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Lecture 2

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Human Biology
Richard Brown

Class 2 - September 20, 2012 Adenovirus into the cell via various surfaces it needs to cross (epithelial; Wiethoff et al paper 2005 - explore how do adenoviruses get out of endosomes - figure 1 - alpha sarcin assay - comparing adenovirus wildtype type 5 vs ts1 (hyper stable because hasn't packaged protease or packaged only little amount so precursor proteins haven't been cleaved) - ts1 goes into cell, but nothing happens - why? - alpha sarcin is toxin - when into cell, cleaves ribosomal RNA - so shuts down protein synthesis - incubating cell with toxin - toxin cannot trigger its uptake, so endocytosed with adenovirus - cannot get out of endosome; while in endoxome, not toxin becuase no access to rRNA; see if toxin is released from endosome - use S35 methionine - look for incorporation of radioactive methionine into newly made proteins - wildtype adenovirus + bafillocymin (inhibits proton pump - protons into interior of endosome, increasing pH as vesicle moves closer to nucleus) - so no reduction in pH, close to neutral - no effect of ad5 - so ad5 needs reduction in pH to permeabilize membrane - if have bafilomycin, need more virus - ts1 - 39 means virions made at 39 degrees, the NON PERMISSIVE temperature, so do not have protease; - Ad5 can disrupt endosomal membrane, releasing alpha sarcin into cytoplasm; while ts1 does not do so efficiently; this says ts1 doesn't effectively infect cells because it gets stuck in endosome - figure 2 - virus particles in presence of fluorescent dye, to see if dye can attach to DNA - at neutral pH, and physiological temperature, virus particles INTACT (no penetration by fluorescent dye) - increase temperature, uptake of fluorescent dye - at pH 4, 4.5, no need for heat to destabilize wildtype Ad5 capsid - low pH is physiological, heat isn't - now ts1: extremely stable regardless of pH or temperature figure 3 - treated virus above a dense material in tube; spin in ultracentrifuge; virus bands with proteins (soluble) staying where virus was can determine proportion that stays with particle and proportion that is soluble - at normal temperature, hexon stay, rest goes into dense material - at neutral pH, takes higher temperature to have same effect as hexon staying - ts1 - all neutral pH - - protein 6 hasn't moved even at 60 degrees figure 4 - ability to interact with membranes - virus outside; but in reality, virus inside the endosome in cell - looking for ability to permeabilize membrane - packaged sulfoB (fluorescent) in high concentration in liposomes (artificial membrane) - fluorescent at low concentrations only - best release at pH 5 - correlation btw particle being able to lose some of its proteins and at the same time, seems to be able to disrupt the membrane - wild typd ad5 can release contents by disrupting artificial membrane - as lon as pH 5, temperature can be low - ts1 - temperature increasing doesn't cause membrane to disintegrate because ts1 at 55 degrees - ts1 does not permeabilize regardless of pH or temperature figure 5 - what is permeabilizing the membrane? - no hexon in part A - protein seven is predominant - soluble fraction better at permeaiblizing membrane liposome under physiological condition - virions in dense material not good at permeabilizing liposome - C part - suggesting permeabilizing due to protein 6 - D square - to prove to get rid of most of penton base figure 6 - protein six is internal protein - known sequence - from nucleotides 36 to 53 - amphipathic helix - conservation of this sequence across multiple species - imply importance - amphipathic helices known to interact with membranes figure 7 - compare precursor protein 6, mature protein 6, protein 6 with 54 nucleotides cleaved - electron microscopy comparing intact membranes and membranes after incubation with protein 6 - if pre-six is exposed, can interact with membrane; if not exposed, then cannot permeabilize - ts1 - the problem is that pre-six protein is NOT EXPOSED 2010 - same group as 2005 - there was amphipatic helix at N-terminus of mature protein - if take that away, reminder of protein lost ability to permeabilize - figure 2 - all except protein six missing amphipathic helix - confirming helix important in permeabilizing membrane -figure 1 - measuring distance between each individual amino acid of helix and tryptophan that's within the - helix does not form A PORE - lies within membrane - figure 4: - giant lipid vesicles - vesicles are fragmented - fragments are tubular - thus suggesting curvature as method of breaking - figure 5 - positive curvature - curve out - negative curvature - curve in - works better when use liposomes prone to positive curvature so interpreting that protein 6 is inserting into membrane and inducing positive curvature and membrane disrupted - NOT permeabilized, it's DISRUPTED - figure 6 - looking at release of sulfo b and dextrane molecule (two dyes) - dextrane large cannot go through pore - if smaller sulfo b released, pores big
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