Protocol

Helicases

Volume 587 of the series Methods in Molecular Biology pp 223-233

Date:

A Method to Simultaneously Monitor Hepatitis C Virus NS3 Helicase and Protease Activities

  • David N FrickAffiliated withDepartment of Biochemistry & Molecular Biology, New York Medical College
  • , Olya GinzburgAffiliated withDepartment of Biochemistry & Molecular Biology, New York Medical College
  • , Angela M.I LamAffiliated withDepartment of Biochemistry & Molecular Biology, New York Medical College

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Abstract

The hepatitis C virus NS3 protein contains an N-terminal serine protease and a C-terminal helicase that unwinds RNA or DNA duplexes. The HCV NS3 protein is the target for several antiviral drugs in clinical trials, which inhibit the protease function. A method is reported to simultaneously monitor the helicase and protease function of the NS3 protein in a single reaction using fluorescence spectroscopy and a single chain recombinant protein where NS3 is fused to its protease activator NS4A. The method monitors both activities together in real time and is amenable to high-throughput screening. This new procedure could be used to identify compounds that inhibit both the helicase and protease activity of NS3.

Key words

Helicase protease ATPase high-throughput screening antiviral agents