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Journal of Virology, December 2005, p. 14945-14955, Vol. 79, No. 23
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.23.14945-14955.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Hydrophobic Domain in the Large Envelope Protein Is Essential for Fusion of Duck Hepatitis B Virus at the Late Endosome

J. Chojnacki,1,2 D. A. Anderson,1 and E. V. L. Grgacic*

Macfarlane Burnet Institute for Medical Research and Public Health, and Australian Centre for Hepatitis Virology, Melbourne, Australia,1 Department of Microbiology and Immunology, University of Melbourne, Melbourne, Australia2

Received 17 June 2005/ Accepted 1 September 2005

The duck hepatitis B virus (DHBV) envelope is comprised of two transmembrane (TM) proteins, the large (L) and the small (S), that assemble into virions and subviral particles. Secondary-structure predictions indicate that L and S have three {alpha}-helical, membrane-spanning domains, with TM1 predicted to act as the fusion peptide following endocytosis of DHBV into the hepatocyte. We used bafilomycin A1 during infection of primary duck hepatocytes to show that DHBV must be trafficked from the early to the late endosome for fusion to occur. Alanine substitution mutations in TM1 of L and S, which lowered TM1 hydrophobicity, were used to examine the role of TM1 in infectivity. The high hydrophobicity of the TM1 domain of L, but not of S, was shown to be essential for virus infection at a step downstream of receptor binding and virus internalization. Using wild-type and mutant synthetic peptides, we demonstrate that the hydrophobicity of this domain is required for the aggregation and the lipid mixing of phospholipid vesicles, supporting the role of TM1 as the fusion peptide. While lipid mixing occurred at pH 7, the kinetics of insertion of the fusion peptide was increased at pH 5, consistent with the location of DHBV in the late-endosome compartment and previous studies of the nonessential role of low pH for infectivity. Exchange of the TM1 of DHBV with that of hepatitis B virus yielded functional, infectious DHBV particles, suggesting that TM1 of all of the hepadnaviruses act similarly in the fusion mechanism.

* Corresponding author. Mailing address: Macfarlane Burnet Institute for Medical Research and Public Health, Hepatitis Research Unit, Commercial Rd., Melbourne 3004, Australia. Phone: 61 3 92822109. Fax: 61 3 92822100. E-mail: grgacic{at}burnet.edu.au.

Journal of Virology, December 2005, p. 14945-14955, Vol. 79, No. 23
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.23.14945-14955.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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