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255 Cards in this Set
- Front
- Back
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What is X-relaed murine RV a cause of?
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Prostate cancer, or chronic fatigue syndrome
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What is the new classification of retroviruses based on?
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Morphology and Genome organization
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What was the old classification of retroviruses based on?
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Pathology related to infection:
-Oncoviruses -Lentiviruses -Spumaviruses (endogenous RVs) |
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What is the shape of a retrovirus particle?
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Spherical
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Do RVs have an envelope?
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Yes! THey have a lipid bilayer that they obtain by budding from infected cells
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What is the retrovirus genome like?
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(+) RNA, 7-20 kbp
Diploid (two copies of the ssRNA genome) |
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What are the three groups of retroviral genes?
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Env, Gag, Pol
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Where are env genes located and what do they include?
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They are on the outer surface or within the lipid bilayer
Env genes encode for: Viral surface glycoprotein (gp120), transmembrane protein (gp41) |
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What is another name for the viral surface glycoprotein gp120, and what is its function?
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SU
Interacts with receptors/ specificty determines cellular tropism for the virus |
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What are the gag proteins for and what do they determine for the virus?
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Gag proteins are the structural proteins that make up the core and the matrix. They determine the morphology of the virus
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What is the morphology of HIV?
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It has a bullet shaped capsid!
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WHat are the gag proteins?
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Matrix (p17) or (MA)
Capsid (p24) or CA Nucleocapsid (NC) |
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What is the pol region responsible for encoding?
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THe proteins that possess the enzymatic activities of the virus
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What are the genes that pol encodes?
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Protease (p9) or (PR)
Reverse transcriptase (p66) or (RT) Integrase (p32) or (IN) |
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What is the protease protein used for in the RV?
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To cleave maturing peptide molecules
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What is the RT used for in RVs?
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To polyermize RNA to DNA!!!!
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Out of gag, pol, and env, which proteins does the virus bring in upon infection and why?
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The pol proteins: PR, RT and IN so that the virus can quickly perform reverses transcription and integration
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What are the (+) strand RNAs like and what can they function as?
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They are capped and polyadenylated, they can act as functional mRNAs
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What do all retroviruses have at their 3' and 5' ends of the RNA?
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A regulatory region that is 1-400 nt in length:
Has the Repeat (R) on both ends U'5 on 5' end U'3 on 3' end PBS: primer binding site on 5' end |
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What are the regulatory regions used for once the DNA is integrated?
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They become transcriptional activators to drive RNA production from the integrated viral DNA
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What is the configuration of RNAs and how is this achieved?
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They are configured in a head to head way. The interaction between two RNAs occurs by a kissing loop at the 5' end, which is mediated through the U5 region.
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What is associated with the PBS of the RNA genome?
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A few tRNA
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What do these tRNA that are associated w/ the PBS for?
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They serve as the initiation for reverse transcription
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How is the association with the PBS and tRNA achieved?
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Through the free 3'OH on the tRNA
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How is entry of RV achieved?
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By viral membrane/cell membrane fusion and release of the core structure into the cytoplasm of the cell
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Where does reverse transcription of the RNA occur?
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This occurs in the cytoplasm, while RNA remains associated with the core
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How is the core altered to allow reverse transcription to occur?
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It is permeabilized to allow necessry substrates for RT to enter and reverse transcribe the core-associated RNA
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What does the reverse transcription process lead to?
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The formation of the pre-integration complex
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Where does integration of the linear dsDNA occur?
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In the nucleus
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How does the DNA obtained by reverse transcription get into te nucleus?
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Once the pre-intregration complex is formed, the core dissociates and the complex can enter the nucleus.
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What happens once the DNA of the retrovirus integrates into the host cell?
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THe DNA remains integrated permanently
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How can viral RNA (transcribed from the integrated DNA) be post-transciptionally modified?
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Splicing, Exported, Translation
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Where does RV assembly occur?
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In the cytosol
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Which RV proteins are targeted to the plasma membrane?
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Env proteins (both surface and transmembrane) as well as nucleic acids which are localized by gag-pol proteins
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How is an RV particle rendered infectious?
