Previous Article | Next Article 
Journal of Virology, November 2009, p. 11926-11939, Vol. 83, No. 22
0022-538X/09/$08.00+0 doi:10.1128/JVI.01008-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Structural and Functional Analysis of Hepatitis C Virus Strain JFH1 Polymerase
Philip Simister ,2,
,
Melanie Schmitt,1,
Matthis Geitmann,3
Oliver Wicht,1
U. Helena Danielson,3
Rahel Klein,1
Stéphane Bressanelli,2* and
Volker Lohmann1*
University of Heidelberg, Department Molecular Virology, Heidelberg, Germany,1 Virologie Moléculaire et Structurale UMR CNRS 2472-INRA 1157, 91198 Gif-sur-Yvette Cedex, France,2 Department of Biochemistry and Organic Chemistry, Uppsala University, SE-751 23 Uppsala, Sweden3
Received 19 May 2009/ Accepted 31 August 2009
The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5- to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains "thumb" and "fingers." These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates.
* Corresponding author. Mailing address for V. Lohmann: Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany. Phone: 49-6221-56-6449. Fax: 49-6221-56-4570. E-mail: Volker.Lohmann{at}med.uni-heidelberg.de. Mailing address for S. Bressanelli: Virologie Moléculaire et Structurale, UMR CNRS 2472-INRA 1157, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. Phone: 33 (0)169823852. Fax: 33 (0)169824308. E-mail: stephane.bressanelli{at}vms.cnrs-gif.fr
Published ahead of print on 9 September 2009.
Present address: Weatherall Institute of Molecular Medicine, Department of Medical Oncology (Cancer Research United Kingdom), University of Oxford, Oxford, United Kingdom.
P.S. and M.S. contributed equally to this study.
Journal of Virology, November 2009, p. 11926-11939, Vol. 83, No. 22
0022-538X/09/$08.00+0 doi:10.1128/JVI.01008-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»