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J. Biol. Chem., Vol. 283, Issue 35, 24089-24102, August 29, 2008

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Functional Characterization of Fingers Subdomain-specific Monoclonal Antibodies Inhibiting the Hepatitis C Virus RNA-dependent RNA Polymerase^*

Andrei Nikonov, Erkki Juronen, and Mart Ustav¹

From the Department of Biomedical Technology, Institute of Technology, University of Tartu, Nooruse Street 1, Tartu 50411 and Laboratory of Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, Ravila Street 19, Tartu 50411, Estonia

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), encoded by nonstructural protein 5B (NS5B), is absolutely essential for the viral replication. Here we describe the development, characterization, and functional properties of the panel of monoclonal antibodies (mAbs) and specifically describe the mechanism of action of two mAbs inhibiting the NS5B RdRp activity. These mAbs recognize and bind to distinct linear epitopes in the fingers subdomain of NS5B. The mAb 8B2 binds the N-terminal epitope of the NS5B and inhibits both primer-dependent and de novo RNA synthesis. mAb 8B2 selectively inhibits elongation of RNA chains and enhances the RNA template binding by NS5B. In contrast, mAb 7G8 binds the epitope that contains motif G conserved in viral RdRps and inhibits only primer-dependent RNA synthesis by specifically targeting the initiation of RNA synthesis, while not interfering with the binding of template RNA by NS5B. To reveal the importance of the residues of mAb 7G8 epitope for the initiation of RNA synthesis, we performed site-directed mutagenesis and extensively characterized the functionality of the HCV RdRp motif G. Comparison of the mutation effects in both in vitro primer-dependent RdRp assay and cellular transient replication assay suggested that mAb 7G8 epitope amino acid residues are involved in the interaction of template-primer or template with HCV RdRp. The data presented here allowed us to describe the functionality of the epitopes of mAbs 8B2 and 7G8 in the HCV RdRp activity and suggest that the epitopes recognized by these mAbs may be useful targets for antiviral drugs.

Received for publication, May 5, 2008 , and in revised form, June 17, 2008.

^* This work was supported by Grants 5998 and 5999 from Estonian Science Foundation and by Target Financial Project SF0182566 from Basic Science Committee. 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.

¹ To whom correspondence should be addressed: Dept. of Biomedical Technology, Institute of Technology, University of Tartu and Estonian Biocentre, Nooruse 1, Tartu 50411, Estonia. Tel.: 372-737-5047; Fax: 372-737-4900; E-mail: mart.ustav{at}ut.ee.

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