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Journal of Virology, October 2000, p. 9732-9737, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Roles of the AX4GKS and Arginine-Rich Motifs of Hepatitis C Virus RNA Helicase in ATP- and Viral RNA-Binding Activity

Shin C. Chang,1,* Ju-Chien Cheng,1,dagger Yi-Hen Kou,1 Chuan-Hong Kao,1 Chiung-Hui Chiu,1 Hung-Yi Wu,2 and Ming-Fu Chang2

Institutes of Microbiology1 and Biochemistry,2 National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China

Received 29 November 1999/Accepted 26 July 2000

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 AX4GKS 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 AX4GKS 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.

* Corresponding author. Mailing address: No. 1, Sec. 1, Jen-Ai Rd., Institute of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan, Republic of China. Phone: 886-2-23123456, ext. 8290. Fax: 886-2-23915293. E-mail: scchang{at}ha.mc.ntu.edu.tw.

dagger Present address: School of Medical Technology, China Medical College, Taichung, Taiwan.

Journal of Virology, October 2000, p. 9732-9737, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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