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Vol. 16, Issue 12, 5502-5513, December 2005

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Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612

Submitted June 7, 2005; Revised August 22, 2005; Accepted September 15, 2005
Monitoring Editor: Guido Guidotti

A method has been developed to follow fusion of individual pseudotyped virus expressing HIV-1 Env to cells by time-resolved fluorescence microscopy. Viral envelopes were labeled with a fluorescent lipid dye (DiD) and virus content was rendered visible by incorporating a Gag-GFP chimera. The Gag-GFP is naturally cleaved to the much smaller NC-GFP fragment in the mature virions. NC-GFP was readily released upon permeabilization of the viral envelope, whereas the capsid was retained. The NC-GFP thus provides a relatively small and mobile aqueous marker to follow viral content transfer. In fusion experiments, virions were bound to cells at low temperature, and fusion was synchronously triggered by a temperature jump. DiD transferred from virions to cells without a significant lag after the temperature jump. Some virions released DiD but retained NC-GFP. Surprisingly, the fraction of lipid mixing events yielding NC-GFP transfer was dependent on the type of target cell: of three infectable cell lines, only one permitted NC-GFP transfer within minutes of raising temperature. NC-GFP release did not correlate with the level of CD4 or coreceptor expression in the target cells. The data indicate that fusion pores formed by HIV-1 Env can remain small for a relatively long time before they enlarge.

Abbreviations used: NC-GFP, Gag-GFP chimera cleaved to nucleocapsid-GFP; CA, capsid protein; MA, matrix protein; DiD, far-red fluorescent lipid analogue.

^* Present address: Institute of Human Virology, UMBI, Baltimore, MD 21201.

Address correspondence to: Grigory B. Melikyan (melikian{at}umbi.umd.edu).

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