Roles of the AX(4)GKS and arginine-rich motifs of hepatitis C virus RNA helicase in ATP- and viral RNA-binding activity.
The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses protease, nucleoside triphosphatase, and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase are not well-characterized. In this study, NS3 proteins with point mutations in the conserved helicase motifs were expressed in Escherichia coli, purified, and analyzed for their effects on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV cross-linking experiments indicate that the lysine residue in the AX(4)GKS motif is directly involved in ATP binding, whereas the NS3(GR1490DT) mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutation at Arg1488 (R1488A) and was also affected by the K1236E substitution in the AX(4)GKS motif and the R1490A and GR1490DT mutations in the arginine-rich motif. Previously, Arg1490 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the gamma-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding.
Chang
S C
SC
Institutes of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China. scchang@ha.mc.ntu.edu.tw
Cheng
J C
JC
Kou
Y H
YH
Kao
C H
CH
Chiu
C H
CH
Wu
H Y
HY
Chang
M F
MF
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001120:Arginine; D008969:Molecular Sequence Data; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D001120:Arginine; D020365:RNA Helicases; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases