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Journal of Virology, November 2001, p. 11205-11217, Vol. 75, No. 22
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.22.11205-11217.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Folding of Hepatitis C Virus E1 Glycoprotein in a Cell-Free System

Marcello Merola,^1,2,* Michela Brazzoli,¹ Fabienne Cocchiarella,^1, Jens M. Heile,^1, Ari Helenius,³ Amy J. Weiner,⁴ Michael Houghton,⁴ and Sergio Abrignani¹

IRIS Research Center, Chiron, 53100 Siena,¹ and Department of Neurological and Vision Sciences, Section of Biochemistry, University of Verona, 37134 Verona,² Italy; Institute of Biochemistry, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland³; and Chiron Corporation, Emeryville, California 94608⁴

Received 10 May 2001/Accepted 16 August 2001

The hepatitis C virus (HCV) envelope proteins, E1 and E2, form noncovalent heterodimers and are leading candidate antigens for a vaccine against HCV. Studies in mammalian cell expression systems have focused primarily on E2 and its folding, whereas knowledge of E1 folding remains fragmentary. We used a cell-free in vitro translation system to study E1 folding and asked whether the flanking proteins, Core and E2, influence this process. We translated the polyprotein precursor, in which the Core is N-terminal to E1, and E2 is C-terminal, and found that when the core protein was present, oxidation of E1 was a slow, E2-independent process. The half-time for E1 oxidation was about 5 h in the presence or absence of E2. In contrast with previous reports, analysis of three constructs of different lengths revealed that the E2 glycoprotein undergoes slow oxidation as well. Unfolded or partially folded E1 bound to the endoplasmic reticulum chaperones calnexin and (with lower efficiency) calreticulin, whereas no binding to BiP/GRP78 or GRP94 could be detected. Release from calnexin and calreticulin was used to assess formation of mature E1. When E1 was expressed in the absence of Core and E2, its oxidation was impaired. We conclude that E1 folding is a process that is affected not only by E2, as previously shown, but also by the Core. The folding of viral proteins can thus depend on complex interactions between neighboring proteins within the polyprotein precursor.

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^* Corresponding author. Mailing address: IRIS Research Center, Chiron, via Fiorentina 1, 53100 Siena, Italy. Phone: (39) 0577 243314. Fax: (39) 0577 243564. E-mail: marcello_merola{at}chiron.it.

Present address: Department of Animal Biology, University of Modena and Reggio Emilia, 41100 Modena, Italy.

Present address: Institute of Biotechnology, Swiss Federal Institute of Technology Zurich, Hànggerberg, HPT, CH-8093 Zurich, Switzerland.

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Journal of Virology, November 2001, p. 11205-11217, Vol. 75, No. 22
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.22.11205-11217.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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