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

## TL;DR

This paper describes a method to control HIV-1 budding in cells using a drug-inducible system involving ESCRT-III proteins and a Hepatitis C virus protease.

## Contribution

A novel protocol for inducible inhibition of HIV-1 budding using ESCRT-III fusion proteins and a protease inhibitor.

## Key findings

- Fusion proteins of ESCRT-III and NS3 protease can be converted into inhibitors with Glecaprevir.
- The protocol allows temporal control of HIV-1 VLP release in cell culture.
- Steps for VLP quantification and live-cell imaging are detailed.

## Abstract

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

•Instructions for drug-inducible temporal control of HIV-1 VLP release in cell culture•Guidelines for titrating CHMP-NS3 plasmid dosage to modulate ESCRT-III inhibition•Procedures for quantifying HIV-1 VLP release using immunoblotting of cell supernatants•Steps for high-resolution live imaging and kinetic analysis of single-cell VLP budding events

Instructions for drug-inducible temporal control of HIV-1 VLP release in cell culture

Guidelines for titrating CHMP-NS3 plasmid dosage to modulate ESCRT-III inhibition

Procedures for quantifying HIV-1 VLP release using immunoblotting of cell supernatants

Steps for high-resolution live imaging and kinetic analysis of single-cell VLP budding events

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.