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Mar 13 - Retroviruses.docx

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York University
BIOL 3155
K.Andrew White

Chapter 28: Retroviruses  Genome is (+) sensed but not directly translated when it goes into cells  Brief History – Table 1.2 o First discovery of retrovirus was by Peyton Rouse (1911). Showed that sarcoma virus could induce tumor formation in chickens. Won nobel prize is 1966. o RT in retroviruses discovered by Temin and Baltimore (1971), nobel prize in 1975. discovered RT, against central dogma. o Dogma was that flow went from DNA to RNA to protein. o But, opposite is true for retroviruses with RT (it converts RNA to DNA) o Cellular oncogenes in retrovirus discovered by Bishop and Varmus, Tumor caused by Rous sarcoma virus acquiring an altered cellular oncogene  Retroviridae (family) o Consists of 7 genera o Grouped based on differences in morphology and genome organization o Table 28.1 o alpha, beta, gamma, delta, epsilon- retroviruses. Lentivirus causes HIV-1 in humans. (missed pic, but basically these r the viruses she mentioned).  Do not need to know example of virus, but know host.  Focus on Lentiviruses, example, and host.  General Structure of Virion o Figure 28.1 o Virus contains envelope. o Icosahedral or conical for capsid (capsid = HIV). o Approximately 100 nm across o Contains RT, integrase (IN) and protease (PR). 3 different enzymes. Protease is outside particle but inside matrix. o Virus contains tRNA which is important because it is used as a primer. o Virus contains two copies of RNA genome. These are diploid and dimerized – have partial base pairing to each other that keeps them together o Both copies may not be identical. May have single or multiple mutations. This is due to RNA-RNA interactions. o Contains transmembrane protein (TM). Surface protein (SU - noncovalently bound to TM). Matrix protein (MA - under envelope; coats inside of the envelope). Capsid protein (CA; the one that actually forms the icosahedral capsid). Nucleocapsid protein (NC - directly coats genome RNA)  General Genome Structure o Positive sense single stranded RNA. (+)ssRNA o Approx 7 – 10 kb o Figure 28.2  Represents simple retroviral genome (more complex retroviruses will contain more genes; e.g. HIV-1 is a complex retrovirus)  All retroviruses have 5’ cap and 3’ poly-A tail (virion genome is not translated, however – this is not how they initiate an infection). Not grouped with (+)RNA viruses.  Repeated sequence (R) at 5’ and 3’ ends.  Unique regions (U5) (contain dimerization RNA hairpin; where the two copies of the genome will interact for dimerization) and U3  Contains primer binding sequence (PBS) – there are tRNAs in particle; these bind to PBS. tRNA type is virus/strain dependent. this is where the tRNAs bind. the tRNA would be used as a primer??  There are also splice sites (SS – 5’ splice site and 3’ splice site).  There is also a packaging signal (psi) – important (must be present)for identifying viral genome as what needs to be packaged and put into particles.  Polypurine tract (a run of As abd Gs) – plays important role during replication cycle.  3 genes involved: (listen here)  Gag (group-specific antigen)  Pol (polymerase) – pol genes are expressed by readthrough or frameshift  Env (envelope) – env genes are expressed from spliced mRNA  Genes are polyproteins – expressed fused to each other. Depending on virus, will have either a readthrough or frameshift event that will extend Gag into Pol.  we have 3 genes but more than 3 proteins.  Overview of Entry o Figure 28.3  Binding of SU to the receptor (virus/cell specific). SU is attachment protein.  Fusion at surface (or endocytosis and pH dependent fusion for some viruses). but in both cases it's mdiated by the black receptor on the pic? Fusion is TM-dependent (TM is involved in fusion).  Capsid is delivered into cytosol. Often, the capsid is referred to as a core.  RT occurs inside the core (in cytosol). RNAs are never released into cytosol; they are used as template for RT while in core.  Net result is ssRNA is converted into dsDNA. Entire core is transported into nucleus through NP (Nuclear pore – occurs for Lentiviruses [i.e. HIV]).  For most retroviruses, core can only be transported after nuclear envelope is broken down. This only occurs during cellular division.  