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Journal of Virology, June 2005, p. 6868-6881, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6868-6881.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Complete Translation of the Hepatitis C Virus Genome In Vitro: Membranes Play a Critical Role in the Maturation of All Virus Proteins except for NS3

Yuri V. Svitkin,1* Arnim Pause,2 Marcelo Lopez-Lastra,3 Sandra Perreault,1 and Nahum Sonenberg1,2

Department of Biochemistry,1 McGill Cancer Center, McGill University, Montreal, Quebec, Canada H3G 1Y6,2 Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Pontificia Universidad Católica de Chile, Santiago, Chile3

Received 2 August 2004/ Accepted 26 January 2005

We developed an in vitro translation extract from Krebs-2 cells that translates the entire open reading frame of the hepatitis C virus (HCV) strain H77 and properly processes the viral protein precursors when supplemented with canine microsomal membranes (CMMs). Translation of the C-terminal portion of the viral polyprotein in this system is documented by the synthesis of NS5B. Evidence for posttranslational modification of the viral proteins, the N-terminal glycosylation of E1 and the E2 precursor (E2-p7), and phosphorylation of NS5A is presented. With the exception of NS3, efficient generation of all virus-specific proteins is CMM dependent. A time course of the appearance of HCV products indicates that the viral polyprotein is cleaved cotranslationally. A competitive inhibitor of the NS3 protease inhibited accumulation of NS3, NS4B, NS5A, and NS5B, but not that of NS2 or structural proteins. CMMs also stabilized HCV mRNA during translation. Finally, the formyl-[35S]methionyl moiety of the initiator tRNAMet was incorporated exclusively into the core protein portion of the polyprotein, demonstrating that translation initiation in this system occurs with high fidelity.

* Corresponding author. Mailing address: Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada H3G 1Y6. Phone: (514) 398-5695. Fax: (514) 398-1287. E-mail: yuri.svitkine{at}mcgill.ca.

Journal of Virology, June 2005, p. 6868-6881, Vol. 79, No. 11
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.11.6868-6881.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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