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80 Cards in this Set
- Front
- Back
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What was the first retrovirus (RV) discovered?
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Rous Sarcoma Virus (RSV)
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What is the most studied enzyme?
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Reverse Transcriptase (RT)
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What is the RSC-Src Oncogene Hypothesis?
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Showed that RSV and RT can take up cellular genes to become transforming oncogenes
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What was the firt human RV discovered?
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HTLV-1 (Human T-cell Leukemia Virus-1)
(1980) |
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When was HIV-1 discovered?
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1983
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1)How are RV classified now?
2)a)How were they classified before? b)What were those classifications? |
1) Now: Morphology and genome organization
2 a)Before: Pathology b) Oncogenic: Causes cancer Lentivirus: Slow virus (like HIV) Spumavirus: Foamy virus, related to human endogenous RV (retroviral seq present in the human genome) |
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Describe a RV organization (genome/virion)
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Spherical
100 nm in diameter 7-10 kb *Is diploid, has 2 copies of the virus genome in its core |
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What are the 3 groups of genes ass't with RV?
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1) Env: on the outer surface/lipid bilayer
-Viral surface glycoptn (gp120) (SU): interacts with host receptor, gives specificity that determines cellular tropism for a particular virus -Transmb ptn (gp41) (TM): required for fusion. Inserts itself into the lipid-bilayer by being localized to the area of budding 2) Gag: structural ptns that make up the matrix and core (morphology changes with each RV) -Matrix (p17) (MA) -Capsid (p24) (CA) -Nucleocapsid (NC) 3)Pol (polymerase region): encodes various enzymatic activities of the virus -Protease (p9)(PR): cleaves maturing peptide molec -Reverse Transcriptase (p66)(RT): polymerizes RNA to DNA activity -Integrase (p32) (IN) |
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Describe the retroviral genome structure.
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-Capped and polyadenylated
-All RV have 5' and 3' ends that have regulatory regions: -> Repeat (R) -> U5 (on 5' end) -> U3 ( on 3' end) -> PBS (Primer Binding Site on 5' end) |
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What is the head-to-head configuraton of the RNAs?
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Interaction between 2 RNAs occurs via a Kissing Loop at the 5' end, mediated through U5 (DIS)
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What regulates the start of RT?
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Interaction btw the free 3'0H gp on tRNAs and PBS.
This interaction and the availability of tRNA regulate the start of RT |
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What steps take place in early phase of the viral cycle?
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1)Binding (Recognition of surface receptors)
2) Fusion of viral env and host cell env (viral core gets deposited in the cytoplasm of the cell) 3) Penetration and Entry 4) Reverse Transcription: occurs in the cytoplasm, while RNA remains ass't with the core. Form the Pre-Integration Complex. The core can then diss't and enter the nucleus 5) Transport (of core) to the nucleus 6)Integration of the Linear ds DNA chromosomes (When the retrovirus integrated into the host cell, it stays there permanently) |
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What steps take place in the late phase of the viral cycle?
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1) Transcription: Occurs only by integrated viral DNA cuz of strong transcriptional enhancer activity at the LTR (LTR is generated in transcription and integration)
-Post-transc. mod varies depending on the RV, but can include: splicing of RNAs, export of RNA and Translation (modification of reading frames/efficiency) 2) Assembly: -Occurs in the cytosol -Gag/Pol ptns are ade in dif amts depending on the cellular demand -Also have Env ptns that pass through the Golgi system to be locaized to the plasma mb 3) Budding: Virus buds off from the surface of the infected cell -Nucleic acid is localized to the mb by Gag-Pol ptns -Virus is not yet infectious 4)Maturation: Protease actiity leads to further cleavage which makes the virus infectious (Usually not a lytic cycle, so cells shouldn't be killed) |
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What are the 2 methods of viral entry into the cell?
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-pH-independent: surface ptns on the virus interact with host cell ptns (HIV, HTLV-1)
-pH-dependent: Involves endocytosis and formation of edocytic vesicles that get acidified and release the viral core (MLV, FV) **All RV need some kind of surface ptn interaction to mediate viral entry** |
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How does HIV-1 enter the cell?
