November 8, 2012.docx

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Department
Molecular Genetics and Microbiology
Course
MGY440H1
Professor
Richard Brown
Semester
Fall

Description
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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