Expression, Isolation, and Characterization of the Hepatitis C Virus ATPase/RNA Helicase

The genome of the hepatitis C virus directs the synthesis of a single polyprotein, which is proteolytically cleaved into at least nine functional proteins. The amino-terminal portion of the polyprotein forms the structural proteins, while the carboxy-terminal region constitutes a variety of viral enzymes. The nonstructural 3 (NS3) protein, consisting of amino acids 1027–1657 of the polyprotein, is believed to be a multifunctional protein with an amino-terminal serine protease domain, which is involved in polyprotein processing, and a carboxy-terminal ATPase/RNA helicase domain, presumably involved in viral replication. We have assembled an expression vector which directs the synthesis of residues 1207–1612 of the polyprotein with an amino-terminal polyhistidine purification tag. This portion of the NS3 protein contains the putative ATPase/helicase domain. The protein has been purified to yield 30–50 mg of enzymatically active protein per liter of culture. The purified NS3 protein has both NTPase and RNA helicase activities. ATP is the preferred substrate for the NTPase; GTP is also utilized; however, UTP is a very poor substrate and CTP is not utilized. The RNA helicase activity is dependent on ATP and divalent cation. Either manganese or magnesium can serve as the divalent cation.