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 HIV-1 env-expressing cells and CD4-positive adjacent cells, monitored by fluorescence video microscopy. This assay demonstrated that the terms cell fusion and syncytia formation can not be interchangeably used. Cell fusion and syncytia formation followed different kinetics. Cell fusion started at 15 min. and reached plateau at 90 min., while syncytia formation first appeared at 2 hr, and reached a plateau at 5-6 hrs. Furthermore, fusion occurred under conditions (i.e. cell ratios) which did not favor syncytia formation (similar to the in vivo situation). This assay was used to re-examine the role played by the adhesion molecules LFA-1/ICAM-1 during HIV-1 env-mediated cell fusion. LFA-1 - deficient B cell lines (from patients with LAD), and T cells (generated in our laboratory by chemical mutagenesis) were used in the study.It was found that the LFA-1 adhesion molecules do not play a role during the early events of HIV-1 env-mediated cell fusion, but do contribute to later events leading to syncytia formation. In a separate project, we studied the fusion between human B cell lines end HIV-1 env-expressing cells. We identified a fusion mechanism which is initiated by CD4-gp120 interactions, but greatly enhanced by the Ig receptors on B cells, providing they bind to the CD4-binding-region on the gp120 envelope. This study provides a mechanism for the selective fusion and elimination of B-cells expressing anti-gp120 Ab receptors. This assay will be used to screen drugs which may block early stages in HIV-1 cell membrane fusion.
