Class Notes (838,930)
Canada (511,155)
HMB265H1 (242)
Lecture 2

Lecture 2 notes.rtf

5 Pages
147 Views
Unlock Document

Department
Human Biology
Course
HMB265H1
Professor
Richard Brown
Semester
Fall

Description
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
More Less

Related notes for HMB265H1

Log In


OR

Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


OR

By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.


Submit