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25 Cards in this Set
- Front
- Back
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What are the 3 genera of Flaviviridae?
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1)Flavivirus: Causes yellow fever (which causes jaundice)
2)Pestivirus: animal plague which causes diarrhea in cows 3) Hepaciviruses: Hepatitis C (affects the liver) |
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What kind of a virus is Hepatitis C (HCV)?
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-ssRNA virus (within the flaviviridae family)
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How is HCV transmitted?
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Percutaneous (Through the blood)
-Can get it via blood transfusion. maternal-neonatal, tattooing, piercing... |
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Therapeutics of HCV? (How mny infected, how many wll resolve infection, therapy available?)
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-250 million ppl affected
--~20% of those affected will be cured -~80% of ppl infected will develop chronic Hep C -Chronic Hep C= progressive liver disease that evolves to end stage liver disease as cirrhosis and hepatocellular carcinoma (eventually destroys the person's liver) -IFN-containing therapy, but limited efficacy (may cure ~40% of cases) and has side-effects |
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What are the approved therapies for Hep C?
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-IFN monotherapy: need daily injectiokns
-Rebetron: Intron A and ribavirin (broadspectrum antibiotic) -ViraferonPeg: pegIFN alpha-2b -ViraferonPeg + Ribavirin |
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What are the limitations of these therapies?
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-Relatively limited efficacy
-Significant side-effects (will have flu-like symptoms for 6 months .: ppl discontinue treatment, virus reemerges) -Very costly ($40,000/yr) |
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Is Hep C a curable virus?
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Yes, but the cure does not work on everyone
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What are the protein coding genes of HCV?
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Structural Proteins:
-E1/E2: Glycoptns that are inserted into the lipid bilayer. Bind receptors and are responsable for liver specificity -Core Ptns: Make viral shell Non-structural Ptns: NS3: Protease at N-term(Similar to 3C in picornaviruses). CAn also act as a RNA helicase at the C-term -NS5B: RNA-dep-RNA-Pol -P7: Ion channel -NS2: Cysteine protease that does 1 cleavage. Also has a role in assembly *These complexes require a lipid bilayer to tether the protein complex together |
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Which enzymatic fcts of HCV are targeted bydrugs?
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1) Protease
2) Helicase 3) Polymerase |
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Model of HCV viral life cycle.
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1) Virus binds 3 or 4 receptors on plasma mb
2) After entry, viral RNA is released and gets translated 3) RNA replication complex NS3-5B, (NS3= protease, N5B= polymerase) forms at ER mb, making even more viral RNA 4) Maturation of the virus at the ER |
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Why were reliable HCV replicative systems lacking?
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-Hard to find a viable cell line that supports infectionand replication
(current model only works with genotype 2A HCV) **Specificity of cell line to subtype of the virus |
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What new developments led to this discovery of the current model?
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1) ID of an infectious seq in chimps
2) Replication of subgenomic HCV RNA in human hepatoma cell line: only used segments of the virus that are for replication (subgenomic RNA). This allowed the study of NS3 and NS4B 3) Hepatitis C virus replication in SCID mice with human livers: mouse liver partly human and has sever immune deficiency 4) Production of infectious Hep C virus in tissue cultures from a cloned viral genome: virus so potent scientists thought it could be culturable |
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How are drug candidates validated?
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1) Find cmpd that might be useful and test them in vitro with a list of possible inhibitors
2) Narrow downthe list using purifications 3) Test remainder in cell culture systems (for the recent discovery, they had to inject it into chimps and then transfer it to human tissue) |
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Which protease is targeted for inhibition?
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NS3
-Responsible for a lot of cleavage in trans (not cis) -NS3 inhibition results in loss of production of 4A, NS4B, NS5A and NS5B -This inhibition leads to a non-fctnal polyptn and inhibition of replication (NS2 only cleaves itself (cis) and is very fast .: hard to inhibit it) |
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How was NS3 studied?
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-Isolate its sequence, express it in a bacteria
-Create a substrate that is a polypeptide that mimics the cleavage site -> Add a fluorescent tag to the polypep. thats blocked by a quencher -> If NS3 cleaves the ubstrate, you'll see the fluorescence -> If NS3 doesn't cleave it, you won't see any color, cuz of quencher |
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What does the cleavage product, NS5A do?
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NS5A naturally inhibits NS3 protease activity
(NS5A/5B DDIVPC is the nat inhibitor,so instead of screening millions of peptides, use this, which will not inhibit the cleavage site. Also, designed peptides can't pass through the mb) |
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What is BILN 2061?
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-Used the knowledge of NS5A structure and sequence to make cmpds (1st Cmpd A: hexapeptide then BILN 2061)
-Tripeptide (3 aa) -Circularized -small enough to pass through plasma mb -can reduce the amount of RNA with time (seemed capable of clearing infection) -As amts of BILN inc, there was more NS3-NS5B and less NS3 .: cmpd could inhibit protease activity -worked in the same way as IFN |
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What was the problem with BILN 2061?
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When tested with long term exposure, showed major sideeffects (cardiovascular problems)
.: stopped production of BILN 2061 |
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What is the current treatment for HCV?
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-New cpmds that are ued in addition to IFN
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Whats the problem with using interferon (IFN)?
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-Even when the simplest form of IFN is used, theviral load is increased
-Virus becomes resistant to this type of therapy -With combination therapy of IFN and drugs, the virus doesn't develop resistance |
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What is the importance of IRES in HCV?
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-Important for translation and replication
-Needed for NS5B (polymerase) to bind to at the 5' end and initiate replication (no primer for HCV, NS5B can replicate on its own) |
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Does HCV have a poly A tail?
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-No polyA tail at the 3' end of the virus
-Instead, 3' end has highly conserved, highly folded region that is important for the initiation of replication |
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What structures are always conserved among dif isolates of this virus? Which ones aren't?
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-Conserved: IRES and 3' UTR
-Not conserved: NS and Structural ptns |
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How does NS4A influence NS3?
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NS3 requires NS4A for full functionality
-1st cleavage takes place btw NS3 and NS4A, then NS4A and NS4B --> releases NS4A -NS4A binds to NS3 and becomes part of NS3 protease -Without NS4A, NS3 is not an efficient protease and it is not attached to the mb |
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What are points of inhibition of HCV?
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-NS2-NS3 or NS3-NS4A protease inhibitors
-Inhibition of mb ass't ptns -NS3-helicase and NS5B polymerase inhibitors (for NS3, can use an ATP analog, since helicases need ATP to fct. For NS5B, could use a nucleoside analog to inhibit the polymerase, such as a chain terminator) -p7 inhibitors -Receptor Inhibitors (inhibit interaction of E1 and E2 with the receptor during entry)*** this is a good target cuz can target the virus extracellularly, using antibodies**** |