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By proteolytic cleavage
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Is the budding process and release of viral particles lytic?
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Not generally, it normally won't kills the cells, resulting in the cells being sustained
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What are the two methods of retroviral entry?
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pH independent entry (surface glycoproteins on the virus interact with host cellular proteins)
pH dependent entry: involves endocytosis and formation of endocytotic vesicles that get acidified and lead to release of viral core |
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Which retroviruses use the pH independent mode of entry?
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HIV, HTLV-1
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Which retroviruses use the PH dependent mode of entry?
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Murine/Feline Leukemia Viruses (MLV, FV's)
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How does HI-V perform its pH independent viral entry?
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Its surface glycoprotein gp120 interacts with the receptor protein CD4. There is also an interaction with the co-receptor CCR5 or CXCR4 which results in a conformational change
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What happens once the conformational change of the virus surface protein occurs?
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The transmembrane domain (gp41) is exposed and inserts itself into the cell membrane
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What type of protein is the TM?
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It forms a hairpin structure that causes the lipid bilayer fusion, resulting in the delivery of the core into the cytoplasm
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What type of activity does RT have?
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RNA dependent and DNA dependent DNA polymerase activity
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In what form is the protein RT?
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A heterodimer (1 subunit 66kDa and the other is 31kDA)
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What is RNAseH?
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It digests RNA present in an RNA-DNA hybrid double-stranded structure
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What is used as a primer for reverse transcriptase and where is it located?
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tRNA lysine present in the core of the virus
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What is minus-strand strong stop DNA?
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(-sss): (-) RNA of the R and U5 region at the 5' end
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What interaction is the first strand jump based on?
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R-R complimentary interaction
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What are the two ways this R-R complimentary interaction is thought to be achieved?
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Either by a jumping of the -sss to the 3' end of the RNA template, or a circularization of the genome involving the translocation of the -sss sequence
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What is resistant to degradation by RNase H?
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The polypurine tract of the RNA template
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What does the undigestable polypurine tract function as?
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A primer for the synthesis of the (+) strand DNA for the second strand of the final dsDNA complex
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What is the plus-strand strong stop?
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Using the ppt as a primer, it is towards the 3' end of the degraded genome RNA
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What is the second strand transfer mediated by?
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PBS-PBS interaction
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What is the creation of duplicate LTRs important for?
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Expression of viral gene products, and future integration of the genome
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What type of protein is HIV-1 integrase?
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32 kDA protein that acts as a dimer or tetramer
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Does integration occur randomly?
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Not necessarily, It is thought that there is selection for where integration occurs, to actively expressed chromatin (CpG islands)
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Why is it thought that integration is selected to the actively expressed chromatin?
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Active chromatin is more open than highly condensed heterochromatin
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Which activities does integration require?
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Integrase
Cellular DNA repair enzymes |
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Why are the cellular DNA repair enzymes needed?
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To repair the free ends generatedby integration
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How is proviral DNA replicated?
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Once it is integrated, within the host genome, as a "cellular gene"
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Where does IN cleave?
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It recognizes highly conserved CA sequences in LTR regions (of the proviral DNA) at the 3' end
It also generates 5' overhangs in host DNA (4-6 nt apart) |
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How does integration occur after IN cleavage of the viral DNA and host DNA?
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The 3'OH of the viral DNA attacks the 5' end of the host, and ligation occurs
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When are the host repair enzymes necessary?
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After integration, to recognize gaps, cleave overhang nts, and fill gaps in genome
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Through integration, what are the two ways that retroviruses can infect cells?
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1. Intregrated replicated their genome with the host genome during cell division. They infect by simply allowing the daughter cells to inherit the DNA
2. Integrated retroviruses can initiate the late phase of its life cycle and actively produce infectious virions |
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What type of infection can the type where the daugher cell inherits the integrated viral DNA result in?
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A "silent infection" or "weak expression"
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Where does viral transcription start?
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There is a TATA box at the U3'R junction
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Which segment encodes the Poly A tail sequences and termination sequences?
