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Journal of Virology, January 2003, p. 571-582, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.571-582.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Structurally Conserved Amino Acid W501 Is Required for RNA Helicase Activity but Is Not Essential for DNA Helicase Activity of Hepatitis C Virus NS3 Protein

Jong Won Kim,1,{dagger} Mi Young Seo,1,2 Anang Shelat,3 Chon Saeng Kim,1 Tae Woo Kwon,1 Hui-hua Lu,2 Demetri Theodore Moustakas,4 Jeonghoon Sun,4 and Jang H. Han1*

Department of Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, Korea,1 Chiron Corporation, Emeryville, California 94608,2 Department of Pharmaceutical Chemistry,4 Chemistry and Chemical Biology Program, University of California, San Francisco, California 941433

Received 16 May 2002/ Accepted 23 September 2002

Hepatitis C virus (HCV) is a positive-strand RNA virus that encodes a helicase required for viral replication. Although HCV does not replicate through a DNA intermediate, HCV helicase unwinds both RNA and DNA duplexes. An X-ray crystal structure of the HCV helicase complexed with (dU)8 has been solved, and the substrate-amino acids interactions within the catalytic pocket were shown. Among these, residues W501 and V432 were reported to have base stacking interactions and to be important for the unwinding function of HCV helicase. It has been hypothesized that specific interactions between the enzyme and substrate in the catalytic pocket are responsible for the substrate specificity phenotype. We therefore mutagenized W501 and V432 to investigate their role in substrate specificity in HCV helicase. Replacement of W501, but not V432, with nonaromatic residues resulted in complete loss of RNA unwinding activity, whereas DNA unwinding activity was largely unaffected. The loss of unwinding activity was fully restored in the W501F mutant, indicating that the aromatic ring is crucial for RNA helicase function. Analysis of ATPase and nucleic acid binding activities in the W501 mutant enzymes revealed that these activities are not directly responsible for the substrate specificity phenotype. Molecular modeling of the enzyme-substrate interaction at W501 revealed a putative {pi}-facial hydrogen bond between the 2'-OH of ribose and the aromatic tryptophan ring. This evidence correlates with biochemical results suggesting that the {pi}-facial bond may play an important role in the RNA unwinding activity of the HCV NS3 protein.

* Corresponding author. Present address: Chiron Corp., 4560 Horton St., Emeryville, CA 94608. Phone: (510) 923-2937. Fax: (510) 923-2586. E-mail: jang_han{at}chiron.com.

{dagger} Present address: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202.

Journal of Virology, January 2003, p. 571-582, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.571-582.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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