A mammalian two-hybrid system-based assay for small-molecular HIV fusion inhibitors targeting gp41

gp41 is a major component of the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) responsible for fusion of the viral envelope with the target cellular membrane. The formation of the trimer-of-hairpins core structure of gp41, via the interaction between its N-terminal heptad repeat (NHR) and its C-terminal heptad repeat (CHR) plays a key role in the membrane fusion process. Hence, inhibitors of trimer-of-hairpins formation have become a promising new class of HIV therapeutics. In the present study, based on the mammalian two-hybrid system, we developed a cell-based assay for detecting small-molecular HIV-1 fusion inhibitors targeting gp41. The optimized assay can be adapted to high-throughput screening in 96- and 384-well microplates with high signal-to-background ratios and acceptable Z′ factors. The known small-molecular gp41 inhibitors, ADS-J1, XTT formazan and tannin acid, tested positive in this assay, with half-maximal inhibitory concentration (IC50) values of 4.9 μM, 5.6 μM and 0.8 μM, respectively. These data suggested that this novel assay is robust, sensitive and specific for identifying small-molecular HIV-1 gp41 inhibitors.