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R
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Which part of transcription gives the virus a "second level" of tropism, aside from the receptor?
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The U3 region, because a particular U3 region will interact with different transcription factors
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What are the transcription factors that interact with U3 in the LTR of HIV?
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NFAT, NF-kB, Sp1 (all T cell specific transcription factors)
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What are the transcription factors that bind to U3 in HTLV-1?
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CREB/ATF and Tax (viral protein!!!!!!)
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Since Mouse Mammary Tumor Virus infects breast epithelial cells, what type of molecule binds its U3?
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Glucocorticoid steroids bind and drive LTR expression of the proviral DNA
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When can promoter interference occur?
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The 3' end U3 could allow binding of transcription factors
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How does the virus avoid this promoter interference?
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Pol III dislodges TF's that bind here, to guarantee that the 5' UTR is what is driving transcription
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What does alternative splicing generate for retroviruses?
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8 different proteins
Gag, env and pol proteins Full length genomes for packaging |
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What is the first thing proviral DNA synthesizes and what is this used for?
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Full length genome mRNA. This can be incorporated into virion or used as a coding region for Gag and Pol proteins
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How are Env proteins generated?
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By splicing out most of Gag and Pol
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What other proteins can alternative splicing yield?
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Auxilliary/regulatory proteins
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How does generation of regulatory proteins compare in HTLV and HIV?
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IN HTLV, a few types of alternatively spliced species generate regulatory proteins
In HIV, there are 50-100 different RNA species which can be multiply alternatively spliced to obtain regulatory proteins |
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How are fully spliced mRNAs transported out of the nucleus?
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By cellular export proteins
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How are full length mRNAs or singly spliced mRNAs transported out of the nucleus?
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Simple retroviruses use Constiutive Transport Elements (CTE)
Complex retroviruses use auxilliary or regulatory proteins |
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What is an example of a retrovirus that uses CTEs?
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Mason-Pfizer monkey virus
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What is the auxilliary protein used by HIV-1 to get the full length mRNA into the cytoplasm?
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Rev
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What is the auxilliary protein used by HTLV-1 to get the full length mRNA into the cytoplasm?
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Rex
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How does Rev/Rex work?
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Binds to viral mRNA and then this complex associatees with cellular export proteins
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What do complex retroviruses used to alternate how they express the Gag-pol polypeptide?
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Ribosomal frameshifting
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What do simple retroviruses do to alternate how they express the gag-pol polypeptide?
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Translational read-through
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How does ribosomal frameshifting work?
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The ribosome will shift x nucleotides backwards, changing the open reading frame
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What is the difference between alpha-retroviruses and beta/delta retriviruses in ribosomal frameshifting?
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Alpha viruses have 1 frameshift because they have two reading frames one with gag, one with pol
Beta/Delta have three ORFS, so two frameshifts in order to synthesize the whole gag-pro-pol |
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What is an example of an alpha virus that only undergoes 1 frameshift?
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Lentiviruses
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How can 10-20x more of the structural proteins be produced than non-structural?
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Normally, after Gag is generated, the ribosome terminates.
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What are the requirements for frame-shifting?
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-Oligo-U or Oligo-A heptamer sequence= "Slippery site"
-secondary hairpin structure on which the ribosome stalls |
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What are some examples of simple retroviruse that use translational read through to generate the gag-pol polypeptide?
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Murine Leukemia Virus and Avian viruses
Gamma and Delta viruses |
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How does translational read-through occur?
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The GAG and POL regions are separated by a stop codon (UAG is sometimes misread as CAG (glutamine)). Consequently, the translation continutes in frame
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What are some requirements for translational readthrough?
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purine-rich sequences
stop codon pseudo-knot structure |
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What are the first few amino acids of the Env protein for?
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A signal peptide for ER localization
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What is a result of this localization of Env to the ER memrane?
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It is heavily glycosylated, folded and oligomerized
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What happens when Env goes through the golgi?
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It is cleaved into TM and SU subunits, which remain loosely associated with each other
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What does the cleavage of Env into TM and SU create for the TM subunit?
