title: 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.
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612.
authors.forename: Ruben M
authors.initials: RM
authors.lastname: Markosyan
authors.forename: Fredric S
authors.initials: FS
authors.lastname: Cohen
authors.forename: Grigory B
authors.initials: GB
authors.lastname: Melikyan
mesh_terms: D015704:CD4 Antigens / Q000032:analysis; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D015686:Gene Products, env / Q000378:metabolism; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008055:Lipids / Q000502:physiology*; D019713:Receptors, CCR5 / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014771:Virion / Q000502:physiology*
chemical_list: D015704:CD4 Antigens; D015686:Gene Products, env; D015683:Gene Products, gag; D008055:Lipids; D019713:Receptors, CCR5; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins