HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 42, 28546-28562, October 17, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/42/28546    most recent M803981200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jirasko, V. Articles by Bartenschlager, R. Search for Related Content PubMed PubMed Citation Articles by Jirasko, V. Articles by Bartenschlager, R. Social Bookmarking                      What's this?

Structural and Functional Characterization of Nonstructural Protein 2 for Its Role in Hepatitis C Virus Assembly^*

Vlastimil Jirasko, Roland Montserret, Nicole Appel¹, Anne Janvier, Leah Eustachi, Christiane Brohm², Eike Steinmann², Thomas Pietschmann², Francois Penin, and Ralf Bartenschlager³

From the Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany and Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS, Université de Lyon, IFR128 BioSciences Gerland-Lyon Sud, F-69367 Lyon Cedex 07, France

The hepatitis C virus (HCV) is a flavivirus replicating in the cytoplasm of infected cells. The HCV genome is a single-stranded RNA encoding a polyprotein that is cleaved by cellular and viral proteases into 10 different products. While the structural proteins core protein, envelope protein 1 (E1) and E2 build up the virus particle, most nonstructural (NS) proteins are required for RNA replication. One of the least studied proteins is NS2, which is composed of a C-terminal cytosolic protease domain and a highly hydrophobic N-terminal domain. It is assumed that the latter is composed of three trans-membrane segments (TMS) that tightly attach NS2 to intracellular membranes. Taking advantage of a system to study HCV assembly in a hepatoma cell line, in this study we performed a detailed characterization of NS2 with respect to its role for virus particle assembly. In agreement with an earlier report ( Jones, C. T., Murray, C. L., Eastman, D. K., Tassello, J., and Rice, C. M. (2007) J. Virol. 81, 8374-8383[Abstract/Free Full Text] ), we demonstrate that the protease domain, but not its enzymatic activity, is required for infectious virus production. We also show that serine residue 168 in NS2, implicated in the phosphorylation and stability of this protein, is dispensable for virion formation. In addition, we determined the NMR structure of the first TMS of NS2 and show that the N-terminal segment (amino acids 3-11) forms a putative flexible helical element connected to a stable -helix (amino acids 12-21) that includes an absolutely conserved helix side in genotype 1b. By using this structure as well as the amino acid conservation as a guide for a functional study, we determined the contribution of individual amino acid residues in TMS1 for HCV assembly. We identified several residues that are critical for virion formation, most notably a central glycine residue at position 10 of TMS1. Finally, we demonstrate that mutations in NS2 blocking HCV assembly can be rescued by trans-complementation.

Received for publication, May 26, 2008 , and in revised form, July 18, 2008.

The atomic coordinates and structure factors (code 2JY0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The proton chemical shifts of all residues have been deposited in the BioMagResBank (BMRB) under the accession number 15579.

^* This work was supported by the Bioquant Ph.D. program, Ministry of Science, Research, and the Arts of Baden-Württemberg Grant Az 23-7532.24-22-21-12/1 (to R. B. and T. P.), German Research Council Grants BA 1505/2-1 (to N. A. and R. B.), SFB 638, Teilprojekt A5 (to R. B.), and grants from the CNRS (to F. P.) and the Agence Nationale pour la Recherche sur le SIDA et les Hépatites Virales (to F. P.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. 1 and 2.

¹ Present address: EMBL Heidelberg, Cell Biology/Cell Biophysics Programme, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

² Present address: Twincore, Center for Experimental and Clinical Infection Research, Department of Experimental Virology, Feodor-Lynen-Str. 7, 30625 Hannover, Germany.

³ To whom correspondence should be addressed: Dept. of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany. Tel.: 49-6221-56-4569; Fax: 49-6221-56-4570; E-mail: ralf.bartenschlager{at}med.uni-heidelberg.de.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				S. Welbourn, V. Jirasko, V. Breton, S. Reiss, F. Penin, R. Bartenschlager, and A. Pause Investigation of a role for lysine residues in non-structural proteins 2 and 2/3 of the hepatitis C virus for their degradation and virus assembly J. Gen. Virol., May 1, 2009; 90(5): 1071 - 1080. [Abstract] [Full Text] [PDF]