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A hydrophobic N terminus that allows it to be inserted into the lipid bi-layer necessary for fusion
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Where does packaging of C-type retroviruses occur?
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At the innerside of the plasmamembrane
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How is packaging driven?
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By gag
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Where does the packaging of D/B type retroviruses occur?
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Core assembly at the cytoplasm
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How is it that only full-length genomic RNA is packaged?
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They contain a Psi sequence, that the nucleocapsid region of GAG binds
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What proteins are involved in packaging and core formation?
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Env, NC, Gag, and Gag-pol
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What other factors are packaged?
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Cellular tRNA (lysine for HIV)
Pol RT enzymes Auxilliary proteins (possible) |
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When does maturation of retroviruses occur?
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After budding, the protease only becomes active after binding and cleaves the gag-pol polyproteins into individual components
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What happens as a result of Gag-pol cleavage?
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The core changes shape and becomes more condensed
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What happens once HIV is cleaved for maturation?
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It goes from having a circular to a conical capsid
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What is the psi sequence required for?
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The genome will not be taken up into the virion without it
It interacts with Gag in order to be taken up |
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How does the position of the psi sequence ensure that only full length mRNA is encapsidaed?
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The 5' splice site is upstream of the psi sequence, and therefore the psi sequence will be spliced out, and the non-full length mRNA will not be able to be targeted to the virions!
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How does the virus ensure that polyadenylation is only at the 3' end of the mRNA?
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The Poly A signal is found at the boundary of U3 and R at the 5' end, and U5 and R at the 3' end
Since the poly A sequence at the 5' end is upstream of the transcriptional start site, it will not be incorporated into the mRNA |
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What are three methods of retrovirus transformation?
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-Acquisition of a mutated cellular gene
-Coding for a viral oncogene -Insertion near a proto-oncogene or tumor suppressor gene |
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How does acquisition of a mutated cellular gene result in transformation?
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A proto-oncogene loses its regulatory elements and is integrated into the viral genome
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What are some examples of retroviruses that transform by acquiring modified cellular genes?
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RSV (Rous Sarcoma Virus), AMV (avian myeloblastosis virus), and Rev-T
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What is an example of a retrovirus that encodes for a viral protein that has transforming potential?
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HTLV-1
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How does insertion near a gene result in transformation?
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Retroviruses can insert themselves near a cellular proto-oncogene, and they alter this proto-oncogene so that it is expressed aberrantly
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What type of transformation is it when a virus inserts itself near a protooncogene?
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A slow transformation
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What is an example of a retrovirus that performs transformation by inserting itself near a proto-oncogene?
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MMTV (Mouse Mammary Tumor Virus)
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How is RSV able to cause transformation?
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It has acquired a mutated form of the cellular Src gene
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What does src encode for?
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Protein tyrosine kinase
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Where does c-Src insert in the RSV genome?
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Into the env sequence of the virus
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What is the result of Src inserting in the Env sequence of the RSV genome?
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RSV becomes replication defective
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What is the difference between v-Src and c-Src?
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-v-Src has lost the introns of the c-Src
-V-Src has conserved the ability to interact with Src partners, as well as the kinase activity -It has lost the regulatory domains found in Src |
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What is a result of v-Src losing the regulatory domains from c-Src?
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The downstream pathways that Src stimulates are always on in RSV-infected cells!
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What type of transformation is caused by this aquirement of a mutated cellular gene in RSV?
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Acute transformation (fast)
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What are some pathways that Src targets?
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-Growth factors that induce mitogenesis
-Activators of the cell cycle -Pathways that are pro-survival -Pathways that induce cellular migration |
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What is a result of turning on all these pathways constantly?
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The cells are continuously growing, proliferating, avoiding apoptosis, and the cancer metastasizes
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What gene does the retrovirus Rev-T steal from the cell?
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C-Rel
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What is C-Rel?
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A transcription factor that is important in developing lymphocytes, and activating cells in the immune system
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What is the difference between v-Rel and c-Rel?
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V-Rel retains the transactivation elements, but deletes the regulatory regions, resulting in Rel being more active
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Which cells does Rev-T infect?
