orbivirus, from HeLa cells up to fivefold and threefold, respectively. Like most other viral proteins which recruit Tsg101, NS3 also harbors a PPXY late-domain motif that allows NS3 to bind NEDD4-like ubiquitin ligases in vitro. However, the late-domain motifs in NS3 do not function as effectively in facilitating the release of mini Gag virus-like particles from 293T cells as the late domains from human immunodeficiency virus type 1, human T-cell leukemia virus, and Ebola virus. A mutagenesis study showed that the arginine residue in the PPRY motif is responsible for the low activity of the NS3 late-domain motifs. Our data suggest that the BTV late-domain motifs either recruit an antagonist that interferes with budding or fail to recruit an agonist which is different from NEDD4.

Expression and functional characterization of bluetongue virus VP5 protein: Role in cellular permeabilization

ABSTRACT Segment 5 of bluetongue virus (BTV) serotype 10, which encodes the outer capsid protein VP5, was tagged with glutathioneS-transferase and expressed by a recombinant baculovirus. The recombinant protein was subsequently purified to homogeneity, and its possible biological role in virus infection was investigated. Purified VP5 was able to bind mammalian cells but was not internalized, which indicates it is not involved in receptor-mediated endocytosis. The purified VP5 protein was shown to be able to permeabilize mammalian and Culicoides insect cells, inducing cytotoxicity. Sequence analysis revealed that VP5 possesses characteristic structural features (including two amino-terminal amphipathic helices) compatible with virus penetration activity. To assess the role of each feature in the observed cytotoxicity, a series of deleted VP5 molecules were generated, and their expression and biological activity was compared with the parental molecule. VP5 derivatives that included the two amphipathic helices exhibited cytotoxicity, while those that omitted these sequences did not. To confirm their role in membrane destabilization two synthetic peptides (amino acids [aa] 1 to 20 and aa 22 to 41) encompassing the two helices and an additional peptide representing the adjacent downstream sequences were also assessed for their effect on the cell membrane. Both helices, but not the downstream VP5 sequence, exhibited cytotoxicity with the most-amino-terminal helix (aa 1 to 20) showing a higher activity than the adjacent peptide (aa 22 to 41). Purified VP5 was shown to readily form trimers in solution, a feature of many proteins involved in membrane penetration. Taken together, these data support a role for VP5 in virus-cell penetration consistent with its revelation in the entry vesicle subsequent to cell binding and endocytosis.

Role of an Arbovirus Nonstructural Protein in Cellular Pathogenesis and Virus Release

ABSTRACT 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.