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pH Independent entry
-SU (gp120) interacts with receptor ptn CD4, get a confor. change. Also need a chemokine surface co-receptor (from macrophage etc) to get infection (CD4/gp120 interaction not sufficient on its own) -SU interacts with a co-receptor (CCR5 or CXCR4)-> conformational change -Conformation change exposes the TM -TM (gp41) inserts itself into the mb -TM forms a hairpin structure which causes the mbs to close tog and form a lipid bilayer fusion -The core is sent to the cytoplasm, where it undergoesmajor changes in composition during its entry process (CORE-> RTC -> PIC) (RTC= RT complex, PIC= pre-integration complex) |
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What is the receptor for HTLV-1?
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HTLV uses glucose-transporter type receptor interacts with the target cells on T-lyphocytes
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What are the highlights of reverse transcription (the main events)?
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-(+) ssRNA --> ds DNA
-generate LTR by duplication of regulatory structures at either end of the RNA -RT has RNA-dep and DNA-dep DNA Pol activity (always 5'->3') -RT is a heterodimer (66kDA and 51kDA subunits) -RNAseH digests only RNA that is part of an RNA-DNA hybrid (ds structure) |
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What are the steps of reverse transcription?
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1)tRNA-lysine (of virus) binds PBS: used as a primer for RT which will rev. transcribe twds to the 5' end of template
2)Synthesis of (-)strand strong stop DNA (-sssDNA): it is produced in xs and comprises of the RNA R and U5 region (at 5' end) 3)Removal of RNA by RNAse H: get rid of the RNA template of original R and U5 (at 5'end) 4) 1st strand jump/transfer by R complimentary interaction through hybridization of 5' and 3' ends: -sssDNA is peeled off since original complementary template was removed and now it is free to jump to the original R end on template 3' end. This creates the 1st LTR 5)Synthesis of full-length DNA (as (-)strand DNA) by RT: this works cuz its now primed by -sssDNA at the new 5'end and it can RT twds new 3' end (which is the template 5' end) 6) Removal of RNA template by RNAse H: except a Poly-Proline tract (PPT) remains and will later be used to prime for the synthesis of (+)strand DNA for the 2nd strand 7)Synthesis of (+)sss DNA: occurs towards the 3' end of the degraded genome by the PPT (primer), get a partially ds DA seq, replicate PBS (since tRNA was originaly linked to a PBS) .: original structures have been reduplicated 8) Removal of tRNA and PPT by RNAse H 9) 2nd strand transfer by PBS-PBS interaction: peeling of on complementary sequence at the 5'end of the template 10)Extension of BOTH DNA strands (using the free 3'OH of each PBS): synthesize Gag, Pol, Env genes on the complementary strand, get 2 LTR equivalents wit a U3--R--U5 that was duplicated |
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What is the subunits of RT?
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p66: Catalytic subunit, has RNAse H activity
p51: Non-catalytic subunit, doesn't have enzymatic activity or an RNAse H domain. Probably has structural/stability role |
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Why are there high lvls of mutations in RV?
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Most RTs don't have proofreading/exonuclease activity --> get error prone mismatches
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What is a Quasi species?
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Large variation of viruses due to high level of mutations
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How are RV imported into the nucleus?
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RT complex is altered by a breakdown of its outer core --> PIC
-This is done by nuclear pore complex ptn interactions with viral ptns, that allow transport of provial DNA into the nucleus |
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What's the difference between simple RV nuclear entry and complex RV nuclear entry?
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-Simple RV (MLV); enter nucleus at mitosis/cell division .: they can only infect actively dividing cells
-Complex RV (HIV/HTLV): active mech to import PIC into the nucleus and .: they can infect terminally differentiated cells (macrophages) |
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What are the components of Proviral ds DNA genome?
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5' cap and 3' tail, Gag (structural/matrix/ nucleocapsi/capsid ptns), Pol (RT/ Integrase/Protease), Env (Surface glycoptn and Transmb domain), LTR regulatory structure (U3--R--U5)
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What is the importance ofLTR?
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Essential for integration step of viral life cycle
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What mediates Proviral DNA integration? Where does it integrate?
