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Journal of Virology, November 2004, p. 12207-12217, Vol. 78, No. 22
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.22.12207-12217.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

C. T. Ranjith-Kumar,1 R. T. Sarisky,2,3,{dagger} L. Gutshall,2,3,{dagger} M. Thomson,2 and C. C. Kao1*

Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas,1 Department of Virology,2 Department of Clinical Virology, Metabolic and Viral Diseases Center of Excellence and Drug Discovery, GlaxoSmithKline, Collegeville, Pennsylvania, and Research Triangle Park, North Carolina3

Received 14 March 2004/ Accepted 20 July 2004

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol. 76:3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a Km for GTP of 3.5 µM, all of the mutations except one had Kms that were three- to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.

* Corresponding author. Mailing address: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Mail Stop 2128, Texas 77843. Phone: (979) 458-2235. Fax: (979) 845-9274. E-mail: ckao{at}tamu.edu.

{dagger} Present address: Centocor Inc., Infectious Disease Research, Malvern, PA 19355.

Journal of Virology, November 2004, p. 12207-12217, Vol. 78, No. 22
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.22.12207-12217.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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