Biomedical subjects

Rob Noad

Publications and source records attributed to Rob Noad.

4 recordsLinked to original sources

PubMed2006

Specific binding of Bluetongue virus NS2 to different viral plus-strand RNAs.

The Reoviridae have double-stranded RNA genomes of 10-12 segments, each in a single copy in the mature virion. The basis of genome segment sorting during virus assembly that ensures each virus particle contains the complete viral genome is unresolved. Bluetongue virus (BTV) NS2 is a single-stranded RNA-binding protein that forms inclusion bodies in infected cells. Here, we demonstrate that the specific interaction between NS2 and a stem-loop structure present in BTV S10 RNA, and phylogenetically conserved in other BTV serotypes, is abolished by mutations predicted to disrupt the structure. Subsequently, we mapped RNA regions in three other genomic segments of BTV that are bound preferentially by NS2. However, structure probing of these RNAs did not reveal secondary structure motifs that obviously resembled the stem-loop implicated in the NS2-S10 interaction. In addition, the specific binding by NS2 to two different viral RNAs was found to occur independently. Together, these data support the hypothesis that the recognition by NS2 of different RNA structures may be the basis for discrimination between viral RNAs during virus assembly.

Animals↗

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

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↗