Development of a Novel Dual CCR5-Dependent and CXCR4-Dependent Cell-Cell Fusion Assay System with Inducible gp160 Expression

Abstract

In the current study, a novel coreceptor-specific cell-cell fusion (CCF) assay system is reported. The system possesses the following features: dual CCR5-dependent and CXCR4-dependent CCF assays, all stable cell lines, inducible expression of gp160 to minimize cytotoxicity, robust luciferase reporter, and 384-well format. These assays have been validated using various known HIV entry inhibitors targeting various stages of the HIV entry/fusion process, including fusion inhibitors, gp120 inhibitors, CCR5 antagonists, CCR5 antibodies, and CXCR4 antagonists. IC ₅₀data generated from this assay system were well correlated to that from the antiviral assays. The effects of DMSOon this assay systemwere assessed, and a 2-to 3-fold increase in luciferase activitywas observed in the presence of 0.05% to2% DMSO. Although cell-cell fusion efficiencywas enhanced, no changes in drug response kinetics for entry inhibitors were found in the presence of 0.1% or 0.5% DMSO. This assay system has been successfully used for the identification and characterization of thousands of CCR5 inhibitors.

Keywords

HIV cell-cell fusion DMSO luciferase reporter entry inhibitor

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