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-Mediated by Integrase (IN)
(HIV-1 IN (p32) is a 32 kDA ptn that acts as a dimer tetramer) -Selection for actively expressed chromatin or CpG islands (cuz active chromatin is more open then heterochromatin, which is highly condensed). Integration occurs randomnly inside these chromatin |
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What 2 enzymatic activities are required for integration?
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-Integrase activity
-Cellular DNA repair enz (repair of free ends that are made during integration) |
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What are the steps of Proviral DNA integration?
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1) IN cleaves 3' end of proviral DNA: recognizes conserved CA seq in LTR region
2) IN makes 5' overhangs on the HOST DNA (4'6 nt apart) and get strand transfer of proviral DNA 3)Recombination: Free OH gps of proviral DNA attack the 5' end of the host and get ligated into the host genome 4) Host repair enz recognize the gaps, cleave overhangs and fill in the gaps) |
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What are the 2 ways that RV can infect cells through integration?
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1) Integrated RV replicate their genome with host genome during cell division, which will get all daughter cells infected. This leads to silent infection or weak expression
2) Integrated RV initiate late phase of viral cycle and actively produce infectious virions |
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What role do LTRs play in RV gene expression?
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-LTRs are promoters
-They assmemble the transcription machinery and drive viral gene epression |
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Where does transcription start? (i.e where is the TATA box)
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The TATA box is at the U3-R junction
-U3 region binds the transcriptional ptns that drive host cell transcription |
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What TFs bind the U3 LTR of HIV or HTLV-1? What is the target of MMTV?
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HIV: NFAT, NF-kB, SP1 (T-cell specific TF)
HTLV-1: CREB/ATF or Tax (viral ptn) -MMTV targets breast epithelial tissue, so it has Glucocorticoid Response Elements (GRE) so glucocorticoid steroids can bind and drive LTR expression |
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What does Pol III do the LTR of proviral DNA? Why?
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-Pol III can dislodge TF that can bind 3'LTR (this is promoter interference/inclusion)
-This ensures that the 5' LTR drives ptn synthesis ofproviral DNA |
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What does the generated mRNA look like?
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Cap-R-U5-PBS----genes---- ppt--U3-R-Poly(A)
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What sequences are coded by R?
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-Poly(A) tail
-Termination sequences |
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What are 2 levels of tropism (specificity) of RV?
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-Specificity of receptor interaction
-Particular U3 region interacts wih differnt (but specific) TF |
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How many ptns need to be generated? How?
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-At least 8 (2 Env, 3 Gag, 3 Pol)
-This is done through alternative splicing, since there is only 1 promoter -> First, proviral DNA makes full-length genome mRNA: This can be incorporated into virions, used as the main coding region of Gag and Pol ptns |
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Which ptns do RV need more?
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-Structural ptns (Gag and Env)
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What does alternative splicing allow?
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Various regulatory/auxiliary ptns that can be generated from differentially spliced RNA
-HTLV: some alt. spliced RNA make regulatory ptns -HIV: can get 100 dif species of RNA which can be alt spliced to get regulatory ptns |
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How is viral mRNA exported from the nucleus?
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Use cellular export ptns
-Simple RV use Constitutive Transport Elements (CTE) -Complex RV use Auxiliary or Regulatory ptns (HIV: Rev, HTLV: Rex) Without Rev or Rex, cannot get unspliced (10kDa) or singly spliced (4 kDa) out of nucleus and into the cytoplasm |
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How do RV get the right amount of ptn?
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Complex RV: Frameshifting (want more capsid and envelope ptns and need less polymerases)
-In frameshifting, the ribosome will move a few nucleotides (usually backwards) to change the ORF Simple RV: Translational Readthrough |
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How many frameshifts do Alpha-RV require? Beta/Delta RV?
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Alpha: 1 framshift
-> Lentivirus:Gag in one ORF and Pol (Pro) in another. -90-95% of the time, generate Gag -5-10% o the time, Gag-Pol fusion ptns are generated Beta/Delta RV: require 2 framshifts, since Gag, Pro and Pol are in 3 dif ORFs |
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What are the requirements for frameshifting?
