The hepatitis E virus ORF1 hypervariable region confers partial cyclophilin dependency

Hepatitis E virus (HEV) is an emerging pathogen responsible for more than 20 million cases of acute hepatitis globally per annum. Healthy individuals typically have a self-limiting infection, but mortality rates in some populations such as pregnant women can reach 30 %. A detailed understanding of the virus lifecycle is lacking, mainly due to limitations in experimental systems. In this regard, the cyclophilins are an important family of proteins that have peptidyl-prolyl isomerase activity and play roles in the replication of a number of positive-sense RNA viruses, including hepatotropic viruses such as hepatitis C virus (HCV). Cyclophilins A and B (CypA/B) are the two most abundant Cyps in hepatocytes and are therefore potential targets for pan-viral therapeutics. Here, we investigated the importance of CypA and CypB for HEV genome replication using sub-genomic replicons. Using a combination of pharmacological inhibition by cyclosporine A (CsA), and silencing by small hairpin RNA we find that CypA and CypB are not essential for HEV replication. However, we find that silencing of CypB reduces replication of some HEV isolates in some cells. Furthermore, sensitivity to Cyp silencing appears to be partly conferred by the sequence within the hypervariable region of the viral polyprotein. These data suggest HEV is atypical in its requirements for cyclophilin for viral genome replication and that this phenomenon could be genotype- and sequence-specific

A highly discriminatory RNA strand-specific assay to facilitate analysis of the role of cis-acting elements in foot-and-mouth disease virus replication

Foot-and-mouth-disease virus (FMDV), the aetiological agent responsible for foot-and-mouth disease (FMD), is a member of the genus Aphthovirus within the family Picornavirus. In common with all picornaviruses, replication of the single-stranded positive-sense RNA genome involves synthesis of a negative-sense complementary strand that serves as a template for the synthesis of multiple positive-sense progeny strands. We have previously employed FMDV replicons to examine viral RNA and protein elements essential to replication, but the factors affecting differential strand production remain unknown. Replicon-based systems require transfection of high levels of RNA, which can overload sensitive techniques such as quantitative PCR, preventing discrimination of specific strands. Here, we describe a method in which replicating RNA is labelled in vivo with 5-ethynyl uridine. The modified base is then linked to a biotin tag using click chemistry, facilitating purification of newly synthesised viral genomes or anti-genomes from input RNA. This selected RNA can then be amplified by strand-specific quantitative PCR, thus enabling investigation of the consequences of defined mutations on the relative synthesis of negative-sense intermediate and positive-strand progeny RNAs. We apply this new approach to investigate the consequence of mutation of viral cis-acting replication elements and provide direct evidence for their roles in negative-strand synthesis

Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers

Sophie Forrest was funded by a BBSRC studentship and by the Wellcome Trust (094898). Zoe Lear is funded by a Wellcome Trust 4-year PhD programme (The Molecular Basis of Biological Mechanisms).We have previously documented the inhibitory activity of RNA aptamers to the RNA-dependent RNA polymerase of foot-and-mouth disease virus (3D). Here we report their modification and use with a subgenomic replicon incorporating GFP (pGFP-PAC replicon), allowing replication to be monitored and quantified in real-time. GFP expression in transfected BHK-21 cells reached a maximum at approximately 8 h post-transfection, at which time change in morphology of the cells was consistent with a virus-induced cytopathic effect. However, transfection of replicon-bearing cells with a 3D aptamer RNA resulted in inhibition of GFP expression and maintenance of normal cell morphology, whereas a control aptamer RNA had little effect. The inhibition was correlated with a reduction in 3D (detected by immunoblotting) and shown to be dose dependent. The 3D aptamers appeared to be more effective than 29-C-methylcytidine (29CMC). Aptamers to components of the replication complex are therefore useful molecular tools for studying viral replication and also have potential as diagnostic molecules in the future.Publisher PDFPeer reviewe

La contribution du Saint-Siège au progrès de l'Europe

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Genetic complementation of hepatitis C virus nonstructural protein functions associated with replication exhibits requirements that differ from those for virion assembly

Within the polyprotein encoded by hepatitis C virus (HCV), the minimum components required for viral RNA replication lie in the NS3-5B region, while virion assembly requires expression of all virus components. Here, we have employed complementation systems to examine the role that HCV polyprotein precursors play in RNA replication and virion assembly. In a trans-complementation assay, an HCV NS3-5A polyprotein precursor was required to facilitate efficient complementation of a replication-defective mutation in NS5A. However, this requirement for precursor expression was partially alleviated when a second functional copy of NS5A was expressed from an additional upstream cistron within the RNA to be rescued. In contrast, rescue of a virion assembly mutation in NS5A was more limited but exhibited little or no requirement for expression of functional NS5A as a precursor, even when produced in the context of a second replicating helper RNA. Furthermore, expression of NS5A alone from an additional cistron within a replicon construct gave greater rescue of virion assembly in cis than in trans. Combined with the findings of confocal microscope analysis examining the extent to which the two copies of NS5A from the various expression systems colocalize, the results point to NS3-5A playing a role in facilitating the integration of nonstructural (NS) proteins into viral membrane-associated foci, with this representing an early stage in the steps leading to replication complex formation. The data further imply that HCV employs a minor virion assembly pathway that is independent of replication.<p></p> IMPORTANCE In hepatitis C virus-infected cells, replication is generally considered an absolute prerequisite for virus particle formation. Here we investigated the role that the viral protein NS5A has in both replication and particle assembly using complementation assays and microscopy. We found that efficient rescue of replication required NS5A to be expressed as part of a larger polyprotein, and this correlated with detection of NS5A at sites where replication occurred. In contrast, rescue of particle assembly did not require expression of NS5A within the context of a polyprotein. Interestingly, although only partial restoration of particle assembly was possible by complementation, that proportion that could be rescued benefitted from expressing NS5A from the same RNA being packaged. Collectively, these findings provide new insight into aspects of polyprotein function. They also support the existence of a minor virion assembly pathway that bypasses replication.<p></p&gt

Amino acid substitutions in norovirus VP1 dictate host dissemination via variations in cellular attachment

Viruses interact with receptors on the cell surface to initiate and coordinate infection. The distribution of receptors on host cells can be a key determinant of viral tropism and host infection. Unravelling the complex nature of virus-receptor interactions is, therefore, of fundamental importance to understanding viral pathogenesis. Noroviruses are non-enveloped, icosahedral, positive-sense RNA viruses of global importance to human health, with no approved vaccine or antiviral agent available. Here, we use murine norovirus as a model to study the molecular mechanisms of virus-receptor interactions. We show that variation at a single amino acid residue in the major viral capsid protein, VP1 301, has a key impact on the interaction between virus and receptor. This variation did not affect virion replication or virus growth kinetics, but a specific amino acid was rapidly selected through evolution experiments and significantly improved cellular attachment when infecting cells in suspension. However, modulating plasma membrane mobility counteracted this phenotype, suggesting a role for membrane fluidity in norovirus cellular attachment. When the infectivity of a panel of recombinant viruses with single amino acid substitutions at this residue was compared in vivo, there were differences in the tissue distribution of viruses in a murine host, suggesting a role for VP1 301 in dissemination in vivo. Overall, these results highlight how capsid evolution can influence infectivity and dissemination in the host

Industrial geography: Industrial restructuring and labour markets

10.1191/0309132502ph375prProgress in Human Geography263367-38