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

Abstract

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. INTRODUCTIONIn the process of membrane fusion, two continuities are created: separate membranes join and distinct aqueous compartments become one. To identify the mechanisms of membrane fusion, both continuities should be followed and the temporal order in which they occur identified. In the case of the HIV-1 fusion protein Env, mechanistic studies have largely relied on fusion of cells expressing the protein to cells expressing CD4 and cognate chemokine receptors (e.g., Munoz-Barroso et al., 1998;Melikyan et al., 2000;Reeves et al., 2002;Abrahamyan et al., 2003;Gallo et al., 2003;Markosyan et al., 2003). But monitoring lipid dye spread in HIV-1 Env-mediated cell-cell fusion has not proved to be very useful in following membrane continuity, for several reasons. The dye does not reliably move between cells, and when it does move, the time course is slow, in large part because the dye segregates nonuniformly over the cell membrane. Also, the dye enters intracellular pools, making it difficult to follow transfer between fused membranes. Furthermore, in cell-cell fusion, the two bound cells are in contact over an appreciable area, so fusion could potentially occur at many sites (Frolov et al., 2000;Leikina and Chernomordik, 2000); the sites of origin for the observed lipid and aqueous dye spread could well be different.By studying fusion of viral particles to cells, these problems can be overcome. The small size of virus minimizes the area of contact and accurately reflects the biological situation. Also, membrane retrieval processes that internalize lipid dye are absent in virions, and the viral envelope is simpler than a cell membrane. But until recently, suitable assays to follow both lipid and contents mixing and to time-order the...

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