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Arch Biochem Biophys. 1995 Oct 20;323(1):47-53.

Expression, isolation, and characterization of the hepatitis C virus ATPase/RNA helicase.

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Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298-0614, USA.


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.

[Indexed for MEDLINE]

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