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-Oligo U or A Heptamer Sequence (Slippery site)
-Hairpin 2ry structure: stalls ribosome. This is at the junction btw Gag and Pol reading frames. Usually have termination after the stall, so get a lot more Gag then Pol, but sometimes, the ribosome will slip back 1 nt and yougenerate a Gag-Pol fusion polyptn with both enzymatic activity. Protease will cleave it into dif ptns |
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How is the Gag-Pol polyptn expressed using Translational Readthrough?
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-Used in beta/delta RV (mostly simple RV like MLV or avain viruses)
-Gag separated from Pro-Pol region by a stop codon -> sometimes the UAG stop is read as CAG (Gln). -After read-through, translation continues in-frame (doesn't frameshift like in complex RV) -90% will be Gag polyptn 10% will be Gag-Pol polyptn that will be cleaved |
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What is required for translational read-through?
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-Purine (G/A) rich sequences
-Stop codon -Pseudo-knot structure: causes termination and release (except when termination is read as CAG and translation continues through the polymerase reading frame, generating Gag-Pol) |
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How is Env expressed?
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Through single-spliced mRNA
Normally translated (no frameshifting or read-through req'd) First few aa are signal peptides for ER localization |
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What happens when Env goes to the ER? Where does it go after?
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-Env is hevaily glycosylated, folded and oligomerized at the ER
-Env goes to the Golgi next -In the golgi, env is cleaved into TM(gp41) and SU (gp120) which are loosely ass't with each other -Cleavage results in a hydrophobic N-terminus of TM that lets it enter into the lipid bilayer req'd for fusion |
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Where is the virus packaged? How is the virus packaged?
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-Virus is packaged in the inner plasma mb
-Packaging is a Gag driven process (NC ptns interact with themselves and viral RNA) -> C-type viruses assemble at plasma mb -> D/B-type viruses: core assembly in the cytoplasm -NC region of Gag binds to Psi seq of RNA genome (Psi is only present in the full length RNA genome. Don't want to pkg spliced RNA) -Gag and Gag-Pol polyptns oligomerize at the inner surface of the plasma mb -cellular tRNA is incorporated with the Pol and RT enz -May also pkg auxiliary ptns -Env glycoptns, NC ptns, Gag, Gag-Pol are involved in pkging and formation of the core sequence -Budding occurs in places with surface glycoptns so there can be an interaction |
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When does maturation occur?
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AFTER budding
-After release from the cell, get condensation in the core -Protease is activated and it cleaves the Gag-Pol poly ptn into its individual components -Core shape is changed and becomes more condensed ** If protease activity is interfered with, the virion will not become infectious** |
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What are the required sequences on the genome to have a fctnal RV?
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-Psi seq: packaging signal, interacts with the Gag ptn
-5' and 3' splice site: needed to make the dif mRNAs ->5' splice site is upstream of the Psi seq (when mRNA is spliced, it can't be encapsidated anymore) ->simple mRNAs have only 1 of each splice sites so only generate 2 mRNAs (full length genome =Gag and Pol, single-spliced = Env) -R-U5 and U3 seq: Make LTR when rev. transcribed -PBS: where tRNA binds to prie for RT -PPT: also req'd for RT |
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Where is the polyadenylation signal?
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-Btw U3 and R (at the 5' end, upstream of start site) and btw R ad U5 (at 3' end)
-Only the 3' PolyA seq ill be transcribed from the template (since the one at teh 5' end is upstream of the start site) so polyadenylation will be on the 5' end of mRNA -Seq that enhance polyA--> in U3 region |
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What are mechanism of transformation for RV?
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1) Aquisition of mutaed cellular gene:Viral piracy. Proto-onc loses regulatory elements, becomes an onc and is integrated into the retroviral genome
ex: RSV, AMV, Rev-T 2) Coding for viral oncogene: retroviral genome encodes purely for viral ptns that also have transforming potential (ex: HTLV-1) 3) Insertion near a proto-oncgene (or a tumour suppressor gene): RV alters the expression of the cellular proto-oncogene, proto-onc will expressed in situations when it shouldn't be. Slow transformation cuz of insertion of the provirus ex: MMTV |
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How can RSV cause transformation?
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Acquired a mutated form of cellular SRC (c-SRC) (causes tumours in chickens), called v-SRC
-c-SRC is inserted into the envelope seq of the virus, which can no longer replicate since the Env is truncated |
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How is v-SRC different from c-SRC?
