HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 52, 52471-52478, December 26, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/52/52471    most recent M308917200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bougie, I. Articles by Bisaillon, M. Search for Related Content PubMed PubMed Citation Articles by Bougie, I. Articles by Bisaillon, M. Social Bookmarking                      What's this?

Initial Binding of the Broad Spectrum Antiviral Nucleoside Ribavirin to the Hepatitis C Virus RNA Polymerase^*

Isabelle Bougie and Martin Bisaillon

From the Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada

Ribavirin is a broad spectrum antiviral nucleoside that displays activity against a variety of RNA and DNA viruses. Ribavirin is currently used in combination with interferon- for the treatment of hepatitis C virus (HCV) infection and was recently shown to be directly incorporated by the HCV RNA polymerase into RNA products. This capacity ultimately leads to increased mutation rates and drastically reduces the viral fitness. As a first step toward elucidating the nature of the specific interaction between ribavirin and the HCV polymerase, we have utilized fluorescence spectroscopy to monitor precisely the binding of ribavirin triphosphate (RTP) to the viral polymerase. This spectroscopic approach allowed us to clearly separate the RTP binding activity from the concomitant catalytic steps. We report here the first detailed study of the binding kinetics and thermodynamic parameters involved in the interaction between RTP and an RNA polymerase. We demonstrate that RTP binds to the same active site as nucleotides. Furthermore, we provide evidence that the HCV polymerase cannot only bind to RTP but also to nonphosphorylated ribavirin, albeit with less affinity. By using various combinations of template-primers, we also demonstrate that base pairing is not involved in the initial binding of RTP to the HCV polymerase. Based on the results of circular dichroism and denaturation studies, we show that the RNA polymerase undergoes subtle conformational changes upon the binding of RTP, although the interaction does not significantly modify the stability of the protein. Finally, although metal ions are required for catalytic activity, they are not required for the initial binding of RTP to the polymerase. Such quantitative analyses are of primary importance for the rational design of new ribavirin analogues of potential therapeutic value and provide crucial insights on the interaction between RTP and the HCV RNA polymerase.

Received for publication, August 12, 2003 , and in revised form, October 16, 2003.

^* This work was supported by grants from the Canadian Institutes for Health Research and Fonds de la Recherche en Santé du Québec. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

New Investigator Scholar from the Canadian Institutes for Health Research. To whom correspondence should be addressed: Dépt. de Biochimie, Faculté de Médecine, Université de Sherbrooke, 3001 12e Ave., Sherbrooke, Québec J1H 5N4, Canada. Tel.: 819-564-5227; Fax: 819-564-5340; E-mail: Martin.Bisaillon{at}USherbrooke.ca.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. Q. Hang, Y. Yang, S. F. Harris, V. Leveque, H. J. Whittington, S. Rajyaguru, G. Ao-Ieong, M. F. McCown, A. Wong, A. M. Giannetti, et al. Slow Binding Inhibition and Mechanism of Resistance of Non-nucleoside Polymerase Inhibitors of Hepatitis C Virus J. Biol. Chem., June 5, 2009; 284(23): 15517 - 15529. [Abstract] [Full Text] [PDF]

				K. D. Ibarra and J. K. Pfeiffer Reduced Ribavirin Antiviral Efficacy via Nucleoside Transporter-Mediated Drug Resistance J. Virol., May 1, 2009; 83(9): 4538 - 4547. [Abstract] [Full Text] [PDF]

				A. Wohnsland, W. P. Hofmann, and C. Sarrazin Viral Determinants of Resistance to Treatment in Patients with Hepatitis C Clin. Microbiol. Rev., January 1, 2007; 20(1): 23 - 38. [Abstract] [Full Text] [PDF]

				M. A. Mir and A. T. Panganiban The Hantavirus Nucleocapsid Protein Recognizes Specific Features of the Viral RNA Panhandle and Is Altered in Conformation upon RNA Binding J. Virol., February 1, 2005; 79(3): 1824 - 1835. [Abstract] [Full Text] [PDF]

				K. K.-S. Ng, N. Pendas-Franco, J. Rojo, J. A. Boga, A. Machin, J. M. M. Alonso, and F. Parra Crystal Structure of Norwalk Virus Polymerase Reveals the Carboxyl Terminus in the Active Site Cleft J. Biol. Chem., April 16, 2004; 279(16): 16638 - 16645. [Abstract] [Full Text] [PDF]