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Journal of Virology, August 2006, p. 7844-7853, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00029-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Conserved Gly436-Trp-Leu-Ala-Gly-Leu-Phe-Tyr Motif in Hepatitis C Virus Glycoprotein E2 Is a Determinant of CD81 Binding and Viral Entry

Heidi E. Drummer,* Irene Boo, Anne L. Maerz, and Pantelis Poumbourios

The Macfarlane Burnet Institute for Medical Research and Public Health Ltd., 85 Commercial Road, Melbourne, Victoria, Australia 3004

Received 5 January 2006/ Accepted 2 June 2006

The hepatitis C virus (HCV) glycoproteins E1 and E2 form a heterodimer that mediates CD81 receptor binding and viral entry. In this study, we used site-directed mutagenesis to examine the functional role of a conserved G436WLAGLFY motif of E2. The mutants could be placed into two groups based on the ability of mature virion-incorporated E1E2 to bind the large extracellular loop (LEL) of CD81 versus the ability to mediate cellular entry of pseudotyped retroviral particles. Group 1 comprised E2 mutants where LEL binding ability largely correlated with viral entry ability, with conservative and nonconservative substitutions (W437 L/A, L438A, L441V/F, and F442A) inhibiting both functions. These data suggest that Trp-437, Leu-438, Leu-441, and Phe-442 directly interact with the LEL. Group 2 comprised E2 glycoproteins with more conservative substitutions that lacked LEL binding but retained between 20% and 60% of wild-type viral entry competence. The viral entry competence displayed by group 2 mutants was explained by residual binding by the E2 receptor binding domain to cellular full-length CD81. A subset of mutants maintained LEL binding ability in the context of intracellular E1E2 forms, but this function was largely lost in virion-incorporated glycoproteins. These data suggest that the CD81 binding site undergoes a conformational transition during glycoprotein maturation through the secretory pathway. The G436P mutant was an outlier, retaining near-wild-type levels of CD81 binding but lacking significant viral entry ability. These findings indicate that the G436WLAGLFY motif of E2 functions in CD81 binding and in pre- or post-CD81-dependent stages of viral entry.

* Corresponding author. Mailing address: The Macfarlane Burnet Institute for Medical Research and Public Health Ltd., GPO Box 2284, Melbourne, Victoria, Australia 3001. Phone: 613 9282 2179. Fax: 613 9282 2100. E-mail: hdrummer{at}burnet.edu.au.

Journal of Virology, August 2006, p. 7844-7853, Vol. 80, No. 16
0022-538X/06/$08.00+0     doi:10.1128/JVI.00029-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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