JVI Accepts, published online ahead of print on 7 October 2009

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J. Virol. doi:10.1128/JVI.01184-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Determinants of hepatitis C virus nonstructural protein 2 protease domain required for production of infectious virus

Thomas G. Dentzer, Ivo C. Lorenz, Matthew J. Evans, and Charles M. Rice^*

Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, 1230 York Avenue, New York, New York 10065; Laboratoire de Rétrovirologie, Centre de Recherche Public-Santé, 84 rue Val Fleuri, L-1526 Luxembourg

^* To whom correspondence should be addressed. Email: ricec{at}rockefeller.edu.

The hepatitis C virus (HCV) non-structural protein 2 (NS2) is a dimeric multifunctional hydrophobic protein with an essential but poorly understood role in infectious virus production. We investigated the determinants of NS2 function in the HCV life cycle. Based on the crystal structure of the post-cleavage form of the NS2 protease domain, we mutated conserved features and analyzed the effects of these changes on polyprotein processing, replication, and infectious virus production. We found that mutations around the protease active site inhibit viral RNA replication, likely by preventing NS2-3 cleavage. In contrast, alterations at the dimer interface or in the C-terminal region did not affect replication, NS2 stability, or NS2 protease activity, but decreased infectious virus production. A comprehensive deletion and mutagenesis analysis of the C-terminal end of NS2 revealed the importance of its C-terminal leucine residue in infectious particle production. The crystal structure of the NS2 protease domain shows that this C-terminal leucine is locked in the active site, and mutation or deletion of this residue could therefore alter the conformation of NS2 and disrupt potential protein-protein interactions important for infectious particle production. These studies begin to dissect the residues of NS2 involved in its multiple essential roles in the HCV life cycle and suggest NS2 as a viable target for HCV-specific inhibitors.