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.
D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004789:Enzyme Activation; D016174:Hepacivirus / Q000201:enzymology*; D008969:Molecular Sequence Data; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D017421:Sequence Analysis; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000251:Adenosine Triphosphatases; D018076:DNA, Complementary; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases