Almost one in every fifty-five Americans have been exposed to the Hepatitis C Virus (HCV), but most are unaware of their infection because the virus causes few acute symptoms. If left untreated, the majority of HCV infections lead to chronic active hepatitis that eventually progresses to cirrhosis, cancer, or liver failure. Current therapies involving the drugs interferon and ribavirin are costly and produce debilitating side effects, frequently worse than the symptoms produced by HCV itself. Newer treatments are, however, quite effective against certain viral genotypes. This proposal will examine the HCV proteins most directly involved in viral replication, the NS3 Helicase and NS5B polymerase, as putative targets for the drug ribavirin and as targets for new antiviral agents. In addition to its established role as a modulator of the immune system, ribavirin has been proposed to eliminate viruses as a mutagen or through direct effects on viral replicative proteins. One popular hypothesis states that ribavirin's enhancement of the already high HCV mutation rate leads to a catastrophe of errors and subsequent virus elimination. Here, ribavirin effects will be examined in vitro, in enzyme assays, and in vivo, using a novel HCV replicon that should allow the assessment of replication fidelity. To attempt to relate ribavirin effects to genotype-specific drug response, all experiments will be repeated with the three most common American HCV genotypes, two that normally do not respond to therapy (la and lb) and one that frequently responds to therapy (2a). The polymerase and helicase proteins from each genotype will also be characterized to define conserved and divergent properties. A rigorous biochemical approach will be used to define enzyme differences because sequence data alone does not accurately predict protein structure or function. Preliminary data show that different genotypes encode enzymes with markedly different properties, hampering current rational drug design efforts. Structure-based site-directed mutagenesis will be used to determine the genetic basis for HCV enzyme variability. The biological consequences (i.e. replication rate, fidelity, protein expression) of HCV genetic variation will then be analyzed in a replicon system. The delineation of genetic variations responsible for certain phenotypes might allow the prediction of patient response to current or future HCV therapies, and the clear identification of conserved HCV enzyme properties will aid future HCV drug development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI052395-05
Application #
7174231
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
Koshy, Rajen
Project Start
2003-02-15
Project End
2009-01-31
Budget Start
2007-02-01
Budget End
2009-01-31
Support Year
5
Fiscal Year
2007
Total Cost
$296,780
Indirect Cost
Name
New York Medical College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041907486
City
Valhalla
State
NY
Country
United States
Zip Code
10595
Mousseau, G; Kota, S; Takahashi, V et al. (2011) Dimerization-driven interaction of hepatitis C virus core protein with NS3 helicase. J Gen Virol 92:101-11
Frick, David N; Ginzburg, Olya; Lam, Angela M I (2010) A method to simultaneously monitor hepatitis C virus NS3 helicase and protease activities. Methods Mol Biol 587:223-33
Belon, Craig A; High, Yoji D; Lin, Tse-I et al. (2010) Mechanism and specificity of a symmetrical benzimidazolephenylcarboxamide helicase inhibitor. Biochemistry 49:1822-32
Meng, Xiao; Zhou, Yajing; Lee, Ernest Y C et al. (2010) The p12 subunit of human polymerase delta modulates the rate and fidelity of DNA synthesis. Biochemistry 49:3545-54
Belon, Craig A; Frick, David N (2009) Helicase inhibitors as specifically targeted antiviral therapy for hepatitis C. Future Virol 4:277-293
Heck, Julie A; Meng, Xiao; Frick, David N (2009) Cyclophilin B stimulates RNA synthesis by the HCV RNA dependent RNA polymerase. Biochem Pharmacol 77:1173-80
Belon, Craig A; Frick, David N (2009) Fuel specificity of the hepatitis C virus NS3 helicase. J Mol Biol 388:851-64
Meng, Xiao; Zhou, Yajing; Zhang, Sufang et al. (2009) DNA damage alters DNA polymerase delta to a form that exhibits increased discrimination against modified template bases and mismatched primers. Nucleic Acids Res 37:647-57
Neumann-Haefelin, Christoph; Frick, David N; Wang, Jing Jing et al. (2008) Analysis of the evolutionary forces in an immunodominant CD8 epitope in hepatitis C virus at a population level. J Virol 82:3438-51
Belon, Craig A; Frick, David N (2008) Monitoring helicase activity with molecular beacons. Biotechniques 45:433-40, 442

Showing the most recent 10 out of 22 publications