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-lost introns of c-SRC
-acquired the ptn from c-SRC that allow it to interact with its partners -has the conserved region from c-SRC that has the tyrosine kinase activity -los the regulatory domains of c-SRC (on the C-term domain of the cellular genome) so v-SRC is always active and the downstream paths are always on -v-SRC can induce acute transformation of chicken cells -c-Src anchored to the cytoplasmic mb, v-Src is not |
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How is SRC activated? What downstream targets does SRC activate?
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-Activated by P
Downstream targets of SRC signalling: -Growth factors that induce mitogenesis (cell growth) -Things that activate the cell cycle and .: inc proliferation -Paths that are pro-survival (.: won'tapoptose) -Paths that induce cellular migration (results in cncers that metastasize) |
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Is RSV an acute or chronic transforming virus?
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RSV= acute transforming virus (i.e it transforms cells very rapidly)
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What kind of transforming virus is Rev-T? What does it steal?What is that importnat for?
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-Acute transfoming virus
-Steals a TF (C-Rel) -Rel-C imp in the dev'l of lymphocytes and activation of other cells in the immune system -Rev-T takes the C-Rel seq, keeps its transactivation elements, but deletes it regulatory regions and accumulated more mutations in C-Rel to make it more active (c-rel part of the NF-kB family of TF, HIV targets this too) |
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What type of cells can Rev-T infect?
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Specific RV, can only infect hematopeitic lineage cells
-Isolated from a turkey with lymphoma -Get expansion of monoblasts which don't respond to growth inhibition signals |
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Where does the c-Rel usually integrate into Rev-T?
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In the Env/Pol sequence area
This makes Rev-T replication deficient .: can only integrate itself into th host's genome, but can't be replicated |
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What is the NF-kB TF family important for?
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-Generation of survival signals
-It triggers growth, blocks apoptosis and makes the cells survive |
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What gene did AMV and E26 both steal?
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v-myb
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What does AMV do with the c-myb gene?
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Puts it right before the end seq of Gag nd replaces Env and Pol
-DNA binding and tansactivation regiond are conserved -Get a truncated negative regulatory region, so loss of fct of c-myb negative regulator in v-myb so constitutively active-> get acute myeloblastosic leukemia |
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What does E26 do to transform?
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-Steals 2 TF: Myb and Ets
-Get a Gag-Myb-Ets fusion ptn, resulting in another type of oncogene which gives acute erythroblastosis |
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What's a replication competent RV? A replication deficient RV?
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Replication competent: Don't cause acute transformation. RV nly encodes for viral genes (Gag, Pol and Env). They can replicate after infecting hosts
Replication deficient: Usually transforming viuses fal into this category. The incoming cellular gene replaces the Gag, Pol or Env gene so the viral particles cannot go and infect other cells |
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Can a transforming virus not be replication defective?
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Yes, RSV can still replicate after SRC integrates, cuz rc integrates right after the Env seq, leaving all the viral seq intact. So RSV cells, upon infection can transform and replicate and make progeny virions
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How can replication-defective tranforming viruses replicate?
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Require a helper virus
Helper Virus = WT RV, .: it is replication competent -When a cell is infected with both the mutated virus and the helper virus, get integration of the normal/helper virus and the mutated virus |
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What happens once both the viral genomes are made?
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They're indistinctly encapsidated
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What is a recombinant virus?
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Has one strand of RNa that encodes all the correct viral ptns and one strand encodes the oncogene (**Remember, RV are diploid**)
-Recombinant viruses are also capable of transforming cells |
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What is the main transforming ptn of HTLV-1?
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Tax
-Tax is an auxiliary ptn req'd for HTLV-1 life cycle -Tax induces transformation of T-lymphocytes by promoting consecutive activation of NF-kB and AP-1 paths |
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Explain cell transformation by HTLV-1.
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1) Tax interacts with NF-kB dimers and IF-kB alpha
2) This interaction triggers protosomal degradation of the inhibitor of the kB's, which free the NF-kB dimers and allows them to transactivate target genes 3)Tax activates NF-kB and AP-1 path -Lymphocytes are verysensitive to constitutive NF-kB activation (.: easy to transform a lymphocyte by permanently turning on NF-kB) -AP-1 activate lymphocytes dev'l by constitutively activating the Jun Kinase |
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What does Tax trigger?
