Fluorescence resonance energy transfer screening assay for the identification of compounds that are capable of abrogating macrophage-tropic hiv-1 cell fusion

Abstract

Previous studies of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein-mediated membrane fusion have focused on laboratory-adapted T-lymphotropic strains of the virus. The goal of this application was to develop a novel screening assay to characterize membrane fusion mediated by a primary HIV-1 isolate in comparison with a laboratory-adapted strain. To this end, a novel fusion assay was developed on the basis of the principle of resonance energy transfer, using HeLa cells stably transfected with gp120/gp41 from the T-lymphotropic isolate HIV-1,or the macrophage-tropic primary isolate HIV-1,. These cells fused with CD4,target cell lines with a tropism mirroring that of infection by the two viruses. Of particular note, HeLa cells expressing HIV-1,gp120/gp41 fused only with PM1 cells, a clonal derivative of HUT 78, and not with other T-cell or macrophage cell lines. These results demonstrate that the envelope glycoproteins of these strains play a major role in mediating viral tropism. Despite significant differences exhibited by HIV-1,and HIV-1,in terms of tropism and sensitivity to neutralization by CD4-based proteins, the present study found that membrane fusion mediated by the envelope glycoproteins of these viruses had remarkably similar properties. In particular, the degree and kinetics of membrane fusion were similar, fusion occurred at neutral pH and was dependent on the presence of divalent cations. The claimed invention will facilitate the screening and identification of novel agents that are capable of inhibiting these interactions.