CLSM analysis of Gag-GFP labelled virus binding to host cell lipids. (A) Incubation of concentrated PFV, VSV-G pseudotyped HIV particles and HIV VLPs (ΔEnv) with extracted HeLa lipids or synthetic lipids (DOPC/DOPS, DOPC). On DOPC (Dioleoyl phosphatidylcholine), a synthetic neutral phospholipid, none of the particles bound. The mixture containing 30% negatively charged DOPS (Dioleoyl phosphatidylserine), which is necessary to mediate VSV-G particle binding, interacted with HIV VSV-G pseudoparticles. Binding to extracted lipids from HeLa cells (Hela lipids) was only detectable for HIV VSV-G pseudoparticles. Scale bars: 5 μm. (B) The total amount of particles bound to the lipid surface was quantified by automated image analysis (average of 3 scanned areas and 3 scans each). (C) Concentrated Gag-GFP labelled PFV particles (grey channel) were incubated with GUVs (Giant Unilamellar Vesicles, red channel), prepared from HeLa lipids and the a far-red lipid dye DiD-C18. No particle binding to the lipid membrane was observed. Images of the same GUV at two different time points (0s, 8s) are shown. Scale bar: 5 μm. (D) Binding of GFP labelled wt (PGwt) or PGM3 derived (PGM3) PFV particles containing (+Env) or lacking (ΔEnv) PFV Env (grey channel, upper panel) to the cell surface of HeLa cells. Nuclei were stained with DAPI (blue channel). The corresponding DIC images are shown below.