An Inducible ESCRT-III Inhibition Tool to Control HIV-1 Budding

Authors:
Open Access
Publisher website
Google Scholar
Abstract
HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated autocleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization, and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins. Notably, upon drug administration, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted no effect but synergized with CHMP2A-NS3. Localization studies demonstrated the relocalization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes.
All Related Versions
Funding Information
  • Agence Nationale de la Recherche (ANR-19-CE11-0002-02, UAR 3518 CNRS-CEA-UGA-EMBL, ANR-10-INBS-05-02, ANR-10-LABX-49-01, ANR-17-EURE-0003, R01AI147869)
  • Grenoble Instruct-ERIC Center (ANR-19-CE11-0002-02, UAR 3518 CNRS-CEA-UGA-EMBL, ANR-10-INBS-05-02, ANR-10-LABX-49-01, ANR-17-EURE-0003, R01AI147869)
  • FRISBI (ANR-19-CE11-0002-02, UAR 3518 CNRS-CEA-UGA-EMBL, ANR-10-INBS-05-02, ANR-10-LABX-49-01, ANR-17-EURE-0003, R01AI147869)
  • GRAL (ANR-19-CE11-0002-02, UAR 3518 CNRS-CEA-UGA-EMBL, ANR-10-INBS-05-02, ANR-10-LABX-49-01, ANR-17-EURE-0003, R01AI147869)
  • Ecoles Universitaires de Recherche (ANR-19-CE11-0002-02, UAR 3518 CNRS-CEA-UGA-EMBL, ANR-10-INBS-05-02, ANR-10-LABX-49-01, ANR-17-EURE-0003, R01AI147869)
  • NIH (ANR-19-CE11-0002-02, UAR 3518 CNRS-CEA-UGA-EMBL, ANR-10-INBS-05-02, ANR-10-LABX-49-01, ANR-17-EURE-0003, R01AI147869)

This publication has 82 references indexed in Scilit: