We have developed a sensitive assay to examine the early stages of HIV-1 env-mediated cell membrane fusion which is based on redistribution of fluorescent dyes between membranes of adjacent cells, monitored by fluorescence video microscopy. This assay demonstrated that membrane fusion between gp120/41 expressing cells and CD4+ cells, can occur under conditions where no synctytia are formed. In general, fusion took place earlier than syncytia formation and at cell ratios which did not favor syncytia formation (similar to the situation in vivo). Also, cell membrane fusion (but not synctia) were sensitive to mechan- ical forces. This assay was used to determine the role of the adhesion molecule LFA-1 in HIV-1 env-mediated cell membrane fusion and syncytia formation. CD4- LFA- EBV transformed B-cell lines from two leukocyte adhesion deficient (LAD) patients, were infected with reombinant vaccinia expressing gp120/41 (HIV-1 IIIB), and co-cultured with CD4+ subclones of the human T cell line CEM, which were generated by chemical mutagenesis and express either normal or low levels of LFA-1. It was found that the LFA-1 (low) T cell clone formed much smaller and fewer syncytia compared to a LFA-1+ revertant subclone. In contrast, both subclones fused equally well with the gp120/41 expressing LFA-1- B cells as monitored by redistribution of fluorescent dyes. Furthermore, monoclonal antibodies agains LFA-1, reduced the number of syncytia formed but had no effect on membrane fusion. These data demonstrate that: I. The adhesion molecule LFA-1 does not play a crucial role in the early events of HIV-1 envmediated cell membrane fusion but contributes to the slower process of syncytia formation. II. The terms fusion and syncytia formation cannot be used interchangeablly. III. This assay can be used to test drugs which may block HIV-1 dependent cell fusion.