There is then integration (integrase protein is involved with this). dsDNA integrated into genome; becomes part of host. Can replicated now along with host genome.  Reverse Transcription (whole thing is mediated by Reverse Transcriptase) o Figure 28.4 o In order for ssRNA genome to be converted into dsDNA, you need RT. RT in its active form is a dimer.  conversion of viral genome into dsDNA. (vRdDp & vDdDp)  Ribonuclease H (RNase H) will degrade RNA only when it is present in a RNA-DNA hybrid. o Viral genome (black) goes from 5’ end (left) to 3’ end (right). o First, tRNA (present in viral particle) base pairs to PBS. tRNA is serving as a primer (will provide 3’ hydroxyl group to which additional nucleotides can be added). o RT will bind to primer and extend it. This is called synthesis of minus-strand strong-stop DNA. (1st step) o DNA is shown in black in the figure. (small DNA sequence) o RNase H now digests RNA in DNA-RNA hybrid. At end of DNA strand, we now have copy of repeated sequence (R’). (2nd step) o First strand transfer now occurs – extend tRNA unprimes, and reprimes at 3’ end of viral genome. (3rd step) o Now RT uses prime template to synthesize a full length genome copy (all the way to PBS). You now have a full-length (-) strand. (4th step) o Now more RNase H digestion occurs. This digests everything but RNase H resistant region (PPT – polypurine tract).(5th step) o PPT is used as primer for synthesis of plus-strand strong-stop DNA. It is extended to bottom of tRNA structure – at the end, this recreates a PBS (because tRNA made a complement to PBS). (6th step) o There is now RNase H digestion which removes RNA present in hybrid. Only PBS left opposite where tRNA used to be. PPT is also digested.(7th step) o There is a second strand transfer of PBS to 3’ end of bottom strand (5’ of top strand). Now there is synthesis of dsDNA in both directions. PBS is priming in one direction, complement of PBS is priming in other direction. (8th step) o You end up with a copy of (+) strand (top) with a complimentary copy (-) strand. o This is referred to as proviral DNA – this is used by integrase to incorporate into viral genome. o The ends of the proviral genome are LTT (long terminal repeats). These correspond to U3 R U5.  RNA genome vs. Proviral genome o Compare RNA genome that entered cell (Step 1) to proviral genome obtained (Step 9).  RT (Reverse Transcriptase) o Has RdDp and DdDp (between 6-7 and 8-9) activity. Both of these activities are primer- dependent (no denovo initiation occurs).  We went from (+)std ssRNA to (-)std DNA which was then made into dsDNA. This ALL occurs in core of particle/in the capsid of the new virion (therefore everything needed needs to be INSIDE particle)  RT lacks 3’ to 5’ exonuclease activity (no proofreading activity). Cannot remove wrong nucleotide if they add it.  Without proofreading activity, there are 1-10 substitutions (of nucleotides) per genome replication (HIGH error rate). You end up with quasispecies.  Quasispecies: virus variants (versions) in a single infection that differ by a small number of mutations.  Quasispecies allows for rapid adaptation to environment. This makes virus good at evading antiviral drugs; achieving drug resistance.  Proviral Integration:  Figure 28.5  Proviral DNA needs to be integrated into the cell’s genome. It does this at random sites.  Integration into cellular genome is catalyzed by the viral integrase (IN),(which remember is inside the capsid)  Integrase binds to the ends of the provirus and brings them together.  IN then removes two (2) 3’-terminal nucleotides at both ends. It then cleaves host DNA and ligates viral 3’ ends to host DNA.  This results in GAPs where nucleotides have no partner sequence (4 bases at each ends that are unpaired). Host repairs the gap with correct nucleotides. You end up with proviral DNA integrated into the host genome. Once provirus is integrated into host cell, it can replicate with genome. Integration is what makes it difficult to cure infections.  Generates a 4-6 bp direct repeat in cellylar DNA. Results in a 2 nt loss from each end of the viral DNA. No effect on progeny virus. (missed pic :( blurry))  Synthesized of RNA genome (transcribed from DNA copy) is shuttled to make particle and some also used as mRNA  Provirus becomes part of host cell chromosome - is replicated along with host genome.  Spread o
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