New reporter cell lines to study macrophage-tropic HIV envelope protein-mediated cell-cell fusion

The infection of human cells by HIV-1 virus can be mimicked by a fusion pro cess between cells expressing the HIV envelope protein (Env) and cells expr essing both human CD4 (huCD4) and appropriate human chemokine receptors. In this study, a macrophage-tropic (M-tropic) HIV cell-cell fusion assay was established that utilized huCD4, human CCR5 (huCCRS), and HIV ADAgp160 as f usion components and a Gal4/VP16-activated luciferase as a reporter system. By combining CHO cells expressing huCD4 and huCCRS with CHO cells expressi ng HIV ADAgp160, a 300-fold increase in luciferase activity could be elicit ed relative to control, No luciferase activity was detected when HXB2gp160 (T-tropic) was used instead of ADAgp160 (M-tropic) as the fusion partner in the assay. Addition of anti-huCD4 (RPA-T4) or anti-huCCR5 (2D7) monoclonal antibodies in the assay significantly inhibited the fusion event; in contr ast, an anti-CXCR4 (12G5) monoclonal antibody had little effect, indicating that the fusion assay was huCD4 and huCCR5 dependent. The cell-cell fusion occurred in a time-dependent manner; the maximum luciferase activity was d etected about 8 hr after mixing the cells. The fusion events could also be monitored by another reporter system in which Gal4/VP16 activated green flu orescent protein (GFP) was used as the reporter instead of luciferase, In c ombination with fluorescence microscopy, the GFP reporter system allowed vi sualization of the fusion events in real time. Compared with previously des cribed HIV fusion models, this system has several advantages, including sim plicity, sensitivity, and the ability to allow continuous monitoring of the HIV cell-cell fusion event. Finally, this cell-cell fusion system is easil y adapted to study other HIV fusion events.