Molecular biology of segment 8 of the influenza A virus genome

The single-stranded RNA genome of influenza A virus is divided into 8 segments, each of which encodes at least one polypeptide product. This work presents investigations into two aspects of the biology of the smallest (8th) segment: 1) a possible new gene product and 2) interactions of the viral non-structural protein translated from this segment – NS1 – with a host cell protein during infection. 1) The genome of influenza A virus is packaged into virions as negative sense viral RNA. To date, all known viral proteins are encoded in the positive sense. A large open reading frame exists on segment 8 of the influenza A genome in the opposite sense to, yet overlapping with, the established proteins expressed from that segment; this hypothetically encodes a protein named Negative Strand Protein or NSP. This thesis presents work that characterised the NSP polypeptide that would be translated from this open reading frame and the effect it may exert on viral replication. Using a reverse genetics system for the A/Puerto Rico/8/34 influenza A strain, mutant viruses were generated containing negative strand open reading frames of different lengths, as well as one with early stop codons that result in a severe truncation. All viruses replicated to similar titres in cell culture and produced similarly-sized plaques as the wild-type. The synthesis and distribution of the major viral structural proteins as well as the positive-strand segment 8 polypeptides were also not affected by alterations to the open reading frame. Negative strand polypeptide produced by in vitro translation was successfully immunoprecipitated by a specific antibody. However, no specific product could be detected from virus-infected cells. In conclusion, if this negative strand protein is indeed expressed by influenza A virus, it is not significant for virus replication in vitro, nor is it expressed to high levels. 2) The nucleolus is a dynamic hub of post-transcriptional RNA processing in eukaryotic cells. The influenza NS1 protein is a multi-functional inhibitor of many cellular anti-viral pathways, such as the interferon response. Prior to this work, a proteomics study found that influenza A virus infection induces the re-localisation of cellular protein ADAR1 to the nucleolus. ADAR1 is an RNA-editing enzyme that converts adenosine residues to inosines, which are then interpreted by cellular machinery as guanosine. There is a potential that ADAR1 activity may affect the influenza virus genome; either negatively through hypermutation, or positively via a specific coding sequence alterations. Evidence for non-specific editing of influenza RNA species has been reported, but the significance of this has not been demonstrated. For other virus families such as vesicular stomatitis, measles, human immunodeficiency and hepatitis delta viruses, ADAR1 has an apparently pro-viral role. The influenza A NS1 protein was determined to be both necessary and sufficient to induce the re-localisation of ADAR1 to cell nucleoli in several common cell lines. Analysis of viruses expressing mutant forms of the NS1 protein showed that lesions in interaction sites for binding to RNA or a cellular ubiquitin ligase – TRIM25 – prevented induction of this re-localisation. This correlated with the inability of these NS1 mutant proteins to suppress phosphorylation of cellular interferon response factor 3. It was also found, using pull-down assays, that ADAR1 interacted with influenza NS1 in an RNA-independent manner, but not with the NS1 mutant deficient in RNA binding. Additionally, a separate interaction was demonstrated between ADAR1 and components of the viral polymerase complex. In cells that had ADAR1 depleted by RNA interference, viral replication was reduced by 3-fold, thus not supporting the hypothesis that ADAR1 is a restriction factor for influenza A virus and instead suggesting a modest pro-viral function. In conclusion, the influenza A virus NS1 protein induces the nucleolar re-localisation of ADAR1, which does not act as an anti-viral factor for the virus.
Wellcome Trust