An inducible ESCRT-III inhibition tool to control HIV-1 budding

Haiyan Wang (1) , Benoit Gallet (2) , Christine Moriscot (2) , Mylène Pezet (3) , Christine Chatellard (4) , Jean-Philippe Kleman (2) , Heinrich Göttlinger (2) , Winfried Weissenhorn (2) , Cécile Boscheron (2)

1 Texas A&M University [College Station]
2 IBS - UMR 5075 - Institut de biologie structurale
3 IAB - Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble)
4 GIN - Groupe d'imagerie neurofonctionnelle

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 auto-cleavable 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 with variable modification of Gag VLP budding upon drug administration. Notably, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted a minor effect and synergized with CHMP2A-NS3. Localization studies demonstrated the re-localization 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.

Domaines: Sciences du Vivant [q-bio]

Haiyan Wang, Benoit Gallet, Christine Moriscot, Mylène Pezet, Christine Chatellard, et al.. An inducible ESCRT-III inhibition tool to control HIV-1 budding. Viruses, 2023, 15 (12), pp.2289. ