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J. Biol. Chem., Vol. 281, Issue 19, 13449-13462, May 12, 2006

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The NS5A Protein of the Hepatitis C Virus Genotype 1a Is Cleaved by Caspases to Produce C-terminal-truncated Forms of the Protein That Reside Mainly in the Cytosol^*

From the Molecular Virology Laboratory, Hellenic Pasteur Institute, 115 21 Athens, Greece

The nonstructural 5A (NS5A) protein of the hepatitis C virus (HCV) is a multifunctional protein that is implicated in viral replication and pathogenesis. We report here that NS5A of HCV-1a is cleaved at multiple sites by caspase proteases in transfected cells. Two cleavage sites at positions Asp¹⁵⁴ and ²⁴⁸DXXD²⁵¹ were mapped. Cleavage at Asp¹⁵⁴ has been previously recognized as one of the caspase cleavage sites for the NS5A protein of HCV genotype 1b (1, 2) and results in the production of a 17-kDa fragment. The sequence ²⁴⁸DXXD²⁵¹ is a novel caspase recognition motif for NS5A and is responsible for the production of a 31-kDa fragment. Furthermore, we show that Arg²¹⁷ is implicated in the production of the previously described 24-kDa product, whose accumulation is affected by both calpain and caspase inhibitors. We also showed that caspase-mediated cleavage occurs in the absence of exogenous proapoptotic stimuli and is not related to the accumulation of the protein in the endoplasmic reticulum. Interestingly, our data indicate that NS5A is targeted by at least two different caspases and suggest that caspase 6 is implicated in the production of the 17-kDa fragment. Most importantly, we report that, all the detectable NS5A fragments following caspase-mediated cleavage are C-terminal-truncated forms of NS5A and are mainly localized in the cytosol. Thus, in sharp contrast to the current view we found no evidence supporting a role for caspase-mediated cleavage in the transport of the NS5A protein to the nucleus, which could lead to transcriptional activation.

Received for publication, February 6, 2006 , and in revised form, March 3, 2006.

^* 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: Molecular Virology Laboratory, Hellenic Pasteur Institute, 127, Vas. Sofias Ave., 115 21 Athens, Greece. Tel./Fax: 30-210-64788777; E-mail: penelopm{at}hol.gr.

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