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-T-cell activation and proliferation: Tax make T cells grow continuously and makes them unresponsive to any checkpoints or apoptotic signals
-Tax ass't with DNA repair machinery and favors genetic instability: makes T-cells accumulate mutations -No apoptosis, so after years of this, get tumorogenic clone which results in transformation and leukemia |
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ow does transformation by insertion near a proto-oncogene occur with MMTV?
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1) Loss of 5' LTR leads to transcription from the 3' LTR: MMTV inserted upstream of an oncogene. When lose activity of 5' LTR (due to mutations) losepromoter occlusion, .: nothing to inhibit 3'LTR from being a promoter. Get high lvls of transcriptin from 3' LTR of the oncogene. (In this case, had a normal cellular gene, but was expressed in such huge amounts that it was disregulated and became transformed)
2)The 3' LTR acts as an enhancer for the expression of the oncogene: No mutation in 5' LTR, but provirus is inserted in such a way that 3' LTR becomes an enhancer (doesn't act directly as a promoter). Drives proto-oncogenic expression to oncogenic lvls--> transformation 3) Provirus integration disrupts theexpression of a tumour suppressor gene: proviral DNA inserts itself into the coding seq of a tumour-suppressor gene, .: can't make tumor suppressors and now prone to cancer dev'l **MMTV is aslow ransforming virus. One of these mechanisms on its own unlikely to trigger cancer. Would also require buildup of mutations |
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What is the goal of gene therapy? How is it done?
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-Replace defective genes by stably introducing a correct copy of it
-Done by replacing the coding sequence of the viral protein with the correct copy of the defective gene causing the disease |
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How can retroviruses be used advantageuosly in gene therapy?
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-New gene will be permanently inserted into the host
-Viral genome is small and easily modified in vitro -Simple strategy to produce infectious, non-replicating particles |
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What are the disadvantages of using RV as gene therapy vectors?
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-They may integrte randomnly into our genomes (don't want them disrupting important cellular processes)
-They may recombine with other RV |
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How can non-replicating retroviral vectors be produced?
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-Use a transgene cassette: encodes gene of interest, which has LTRs, PBS, Psi seq and Ppt -> will make a replication-defective particle
-Include 2 other plasmids: one has Gag-Pol ptns, the other expresses Env ptns -> these 2 plasmids will form the viral ptns req'd to encapsidate our genome of interest -Transfect the plasmids into a pkging cell -Produce a replication-defective virus that contains the transgene cassette, so they can infect and integrate into the genome, but they can't replicate cuz don't have any structural ptns (no Gag on its own) -Purify the replication defective cells and put them in target cells to correct the genetic defect -LTRs will drive expression of the transgene cassette in the target cells |
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What causes SCID?
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Mutations in the gamma common cytokine receptor chain or mutations in ADA
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What happened to patients that were treated with retroviral gene therapy?
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Many of the patients dev'l leukemia (mostly Acute Lymphocytic Leukemia (T-ALL)
This is because the RV vector did not integrate randomnly but 20-25% of the time integrated within 10 kb of the promoter. Insertion gave survival adv. to the cell so that it could divided faster and it wouldn't respond to apoptotic signals: cells that divided faster (transformed cells) were repopulating the patient: clonal expansion => leukemia |
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What are Endogenous Retroviruses (ER)?
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-Make up 8% of our genome
-Infect germline cells and are transmitted to progeny in a mendelian fashion -Positive role: in pregnancy, placenta formation, immunosuppression -Negative role: ass't with multiple sclerosis, schizophrenia and certain types of cancers (but not sure if there is a cause and effect relationship btw ER and these diseases) |
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What is the strategy to correct immunodeficiencies?
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1) Hemaopoeitic stem cells are removed from patients
2) Generate replicative defective particles that contain a correct version of the gene that was causing the immunodeficiency 3) Patient stem cells were infected with these viral particles 4) The gene was corrected in these stem cells 5) These cells were putback in the patient |
