The NS3 Helicase RNA Unwinding Activity Is Critical for Hepatitis C Virus Replication

Abstract

The Hepatitis C Virus (HCV) non-structural (NS) 3 helicase is a promising anti-viral drug target. In order to examine the specific role of helicase during viral replication, mutations (R393A, F438A, T450I, E493K, W501A, and W501F) affecting the NS3 RNA unwinding without abolishing its ability to unwind DNA, bind nucleic acids, or hydrolyze ATP, were introduced into the helicase region of a subgenomic replicon. One replicon lacks two-thirds of the NS3 helicase (Δhel) was included as a negative control. Previous data has shown that purified R393A, F438A and W501A proteins do not unwind RNA, while purified W501F, T450I, and E493K proteins possess unwinding abilities similar to or better than wild type NS3. Results showed that hepatoma cells containing the Δhel replicon encoded and processed a HCV polyprotein, but did not synthesize additional viral RNA. The same phenotype was seen for R393A, F438A, W501A, and surprisingly the E493K replicons. Only T450I and W501F replicons synthesized both minus- and plus-sense viral RNA and formed colonies after selection with similar efficiencies as the parent replicon. Because these residues are unique for HCV and not cellular helicases, they represent novel sites for developing anti-viral drugs.