Protocol for HIV-1 budding control by inducible inhibition of ESCRT-III.

We present a protocol for temporal inhibition of HIV-1 virus-like particle (VLP) release using ESCRT-III proteins fused to the Hepatitis C virus NS3 protease. These fusion proteins function like wild-type ESCRT-III but convert into dominant-negative inhibitors upon addition of the NS3 inhibitor Glecaprevir. The procedure involves co-transfection of Gag and CHMP-NS3-Green plasmids into HEK293 or HeLa cells, followed by drug treatment. Steps for protein expression analysis, VLP quantification by immunoblotting, and live-cell imaging of VLP release kinetics are included. For complete details on the use and execution of this protocol, please refer to Wang et al.(1).