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Those of a hematopoietic lineage
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Where does v-Rel insert and what is a result of this?
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v-Rel inserts within the Pol and Env sequences of Rev-T, resulting in Rev-T usually being replication deficient
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How fast does Rev-T transform target cells?
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Acutely
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Which family of transcription factors does c-Rel belong to?
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NF-kB
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What is the NF-kB family important for?
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Generation of survival signals- triggers growth, blocks apoptosis, makes cells survive
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Which gene did both AMV and E26 viruses steal?
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c-Myb
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What disease does AMV cause?
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Acute leukemias
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What disease does E26 cause?
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Acute erythroblastosis
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Where does AMV insert the c-Myb sequence?
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Right before the end of the gag sequence, replacing pol and env
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Which regions of c-myb are conserved in the v-myb of AMV?
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DNA-binding, transactivation
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What regions of c-myb are lost in the v-myb of AMV?
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The negative regulatory regions
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Which TF's does E26 steal?
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myb and ets
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What type of transformation do replication competent viruses cause?
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Slow transformation (not acute!)
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How are replication-defective transforming retroviruses able to replicate?
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By helper viruses
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What type of viruses are helper viruses?
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Wild-type retroviruses with intact structural proteins and no oncogenes within the sequence
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How does the helper virus "help" the replication defective virus?
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By providing it with the structural proteins
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What type of virions can result when cells are co-infected with the helper and mutant retroviruses?
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Capsids can be formed with both genomes, or just of one of the virus. The recombinant virus that contains both types of genomes is replication competent
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What is the helper virus for RSV?
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Rouse Associated Virus
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What is the helper virus for AMV?
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Myeloblastosis-Associated Virus-1 or Myeloblastosis-Associated Virus-2
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What is the main transforming protein of HTLV-1?
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TAX!!
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How does Tax allow HTLV-1 to induce transformation?
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Tax promotes constitutive activation of NF-kB and AP-1 pathways
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What type of transformation does HTLV-1 cause?
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Slow transformation
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What percentage of HTLV-1 infected individuals develop ATL?
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Only 2-5%
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What molecules does Tax interact with?
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A complex consisting of NF-kB dimers and IF-kappaB alpha
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What does the interaction of Tax with this complex of NFkB dimers and IFkappaB alpha trigger?
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-Protosomal degradation of inhibitor of both these compounds
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Which pathways does Tax activate?
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NF-kB and AP1
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Which TF is associated with the NF-kB pathway?
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cFos
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Which TF is associated with the AP-1 pathway?
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cJun
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How are lymphocytes able to be transformed so easily by Tax?
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Lymphocytes are extremely sensitive to NF-kabbaB activation!!!
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What happens when Tax is expressed in a T lymphocyte?
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The T lymphocytes mimics an acitvated states
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What happens when Tax associates with DNA repair machinery?
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Results in genetic instability and a slow accumulation of DNA mutations through the replication cycles, eventually resulting in an tumorigenic gene
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What are the three mechanisms that transformation can occur by MMTV?
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-Loses its 5' LTR promoter activity
-3'LTR acts as an enhancer of the proto-oncogene -Integration disrupts expression of tumor-suppresive gene |
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How does loss of function of 5'LTR of the MMTV result in transformation?
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5'LTR is responsible for promoter occulusion of the 3'LTR, and since this occulusion is lost, 3'LTR is active and a power promoter and will result in high levels of the proto-oncogene, resulting in transformation
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How does the 3'LTR act as an enhancer?
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When the 5'LTR is not disrupted, the 3' LTR can act as an enhancer (not directly as a promoter)
Enhancer activity is sufficient to drive proto-oncogene levels to a level where they can cause transformation |
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What is an example of a tumor suppessor gene that can be disrupted?
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Rb (retinoblastoma) protein
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What type of transformation does MMTV cause?
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Slow-transforming
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What type of retroviruses are used in gene therapy?
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Replication defective
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What is an example of a disease that retroviral gene therapy is used for?
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SCID (Severe combined immunodeficiency)
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What is SCID caused by?
