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Journal of Virology, November 2009, p. 11682-11693, Vol. 83, No. 22
0022-538X/09/$08.00+0 doi:10.1128/JVI.00691-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Characterization of Determinants Important for Hepatitis C Virus p7 Function in Morphogenesis by Using trans-Complementation
Christiane Brohm,1,5,
Eike Steinmann,1,5,
Martina Friesland,5
Ivo C. Lorenz,2,
Arvind Patel,3
Francois Penin,4
Ralf Bartenschlager,1 and
Thomas Pietschmann1,5*
Department of Molecular Virology, University Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany,1 Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, 1230 York Avenue, New York, New York 10065,2 MRC Virology Unit, Institute of Virology, University of Glasgow, Church Street, Glasgow G11 5JR, United Kingdom,3 Institut et Chimie des Proteines, UMR 5086 CNRS, Universite Lyon, IFR 128 BioSciences Lyon-Gerland, Lyon, France,4 Division of Experimental Virology, Twincore, Centre for Experimental and Clinical Infection Research, Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Feodor-Lynen-Str. 7, 30625 Hannover, Germany5
Received 3 April 2009/ Accepted 26 August 2009
Hepatitis C virus (HCV) p7 is an integral membrane protein that forms ion channels in vitro and that is crucial for the efficient assembly and release of infectious virions. Due to these properties, p7 was included in the family of viroporins that comprises proteins like influenza A virus M2 and human immunodeficiency virus type 1 (HIV-1) vpu, which alter membrane permeability and facilitate the release of infectious viruses. p7 from different HCV isolates sustains virus production with variable efficiency. Moreover, p7 determinants modulate processing at the E2/p7 and the p7/NS2 signal peptidase cleavage sites, and E2/p7 cleavage is incomplete. Consequently, it was unclear if a differential ability to sustain virus production was due to variable ion channel activity or due to alternate processing at these sites. Therefore, we developed a trans-complementation assay permitting the analysis of p7 outside of the HCV polyprotein and thus independently of processing. The rescue of p7-defective HCV genomes was accomplished by providing E2, p7, and NS2, or, in some cases, by p7 alone both in a transient complementation assay as well as in stable cell lines. In contrast, neither influenza A virus M2 nor HIV-1 vpu compensated for defective p7 in HCV morphogenesis. Thus, p7 is absolutely essential for the production of infectious HCV particles. Moreover, our data indicate that p7 can operate independently of an upstream signal sequence, and that a tyrosine residue close to the conserved dibasic motif of p7 is important for optimal virus production in the context of genotype 2a viruses. The experimental system described here should be helpful to investigate further key determinants of p7 that are essential for its structure and function in the absence of secondary effects caused by altered polyprotein processing.
* Corresponding author. Mailing address: Twincore Center, Department of Experimental Virology, Feodor-Lynen-Str. 7, Hannover 30625, Germany. Phone: 49 511 220027 130. Fax: 49 511 220027 186. E-mail: thomas.pietschmann{at}twincore.de
Published ahead of print on 2 September 2009.
These authors contributed equally to this work.
Present address: International AIDS Vaccine Initiative, AIDS Vaccine Design & Development Laboratory, Brooklyn, New York 11220.
Journal of Virology, November 2009, p. 11682-11693, Vol. 83, No. 22
0022-538X/09/$08.00+0 doi:10.1128/JVI.00691-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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