November 8, 2012.docx

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Molecular Genetics and Microbiology
Richard Brown

Lecture November 8, 2012 For normal influenza viruses, NS1 important For the other influenza viruses, NS1 associated with virulence READ: Jackson et al 2008 PNAS 108 FOR NEXT WEEK: Zielecki et al J Virol 84 2010 Reverse genetics = looking at the effect of a mutation you generate - Transfecting cells with plasmids - In the context of influenza virus – a negative strand RNA virus – cell cannot process negative- sense RNA - Two main systems - 12 plasmid system and 8 plasmid system o Initially 17 plasmid system – did not need the structural proteins (in square brackets) – just needed polymerase proteins 12 plasmid system - Consist of set of plasmids that express the virion RNA – all have RNA polymerase I promoter – to give virion RNA in cell - Another set of plasmids – each expresses the particular protein PA, PB1, PB2, and NP o Promoter is RNA polymerase II promoter – and polyA signal o Transcript from these four are regular mRNA molecule to be translated by the CELL to give the two proteins - Cell transfected with mixture of these plasmids (8 + 4) o 8 virion RNAs and get polymerase proteins which copy the virion RNA into mRNA = giving rise to 10 mRNA (two are spliced)  Resulting in an infected cell 8 plasmid system - E Hoffmann et al PNAS 2000 97 - Each particular plasmid has cDNA to virion RNA and it is transcribed in two directions – carries both promoters and transcribed in one direction – transcribed from RNAse polymerase 2 promoter and polyA signal on other end – polymerase 1 promoter that activates transcription in opposite direction and that gives you the virion RNA - Virion RNA copied by appropriate polymerase to give mrNA – translated into more proteins - Produce “designer” viruses Mix and match segments - Not all segments are equally compatible with each other – Jackson et al paper - Terminology o Pathogenicity – ability to cause disease o Pathogenesis – mechanism for disease development o Virulence – severity of disease - Avian host, low vs high pathogenic – low pathogenic avian strains have monobasic cleavage site in hemagglutinin (cleaved by specific trypsin enzymes in specific locations); high pathogenic avian strains have polybasic cleavage sites (can be cleaved by ubiquitous enzymes) Jackson et al. paper 2008 - Highly virulent strains can be transmitted from birds to people – not efficient transmission from avian to human - H5N1 – 60% of patients (with disease) are killed - Particular motif modulating VIRULENCE in /mouse/ model - Several genes associated with virulence of H5N1 o Use reverse genetics system – make designer virus which most people call “recombinant” virus - NS1 associated with virulence – comes from kinds of studies where using reverse genetics, mix and match plasmids – NS1 of a virulent strain plus other genes from less virulent system, seemed to cause more disease - Virulence signature for c-terminal amino acids – nineteen-eighteen flu and H1N1 strain isolated from extremely ill patients o These c-terminal resides bind to PDZ domains (found in proteins that are involved as part of scaffolding structu
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