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Mutations in the gamma common cytokine receptor chain or mutations in ADA
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Why did patients develop acute lymphocytic leukemia when using retroviral gene therapy?
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Integration was not random, but within a promoter element of a transcriptionally active gene, giving a survival advantage to the cell and the development of transformed cells leading to leukemia
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How much of the human genome is composed of endogenous retroviruses?
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8%
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What type of cells do endogenous retroviruses infect?
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Germline cells (eggs, sperm)
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What are some positive roles of endogenous retroviruses?
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In pregnancy:
Helps with placenta formation, immunosuppression (so mother doesn't reject the baby) |
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How is Src activation controlled?
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Through positive and negative phosphorylation events
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What happens when v-Src loses some of the phosphorylation sites of c-Src?
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The protein will no longer be anchored on the cytoplasmic membrane
Protein will be continuously active |
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What are some diseases that HTLV-1 can cause?
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Adult T-cell leukemia, or Tropical spastic parapesis (HTLV-1 associated myelopathy)
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What type of T cells does HTLV-1 infect?
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Multiple T cell types
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What type of T cell can become transformed after HTLV-1 infection?
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CD4+ and CD25+
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What is the difference in HTLV-1 effect on T cells and HIV's effecT?
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HIV causes a decrease in the number of T cells, while HTLV-1 causes a dramatic increase
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What is Tropical Spastic Parapesis?
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A demyelinating neuropathy disease
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Where is HTLV-1 particularily present?
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Southern Japan, South America, and Central Africa
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Where does HTLV-1 induced neuropathy disease tend to occur?
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South America
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What type of virus is HTLV-1?
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Blood borne
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How is HTLV-1 transmitted vertically?
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Breast milk (by passing infected lymphocytes)
Pregnancy (transplacental passage of lymphocytes) Blood (whole blood transfer of infected cells) |
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How is HTLV-1 transmitted horizontally?
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Sexual contacts
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What type of receptor does HTLV-1 use upon entering the cell?
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A glucose transporter
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What is special about the 5' end of the HTLV-1 genome?
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There are a number of DNA elements known as "Tax responsive elements"
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What type of interaction is that of Tax with the LTR?
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INDIRECT!
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How is this indirect interaction achieved?
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Tax interfaces with cellular transcription factors, resulting in the increase of these TF affinity for the binding to TREs
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What is an example of a TF that Tax interacts with?
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CREB
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What type of mRNA encodes Tax and Rex?
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A doubly spliced mRNA
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How does Tax induce T cell activation and proliferation?
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Through an IL-2 autocrine loop
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What is IL-2?
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A T cell growth factor
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How does Tax continue this continuous stimulatory loop?
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By upregulating the IL-2 receptor
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What type of T cell proliferation occurs in the early phase of HTLV-1 induced cell transformation?
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Polyclonal T-cell proliferation (multiple types of T cells proliferate)
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What is the phase after this polyclonal T-cell proliferation phase?
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As proliferation occurs, mutations are accumulated that allow them to grow in the absence of normal stimuli required for T-cell growth. The T cells enter an IL-2 independent growth phase, and there is the emergence of monoclonal leukemic T-cells
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How is ATL diagnosed?
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-Multi-lobulated nuclei with T-cell surface antigens (CD4+ and CD25+)
-Presence of anti-HTLV-1 antibodies in the sera of the patient |
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What are CREBS?
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cAMP response element proteins
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What other proteins does Tax interact with?
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It will dimerize with NF-kB transcription factors
Tax also has a transactivating domain |
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What type of effect does Tax have on transcription factors?
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-Increases their affinity for each other or for DNA
-Increases nuclear translocation of the transcription factors |
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How does Tax interact with CBP? (CREB Binding Protein)
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It binds CBP
It uses the properties of CBP to stimulate transcription of the VIRAL genome (histone acetyltion activity, recruitment of transcription machinery) |
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What are some NF-kB members?
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p65/RelA, RelB, c-Rel, p50, p52
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How are NF-kB TF's held in the cytokplasm?
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Inactive by inhibitor IkB
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How is the inhibition of NF-kB TF's relieved?
