Development of an in vitro assay system for screening of gp41 inhibitory compounds

The transmembrane protein of HIV-1, gp41, mediates fusion between membranes of the virus and target cell. Strong interaction between the helical regio ns in the ectodomain of gp41 has been exploited to develop a method that ca n detect a potential inhibitor against gp41, The N-terminus coiled-coil or the C-terminus helical sequences within the ectodomain of gp41 were inserte d into the C-terminus of thioredoxin (Trx) or glutathione S-transferase (GS T) to generate the fusion proteins, Trx-N and GST-C, respectively. The inse rted sequences of GST-C and Trx-N cause the two proteins to interact with e ach other and to form a complex, Furthermore, GST-C binds specifically to t he surface-coated Trx-N, and the amount of attached GST-C is detected by an ELISA assay using anti-GST antibodies, Peptides derived from the helical r egions of gp41 compete with GST-C for binding to Trx-N as well as prevent t he gp41-mediated cell fusion, This in vitro assay system can be applied to screening compounds that have an inhibitory activity against gp41.