Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 102, Issue 24, Pages 8728-8733Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0501864102
Keywords
fluorescence microscopy; membrane fusion; retroviral fusion protein; single virus imaging; pore flicker
Categories
Funding
- NCI NIH HHS [CA098727, R01 CA098727] Funding Source: Medline
- NIAID NIH HHS [AI053668, R01 AI053668] Funding Source: Medline
Ask authors/readers for more resources
Viral fusion proteins catalyze merger of viral and cell membranes through a series of steps that have not yet been well defined. To elucidate the mechanism of virus entry, we have imaged fusion between single virions bearing avian sarcoma and leukosis virus (ASLV) envelope glycoprotein (Env) and the cell membrane. Viral particles were labeled with a lipophilic dye and with palmitylated enhanced YFP that was incorporated into the inner leaflet of the viral membrane. When individual virions were bound to target cells expressing cognate receptors, they transferred their lipids and contents only when exposed to low, but not neutral, pH. These data are consistent with the proposed two-step mechanism of ASLV entry that involves receptor-priming followed by low pH activation. Most importantly, lipid mixing commonly occurred before formation of a small fusion pore that was quickly and sensitively detected by pH-dependent changes in palmitylated enhanced YFP fluorescence. Nascent fusion pores were metastable and irreversibly closed, remained small, or fully enlarged, permitting nucleocapsid delivery into the cytosol. These findings strongly imply that hemifusion and a small pore are the key intermediates of ASLV fusion. When added before low pH treatment, a peptide designed to prevent Env from folding into a final helical-bundle conformation abolished virus-cell fusion and infection. Therefore, we conclude that, after receptor-activation, Env undergoes low pH-dependent refolding into a six-helix bundle and, in doing so, sequentially catalyzes hemifusion, fusion pore opening, and enlargement.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available