14 - HIV - notes II.docx

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University of Toronto St. George
Molecular Genetics and Microbiology
Alan Cochrane

Integration of virus renders cure of the aIDS infection complicated b/c half life of quiestent T cells By time you are diagnosed, assuming perfect adherence to therpy, 60 years before cured of infection by straight drug treatment Pools are established early on in infection - LTR lets virus go in and out of latency easily Fluctuate activity of virus with activity of T cell – if T cell active, virus active; if T cell dormant, virus dormant Virus has two states potentially in cell – latent; active phase when producing virus - Immune system cannot see latent virus pool - Activating all T cells – can kill the patient - Recently, developed integrase inhibitors – hit two sites of activity o Three prime end cleaveage – cleave in staggered fashion the ends of proviral DNA o Attack of viral dNA and drug that is currently on market is raltegravir Role of HIV-1 non-structural genes - Six addition reading frames – all retained in virus and in infectious system - All ahve role to play in maintaining replication edge of virus - In culture, can do away with Nef, Vif, VPR, and VPU in CULTURE – suggesting they are not required at cellular level, but at immunological level - TAT and REV absolutely essential – attractive targets TAT – involved in enhancing transcription from viral provirus - Through mechanism of - In absence of TAT, little viral RNA – operating at transcriptional level – enhancing initiation or elongation o Elongation o HIV promoters in absence of Tat intiate Fin o Polymerase load onto promoter, transcribe little RNA – carboxy domain rich in serine residues generally phosphorylated to form elongation competent complex and in case of HIV, they don’t do it in absence of Tat  In absence of Tat, inhibiate polymerase, transcribe a little bit, polymerase falls off – wasted effort  At low frequency, stick around to end of genome, make RNA that is used  Small amount of RNA driven in absence of Tat – Tat works in different way – most transcription activator proteins activate through DNA – that’s why have enhancers that – double stranded – have sequence to bind to – Tat is different – recognizes element called TAR, which operates in position and orientation dependent fashion  Acted like enhancer but behaved differently – TAR operating through RNA, NOT DNA – RNA is single stranded, so sequence only transcribed in certain direction, certain polarity, so must be positioned correctly and oriented correctly  Recruits two proteins – CDK9 and cyclin T o CDK9 is kinase- phosphorylate CTD of polymerase – from elongation inefficient to efficient polymerase so TAT binds to TAR and essentially phosphorylates CTD elongates efficiently to end of genome o TAR required in position dependent fashion -0 must be right near promoter – o TAR must be positioned right after promoter, must modify polymerase it just initated  Cannot be upstream b/c nust be transcribed by polymerase  Recognized by RNA  Enhancer operates in position and orientation dependent fashion  Must modify polymerase soon after initaiton to allow it to continue on and transcribe the genome  KNOW, TAR, TAT, CDK9, cYCLIN T, ELONGATION EFFICIENCY,  Cyclin T binds to loop sequence and brings in CDK9  b/c RNA – so orientation dpendent – recognizing single stranded sequence virus favours latency b/c of the way it is transmitted – sexually transmitted – how many partners? - Keep host alive long enough to have enough episodes to transmit the virus - Needs latency to maintain the infection Make nine proteins – gag, pol, env, etc. - Gag and gagpol – singly spliced – - Depending on where spliced, make env, vpr, vif, vpu - Then can be spliced to make fully spliced RNA to produce tat, rev, nef – depending on which splice site used - GENERATE FORTY mRNA – complexity increases - In early phase of replication, cell makes one RNA which is processed into forty mRNA o Cell only exports the multiply spliced RNA into cytoplasm – make Tat, which goes back in to activate TAR, Ref goes back in to bind to Rev response element to get them out into cytoplasm, and in lte phase, produce all other genes of the virus o Early phase makes Tat, Nev, Ref  Engough Rev, activate late phase of expression – gives all structural genes of virus Rev produced in cytoplasm, can enter nucleus – dynamic protein - Depending on various set of interactions o Rev produced in cytoplasm, binds importer Beta, which brings it into nucleus, affected by charge on Ran  Ran GDP – stable interactions of the complex, import IN  In nucleus, high GTP state so exchange GDP off RAN to GTP bound state – Rev now free to bind RNA through IRE – to form complex that docks RNA onto pore that allows it to come back out into cytoplasm – hydrolyze Ran back to GDP – complex breaks apart, Rev free to be imported back and translated by the cell o Rev bind to importer beta – complex enters nucleus – GTP exchange – Importer beta off – Rev free – docks RNA in nuclear pore, transports it through – RNA enters cytoplasm –  CRM1 goes back into the nucleus, a shuttling protein like importer beta RNA out in cytoplasm – assemble the virus - Gag and viral RNA traffick along vesicles to plasma membrane – viral RNA move on vesicles through cytoplasm to engage plasma membrane - Structure of gag o Matrix, capsid, nucleocapsid, with a tail called P6  Matrix involved in targeting gag to membrane  Capsid drives assembly of virus forming moltomeres  Nucleocapsid binds RNA through psi sequence  P6 function?  Delete it – intermediate assemblies – stalk tethers the progeny to the cell – almost at point of release, but cannot be released  Involved in late stage of viral assembly  Hijacking components of normal vesicle formation machinery o
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