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J. Biol. Chem., Vol. 282, Issue 48, 34913-34920, November 30, 2007

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The Serine Protease Domain of Hepatitis C Viral NS3 Activates RNA Helicase Activity by Promoting the Binding of RNA Substrate^*

Rudolf K. F. Beran¹, Victor Serebrov, and Anna Marie Pyle, Investigator with Yale University and the Howard Hughes Medical Institute²

From the Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520 and The Howard Hughes Medical Institute, Chevy Chase, Maryland 20815

Nonstructural (NS) protein 3 is a DEXH/D-box motor protein that is an essential component of the hepatitis C viral (HCV) replicative complex. The full-length NS3 protein contains two functional modules, both of which are essential in the life cycle of HCV: a serine protease domain at the N terminus and an ATPase/helicase domain (NS3hel) at the C terminus. Truncated NS3hel constructs have been studied extensively; the ATPase, nucleic acid binding, and helicase activities have been examined and NS3hel has been used as a target in the development of antivirals. However, a comprehensive comparison of NS3 and NS3hel activities has not been performed, so it remains unclear whether the protease domain plays a vital role in NS3 helicase function. Given that many DEXH/D-box proteins are activated upon interaction with cofactor proteins, it is important to establish if the protease domain acts as the cofactor for stimulating NS3 helicase function. Here we show that the protease domain greatly enhances both the direct and functional binding of RNA to NS3. Whereas electrostatics plays an important role in this process, there is a specific allosteric contribution from the interaction interface between NS3hel and the protease domain. Most importantly, we establish that the protease domain is required for RNA unwinding by NS3. Our results suggest that, in addition to its role in cleavage of host and viral proteins, the NS3 protease domain is essential for the process of viral RNA replication and, given its electrostatic contribution to RNA binding, it may also assist in packaging of the viral RNA.

Received for publication, August 27, 2007 , and in revised form, October 4, 2007.

^* This work was supported in part by National Institutes of Health Grant GM60620. 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 Supported by Ruth Kirschstein Post-doctoral Fellowship F32 GM071120-01A1 from the National Institutes of Health.

² To whom correspondence should be addressed: 266 Whitney Ave., Bass Bldg., Rm. 334, Yale University, New Haven, CT 06520. Tel.: 203-432-5633; Fax: 203-432-5316; E-mail: anna.pyle{at}yale.edu.

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