Figure 1

Characterization of the fluorescently labelled virus derivatives. (A) Map showing the insertion of the eyfp gene within the gag open reading frame of pCHIV. Arrowheads indicate cleavage sites for HIV protease. (B) Fluorescence micrographs of HeLaP4 cells transfected with plasmids peYFP-C1, pCHIV^eYFP or pCHIV^eYFPlate(-), respectively, recorded at 40 h post transfection. Note that a shorter exposure time was used for cells transfected with peYFP-C1 to adjust for the higher cytoplasmatic fluorescence intensity. (C) Immunoblot analysis of cell lysates and VLPs purified from the supernatant of transfected cells. At 36 h post transfection with peYFP-C1 (lanes 1 and 5), pCHIV (lanes 2 and 6), pCHIV^eYFP (lanes 3 and 7) or pCHIV^eYFPlate(-) (lanes 4 and 8), respectively, cells were harvested and particles were prepared from the medium by ultracentrifugation through a sucrose cushion. Samples corresponding to equal cell numbers or equal amounts of supernatant were subjected to SDS-PAGE, transferred to a nitrocellulose membrane and proteins were detected by enhanced chemiluminescence using the indicated antisera. Antiserum raised against GFP displayed cross-reactivity against eYFP. (D) Relative release efficiencies of eYFP, HIV^eYFP and HIV^eYFPlate(-) particles. Relative eYFP fluorescence intensities of tissue culture supernatants and lysates of cells transfected with the indicated plasmids were determined at 44 h post transfection using a TECAN Safire multi-well reader. Values shown here were obtained by subtracting the background derived from untransfected cells and dividing extracellular by total (intracellular plus extracellular) fluorescence. Data shown represent mean values and standard deviations from three parallel transfections.