Lecture notes - Oct 4, 2012.rtf

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University of Toronto St. George
Molecular Genetics and Microbiology
Richard Brown

Lecture 4 notes - October 4, 2012 SRC KINASE MEDIATES PRODUCTIVE ENDOCYTIC SORTING OF REOVIRUS DURING CELL ENTRY Figure 4 - bind to JAMA and integrins, endocytosis, proteolytically cleaved, outer capsid off, Mu 1 gets cleaved twice, endosome breaks, release Figure 1 of paper - genistein inhibits all tyrpsine kinases; PP2 inhibits src (tyrosine kinase) - fluorescent focus assay - stain cells for reovirus by antibody - if cells lit up, infected cell - A and B shows that src required for infectivity - C - measure viral titre - 10x decrease comparig DMSO and genistein and DMSO and PP2 - - D - to prove not cytotoxicity of compounds - tryphan blue assay - PP2 number of cells comparable to DMSO; genistein decreased number of cells due to cell proliferation injured - genistein inhibits multiple tyrosine kinases, perhaps the reason for decreased not cytotoxic FIGURE 2 - src involved in which step of reovirus infection? - A - twenty hours = one replication cycle - after one hour of adsorption, the virus infectivity unchanged from untreated cells one hour adsoprtion =?? - B - ISVPs show no increase in infectivity - note that virion entry and ISVP entry are not the same FIGURE 3 - is virus attaching to cells? - use flow cytometry - A - same JAMA as on cells treated with DMSO and the compounds - same number of receptors - B- same peak for beta 1 integrin - C - antibody against reovirus - same amount of virus attaching to the cells SO virus can attach FIGURE 4 - nucleus, actin, reovirus stained - virus in the cells even in treated cells - so reovirus can also enter the cells - give the virus time to get in (20 minutes after adsorption period) but that's it - D - comparable number of cells in the DMSO-treated, genistein-treated, and PP2-treated cells FIGURE 5 - lam1 = lysosome marker - more yellow in part B than A - C and D - walked along the white line - measured the intensity of each _______ - investigate colocalization - - almost no colocalization of virus particles and lysosomes in DMSO treated cells but a lot of colocalization of these two in genistein and PP2 treated cells THUS absence of inhibition of src may lead to degradation of virus particle in lysosomes Figure 6 - src = tyrosine kinase, as two tryosine residues that can be phosphorylated - - increase in phosphorylation of 416 tyrosine argue local src tyrosine activation by src particles - NO CLUE ABOUT THIS FIGURE Figure 7 - colocalization of src and reovirus - endogenous level of src low in HeLA cells - not detected by immunoflourescence, thus use GFP - increase in colocolaization of virus particles and src in B,quantification of increasing trend - src and virus particles colocalize over time FIGURE 9 - knockdown - B part - JAMA knocked down, signficant decrease in infectivity; src knockdown CONCLUSION Src inhibition does not affect entry of virus particles into cell, but does affect percent infectivity likely due to trafficking of endosome with virus particle charges and led to lysosomes so virus particles get degraded in lysosomes NOTE PFU/CELL; TIME OF EXPERIMENTS; - particle =/= PFU - PFU tends to be used for infectivity - particles - figure 5, 20 000 particles/cell MOI - how do you know that what is being visualized is real? is natural? could be the product of a lot of particles - needs to assume - infectivity assay - need to wait - hence the different lenghts of time of experiments L929 cells - why use this for purification and titration? Why use HeLa cells? At 100 PFU/cell, all cells should be infected most cells will have at least one particle FIGURE 1 D - FIGURES 7 8 - a trend - figure 8 - MOI different between virions and ISVPs SECOND PAPER - first paper = src signalling important for reovirus infectivity - endocytic proces
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