November 1, 2012 - class notes.docx

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Molecular Genetics and Microbiology
Richard Brown

Lecture Notes - November 1, 2012 Paper: Influenza H5N1 and H1N1 virus replication and innate immune responses…. - Looking at virulence of these two viruses - Cells in differentiated and undifferentiated states – may account for conflicting results in previous papers - NHBE – cells grown on transwell inserts o Cells with medium below, and air above? o To infect, virus on top Figure 1 - Looking at differentiation of cells o Day 21 – see cilia and pseudocolumnar cells unlike at day 7 o Well-differentiated (21 days) can be used as good model to model human bronchial tissue - WHY IS H5N1 so virulent? o Even in the same cells, using cells that CAN differentiate, state of differentiation makes a difference - HAT protease – use trypsin to cleave hemagglutin to infect cells in culture to make sure the influenza hemagglutinatin is cleaved o HAT is produced in normal airway – cleaves hemagglutination of influenza virus, helping virus infect cells o Want to show that HAT is expressed at high enough levels to justify not adding trypsin Figure 2 - Sia receptor where? How abundant? - First two rows are undifferentiated epithelium o MAL1 – unpreferred – H5N1 – sialic receptor 2-3 Gal B1-4 GlcNAc o MAL2 – preferred – H5N1 – sialic 1-3 Gal o SNA – Sia 2-6 - Receptors required for infection of differentiated and undifferentiated tissues are present but in varying quantities but present in both types Figure 3 - Influenza infection in both differentiated and undifferentiated cells - Measured influenza matrix expression using qPCR - MOI 2 - Well differentiated cells have goblet cells that secrete mucin that have alpha two three receptors so can be used as decoy receptors to compete with the actual receptors on cells o Hence decreased infection as determined by amount of gene present - In the first round of replication – in both ud and wd cells – H1N1 preferred to bind to unciliated cells – H5N1 preferred to bind to ciliated cells o Explain the similar levels of mrNA – since balance of what virus wants to bind to and what the cells have Figure 4 - Immunoflourescent staining - Target cells to be infected – staining of viruses - Percentage of cells infected high in ud; decrease in wd cells o Lower virulence? Figure 5 - Compare infectious virus yields and replication kinetics - Part A o Higher TCID in well-differntiated cells - Part C o H5N1 + wd – no productive infection of virus o Other three, significant statistically Figure 6+7 - Cytokines – interferon beta, RANTES, IP10, and TNF alpha o IP10 induced in response to interferon gamma – attracts macrophages, NK cells, dendritic cells – promote T cell adhesion to endothelial cells o RANTES – mediates inflammation – chemoattractants o Interferon beta – involved in early innate response – decrease viral replication and activates other genes cause cell apoptosis - Undifferentiated epithelium o qPCR – examine mrNA expression o H5N1 induces greater responses in Interferon beta, RANTES, IP10 –nothing for interferon gamma detected  Response induced by H5N1 greater than that of H1N1 o H5N1 – shown to have lower replication - ELISA – use apical surface o Issue because in ud cells, no apical surface o Also some cytokines preferentially secreted from certain apical, basal lateral, etc surfaces - IP10 – H5N1 produces more cytokines – does so at both time points – IP10 produced at higher level when looking at protein levels - Now looking at WELL DIFFERENTIATED MULTIFACTORIAL ISSUE NOTE! Virulent = severity of disease Pathogenic = causes disease - These two do not correlate – virus replication does not correlate with amount of disease - Both H5N1 and H1N1 are pathogenic; H5N1 is virulent, because it causes severe disease (~60% of patients die) Characteristics - H1N1 - Found in respiratory tract - H5N1 – multiple sites – multiple organs so not unusual for H5N1 virus to spread to multiple organs o Contributing to virulence Hemagluttinin folded up – taken into endosome – proton pumps – pH decrease – globular heads move out of the way – fusion peptide – reorganization of hemaglutin with globular head move out of the way – middle, fusion pepetide, pops out and inserts into membrane – - This cannot be done unless molecule is cleaved with trypsin Hemaglutinin made as precursor, called HA0 – cleaved by trypsin or a trypsin-like enzyme into HA1 and HA2 - The molecule in figure 5.39 – if cleaved – when it’s exposed to low pH, get conformational change and fusion peptide is exposed - If cleavage has never taken place, then do not get conformational change and that peptide canot be exposed and free N does not get exposed – where does virus gets up – so virus stuck in cell, STUCK IN ENDOSOME - The part that ultimately needs to be exposed to insert into endosomal membrane cannot be exposed, so stuck in centre, so virus stuck in endosome - HAT is important to act on hemagglutinin – important for infection – THIS IS WHY******** Adenovirus – it’s protein 6 that disrupts endosome – protein 6 normally on inside Reovirus – it’s mu one that puts pores in endosmome – needs conformational change – cleaveage step that Influenza – the bit that inserts into endosome is buried inside hemaglutinin molecule MAKES SENESE because needs to interact with membrane in order to deliver genome If this done prematurely (like these molecules on outside membrane of cell) – makes sense that the key protein that is required to get the viral genome or the contents of the virus across the membrane (usually endosomal membrane), what protein needed, is on the inside of virus and IS SELECTIVELY EXPOSED as result of certain trigger (low pH for instance) BUT prior to step, hemagglutinin must already be cleaved (like as being done by trypsin) – in reality, in human body, HAT seems important When is HAT working? Reference: E. Bottcher J Virol 84 2010 - Different proteases cleave at different times - HAT cleaves newly synthesized hemagglutin before or during release of progeny
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