Fusion of Sendai virus and individual host cells and inhibition of fusion by lipophosphoglycan measured with image correlation spectroscopy
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Fusion between Sendai virus (SV) and individual host cells was investigated with confocal laser scanning microscopy (CLSM) and image correlation spectroscopy (ICS). SV was labeled with the fluorescent probe 7-octadecylamino-4-nitrobenz-2-oxa-1,3-diazole (NBD-NH-C18) and was allowed to bind to host cells (HEp-2, BALB-3T3) at 4 degrees C. The effect of lipophosphoglycan (LPG), isolated from Leishmania donovani, on virus fusion was investigated by incorporation of LPG (0, 5, 10 or 20 microM) into the host cell membrane (HEp-2) before addition of SV. LPG did not affect the number of SV bound per cell. After incubation at 37 degrees C for 15 min without LPG, CLSM revealed a redistribution of NBD-NH-C18 from the SV envelope to the host cell membrane and an increase in average fluorescence intensity, indicating dequenching. ICS analysis of images obtained after incubation at 37 degrees C showed an increased mean cluster density to 260% of the value at 4 degrees C, reflecting the disappearance of labeled SV from the cell surface and diffusion of NBD-NH-C18 into the host cell membrane. Preincubation of the cells with LPG inhibited the temperature-induced redistribution and dequenching of NBD-NH-C18 in a concentration-dependent manner, with a total inhibition of fusion at 20 microM LPG. Together, the results demonstrate that CLSM combined with ICS is a powerful tool for studies of fusion of enveloped viruses with individual host cells and that LPG inhibits the fusion process at or before the hemifusion (lipid mixing) stage of SV interaction with cells.
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