(1) Goals of project: - To develop sensitive assay for monitoring HIV-1 envelope-mediated cell fusion. - To identify surface receptors(in addition to the CD4 receptors) involved in HIV-1 fusion and cell entry. - To apply the knowledge gained towards development of agents capable of blocking infection by cell-free or cell-associated HIV-1. (2) Experimental approach: 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. (3) Major Findings: - Our study demonstrated that HIV-1 mediated cell fusion and syncytia formation followed different kinetics. Furthermore, fusion occurred unde 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. It was foun 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. - We also 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. - Currently, we are studying a new receptor required for HIV-1 fusion which can be down-modulated by the PKC activator, phorbol ester (PMA).
