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Journal of Virology, August 1999, p. 6782-6790, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Functional Analysis of Cell Surface-Expressed Hepatitis C Virus E2 Glycoprotein

Mike Flint,1 Joanne M. Thomas,1 Catherine M. Maidens,1 Christine Shotton,2 Shoshana Levy,3 Wendy S. Barclay,1 and Jane A. McKeating1,*

School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ,1 and Institute for Cancer Research, Sutton SM2 5NG,2 United Kingdom, and Department of Medicine, Division of Oncology, Stanford University Medical Center, Stanford, California 943053

Received 6 November 1998/Accepted 29 March 1999

Hepatitis C virus (HCV) glycoproteins E1 and E2, when expressed in eukaryotic cells, are retained in the endoplasmic reticulum (ER). C-terminal truncation of E2 at residue 661 or 715 (position on the polyprotein) leads to secretion, consistent with deletion of a proposed hydrophobic transmembrane anchor sequence. We demonstrate cell surface expression of a chimeric glycoprotein consisting of E2 residues 384 to 661 fused to the transmembrane and cytoplasmic domains of influenza A virus hemagglutinin (HA), termed E2661-HATMCT. The E2661-HATMCT chimeric glycoprotein was able to bind a number of conformation-dependent monoclonal antibodies and a recombinant soluble form of CD81, suggesting that it was folded in a manner comparable to "native" E2. Furthermore, cell surface-expressed E2661-HATMCT demonstrated pH-dependent changes in antigen conformation, consistent with an acid-mediated fusion mechanism. However, E2661-HATMCT was unable to induce cell fusion of CD81-positive HEK cells after neutral- or low-pH treatment. We propose that a stretch of conserved, hydrophobic amino acids within the E1 glycoprotein, displaying similarities to flavivirus and paramyxovirus fusion peptides, may constitute the HCV fusion peptide. We demonstrate that influenza virus can incorporate E2661-HATMCT into particles and discuss experiments to address the relevance of the E2-CD81 interaction for HCV attachment and entry.

* Corresponding author. Mailing address: School of Animal and Microbial Sciences, University of Reading, Whiteknights, P.O. Box 228, Reading RG6 6AJ, United Kingdom. Phone: (44) 1189 875 123, ext. 7892/4275. Fax: (44) 1189 316 671. E-mail: j.a.mckeating{at}reading.ac.uk.

Journal of Virology, August 1999, p. 6782-6790, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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