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Viral infection results in phosphoryltion of IkB, targeting it for degradation
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What happens once NF-kB is no longer inhibited?
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It can translocate into the nucleus, activating gene expression
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How does Tax affect NFkB?
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Interacts with the DNA binding domains of NFkB
Interfaces with IKK complex (phosphoyrlates IKB) Increases ability of NFkB to recruit cofactors |
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What are some types of molecules that Tax interacts with?
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Cytokines, Receptors, Surface proteins, Oncogene products, Growth factors
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What happens when Tax interacts with CBP?
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Causes ATF/CREB to intitiate viral genome/ LTR transcription
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What happens when Tax interacts with NF-kB/p300?
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Affects immune and inflammatory pathways, and cellular proliferation
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What happens when Tax associates with SRF?
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Induces cellular proliferation
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What can the various manifestations of HTL-1 infection be due to?
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-Differences in genetic background of the individuals
-Infection with variants of HTLV-1 -different host immune responses to the virus |
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WHat is HTLV-1 infection characterized by?
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Enlarged lymph nodes,
Abnormal (flower) and elevated T lymphocytes Prominent eosinophilia |
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At what stge of ATL development do people usually notice?
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At the lymphoma stage, but at this stage treatment is often ineffective
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What happens after the lymphoma stage of ATL development?
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The acute phase of the disease leading to an agressive and fatal leukemia of the T cells
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What are the four clinical phases of ATL development?
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Smoldering, Chronic, Lymphoma, and Acute
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How is Tropical Spastic Parapesis caused?
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When HTLV-1 infected T cells infiltrate the spinal cord
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What are the three models of of the pathogenesis of TSP?
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Autoimmune model, Direct infection, Bystander damage
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What is the autoimmune model?
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HTLV-1 infection activates autoreactive T cells, which will migrate to the nervous system, recognize antigens on CNS cells, and destroy them
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What is the direct infection model?
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Persistent HTLV-1 infection and activation in the CNS leads to tissue destruction
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What is the bystander damage model?
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Infected T cells flow into CNS, release alot of growth factors, cytolines, inflammatory molecules, etc...
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What are the three constructs needed to produce a non-replicating retroviral vector?
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1) Transgene cassete: gene of interest, LTRs, PBS,Psi Packaging sequence
2) Gag-pol 3) Env |
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What is the thymidine Kinase + gancyclovir strategy?
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Retroviral gene therapy is used to treat brain cancer: cells are infected with a retroviral vector containing a thymidine kinase, and are also given gancyclovir,which will stimulate death of these brain cells (in excess due to cancer!)
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What is retroviral pseudotyping?
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Substituting a retroviral envelope glycoprotein with a different envelope, encoding a viral receptor with a wider target host range
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What is an example of retroviral pseudotyping?
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Replacing the glycoproteins with the VSV (Vesicular Stomatitis Virus) G protein
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What are some limitations of retroviral gene therapy?
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-Efficient delivery
-Necessity to transduce into dividing cells -Long term gene expression (does not occur, usually tunrs off) -Cost-effective manufacturing |
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What is oncolytic virotherapy?
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Harnesses properties of viruses to fight cancer but leaves normal cells unaffected
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How does oncolytic virotherapy compare to chemotherapy?
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It has a higher therapeutic index (10000:1 compared to 6:1) and has fewer side effects
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What is a drawback of oncolytic virotherapy?
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The long term effect is that the tumors come back!
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What are sone KEY advantages of using VSV for oncolytic virotherapy?
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IT is not a human pathogen
-It does not integrate -It is genetically stable -replicates in cytoplasm |
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How are normal cells protected?
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Treat with interferon
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What is the mechanism of oncolysis by virotherapy?
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Virus induced apoptosis, indirect apoptosis caused by inflammatory cytokine release, shutdown of tumor vasculature, adaptive immune response against tumor antigens
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What is the general process of virus therapy?
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The virus enters, multiplies rapdily and with deadly effect to cancer cells, but is self-limiting and shuts off
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What are histone-deacetylase inhibitors?
