application abstract): Hepatitis C virus (HCV) infects 3.9 million people in the U.S. and is a major cause of chronic hepatitis, leading to liver failure and hepatocellular carcinoma. The viral factors that are responsible for replication of the virus are poorly understood, and detailed biochemical characterization of these factors will assist rational development of anti-HCV therapies. The HCV RNA helicase, NS3, is believed to be essential for removing the RNA secondary structure during viral replication. Currently, no rigorous, biochemical models for RNA helicase activity exist, making this class of enzymes one of the least understood viral factors. The goal of this research is to characterize the biochemical mechanism of NS3 using new approaches that have recently been developed in the applicant's laboratory. RNA unwinding will be studied using a new pre-steady-state assay in which the enzyme will be assembled in a stoichiometric fashion on well-defined oligonucleotide substrates. Translocation of NS3 on single-stranded RNA will be evaluated using a second new assay to determine whether a directional bias in translocation exists. The results from these experiments will provide the frame work in which to develop a minimal kinetic mechanism of NS3 which is necessary for a quantitative understanding of the function of this enzyme. Binding of NS3 to nucleic acids will be examined using equilibrium binding experiments. The intrinsic fluorescence of NS3 will be measured as a function of nucleotide concentration to determine the binding site size of the enzyme and the dissociation constants of various nucleic acids.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Small Research Grants (R03)
Project #
5R03AI043352-02
Application #
2887779
Study Section
Microbiology and Infectious Diseases B Subcommittee (MID)
Program Officer
Johnson, Leslye D
Project Start
1998-07-01
Project End
1999-09-30
Budget Start
1999-07-01
Budget End
1999-09-30
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Arkansas for Medical Sciences
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Little Rock
State
AR
Country
United States
Zip Code
72205