Mutational analysis of the hepatitis C virus RNA helicase.
The carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to possess an RNA helicase activity, typical of members of the DEAD box family of RNA helicases. In addition, the NS3 protein contains four amino acid motifs conserved in DEAD box proteins. In order to inspect the roles of individual amino acid residues in the four conserved motifs (AXXXXGKS, DECH, TAT, and QRRGRTGR) of the NS3 protein, mutational analysis was used in this study. Thirteen mutant proteins were constructed, and their biochemical activities were examined. Lys1235 in the AXXXXGKS motif was important for basal nucleoside triphosphatase (NTPase) activity in the absence of polynucleotide cofactor. A serine in the X position of the DEXH motif disrupted the NTPase and RNA helicase activities. Alanine substitution at His1318 of the DEXH motif made the protein possess high NTPase activity. In addition, we now report inhibition of NTPase activity of NS3 by polynucleotide cofactor. Gln1486 was indispensable for the enzyme activity, and this residue represents a distinguishing feature between DEAD box and DEXH proteins. There are four Arg residues in the QRRGRTGR motif of the HCV NS3 protein, and the second, Arg1488, was important for RNA binding and enzyme activity, even though it is less well conserved than other Arg residues. Arg1490 and Arg1493 were essential for the enzymatic activity. As the various enzymatic activities were altered by mutation, the enzyme characteristics were also changed.
Kim
D W
DW
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon.
Kim
J
J
Gwack
Y
Y
Han
J H
JH
Choe
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D011755:Pyrophosphatases / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000251:Adenosine Triphosphatases; D011755:Pyrophosphatases; D012313:RNA; 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; C019950:CTPase