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THey are combined with viral therapy
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What is the purpose of the HDIS?
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Reducing patient resistance to the oncolysis and oncolytic viruses
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How do HDI work?
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Inhibit histone de-acytelases, and therefore increases gene transcription
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How do HDIs senstitive cancer cells to the oncolytic effets of VSV?
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Through their capacity to inhibit residual innate response (IFN) in tumor cells
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What are the seven Retrovirus genera?
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Alpharetrovirus (RSV)
Betaretrovirus (MMTV) Gammaretrovirus (MLV) Deltaretrovirus (HTLV) Epsilonretrovirus (WDSV) Lentivirus (HIV) Spumavirus (Simian foamy virus) |
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When can retroviruses normally productively infect cells? Which retroviruses are an exception to this?
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Normally only during mitosis: because the nuclear membrane is dissolved and normally the PREIC can't enter. Lentiviruses such as HIV encode for proteins that mediate the nuclear transport of the viral DNA complex
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How are Gag/Pol proteins incorporated into the assembling virus core?
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By an interaction between its CA region and the corresponding region of Gag
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How is the tRNA incorporated into the assembling core?
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Binding to RT and NC portions of the Gag/pol polyprotein
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In gene therapy, how do you ensure that target cells have been integrated with provirus DNA?
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Vectors can incorporate genes that confer resistance to toxic drugs such as G418 (neomycin), hygromycin B, or puromycin.
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In HTLV, what is the protease encoded by?
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A reading frame that overlaps part of the gag region and part of the pol region
To get the gag/pro/pol protein, 2 ribosomal frameshifts are required |
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Where is the Tax/Rex/p12 ORF of HTLV?
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Between Env and U3 region
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In addition to nuclear export of singly spliced and full length mRNA, what are some additional roles of the HTLV protein Rex?
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Processing of 3' end of viral transcripts: formation of the correct secondary structure of the mRNA in the Rex responsive element brings the cleavage site closer to the AAUAAA, permitting cleavage and polyadenylation
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What are the two essential regions of Rex?
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-Amino terminus- mediates binding to RRE, also is an NLS for Rex
-Leucine rich activation domain that functions as a nuclear export signal by binding to the cellular cofactor exportin 1. |
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How does Rex function?
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Since only doubly spliced mRNAs are exproted to the cytoplasm, Rex binds and prevents further splicing of singly spliced and unspliced mRNA, and mediates their eport.
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How is import of cellular proteins into the nucleus mediated?
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NLS that bind to importin alpha, which binds to importin beta, which targets the resultant protein complex to the nucleus via pores.
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How do Rex and Rev differ in the import of cellular proteins?
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Rex and Rev get into the nucleus by binding directly to importin beta, bypassing the importin alpha.
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What is the HTLV protein p12 for?
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Binds and dimerizes chains on the IL-2 receptor, mimicing the action of IL2 on the receptor, this results in Jak-stat activation and leading to gene activation of cell proliferation. This results in IL-2 independent growth
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How does Tax play a role in cell cycle progression?
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Tax bings to cell cycle inhibitor, p16INK4A, which is a tumor suppressor. This tumor suppressor is responsible for binding the cdc kinases and inhibiting their activity. Binding of tax reverses this inhibition. This results in RB phosphorylation and E2F activation, the TF that allows transformation from G1 to S and cellular proliferation
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How does Tax result in loss of cell cycle checkpoints?
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Binds to human protein MAD1, preventing the necessary dimerization MAD1 is necessary for allignment of chromosomes before anaphase. The disruption of its function results in chromosome abnormalities in ATL cells.
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What is the difference in the latency period with HTLV associated ATL and TSP?
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TSP latency is alot shorter, 1-3 years.
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What is the most successful therapy in treating HTLV?
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Azidothymidine and interferon alpha. Allows remission to occur within a few months. But if removed from treatment will relapse
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What is the role of Vpr?
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Allows HIV to infect cells that are not dividing
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What is the role of Nef?
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In HIV pathogenisis
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What is the role of Vpu?
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ENhances release of progeny virions from infected cells
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