Towards defining a minimal functional domain for NTPase and RNA helicase activities of the hepatitis C virus NS3 protein.
Hepatitis C virus (HCV) possesses two separate enzymatic functions in the NS3 protein: a protease and an NTPase/RNA helicase. In order to determine the minimal domain for NTPase and RNA helicase activities of the HCV NS3 protein, serial deletion mutants were constructed. The NS3H protein a fusion protein of 25 amino acids (aa) from an expression vector and the C-terminal 466 aa of the HCV NS3 protein, contains an NTPase/RNA helicase activity. We made deletion mutants of 10, 30, 50, 97, and 135 aa from the C-terminus and 16 and 32 aa from the N-terminus of the NS3H protein. The deleted protein lacking 50 aa from the C-terminus still possessed both activities, while the protein lacking 97 aa from the C-terminus lost an RNA helicase activity. The mutant lacking 16 amino acids from the N-terminus retained the enzymatic activities and the N-terminal 32 aa deleted mutant lost an NTPase/RNA helicase activity. A combinational deletion mutant lacking 16 aa the N-terminus and 50 aa from the C-terminus retained the enzymatic activities. These results show that the functional domain of the HCV NTPase/ RNA helicase is about 400 aa in length and maps between NS3 residues 1209 and 1608.
Kim
D W
DW
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea.
Gwack
Y
Y
Han
J H
JH
Choe
J
J
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D016174:Hepacivirus / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D043583:Nucleoside-Triphosphatase; D016133:Polymerase Chain Reaction; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D017766:Acid Anhydride Hydrolases; D017931:DNA Primers; D043583:Nucleoside-Triphosphatase; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases