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Originally published In Press as doi:10.1074/jbc.M407787200 on August 31, 2004
J. Biol. Chem., Vol. 279, Issue 47, 48576-48587, November 19, 2004
The NS5A Protein of Hepatitis C Virus Is a Zinc Metalloprotein*
Timothy L. Tellinghuisen  ,
Joseph Marcotrigiano¶,
Alexander E. Gorbalenya||, and
Charles M. Rice**
From the
Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, New York 10021 and the ||Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, P. O. Box 9600, E4-P, 2300 RC Leiden, The Netherlands
The NS5A protein of hepatitis C virus is believed to be an integral part of the viral replicase. Despite extensive investigation, the role of this protein remains elusive. Only limited biochemical characterization of NS5A has been performed, with most research to date involving the myriad of host proteins and signaling cascades that interact with NS5A. The need for better characterization of NS5A is paramount for elucidating the role of this protein in the virus life cycle. Examination of NS5A using bioinformatics tools suggested the protein consisted of three domains and contained an unconventional zinc binding motif within the N-terminal domain. We have developed a method to produce NS5A and performed limited proteolysis to confirm the domain organization model. The zinc content of purified NS5A and the N-terminal domain of NS5A was determined, and each of these proteins was found to coordinate one zinc atom per protein. The predicted zinc binding motif consists of four cysteine residues, conserved among the Hepacivirus and Pestivirus genera, fitting the formula of CX17CXCX20C. Mutation of any of the four cysteine components of this motif reduced NS5A zinc coordination and led to a lethal phenotype for HCV RNA replication, whereas mutation of other potential metal coordination residues in the N-terminal domain of NS5A, but outside the zinc binding motif, had little effect on zinc binding and, aside from one exception, were tolerated for replication. Collectively, these results indicate that NS5A is a zinc metalloprotein and that zinc coordination is likely required for NS5A function in the hepatitis C replicase.
Received for publication, July 12, 2004
, and in revised form, August 30, 2004.
* This work was supported in part by Grant 5 R01 CA57973-12 from the National Institutes of Health and the Greenberg Medical Research Institute (to C. M. R.). 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.
 Recipient of fellowships from the Charles H. Revson Foundation for Biomedical Research and the National Institutes of Health Ruth L. Kirschstein National Research Service Award (5F32 AI51820-03) granted through the NIAID, National Institutes of Health.
¶ Recipient of a Merck Fellow of the Life Sciences Research Foundation.
** To whom correspondence should be addressed. Tel.: 212-327-7046; Fax: 212-327-7048; E-mail: ricec{at}rockefeller.edu.
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Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
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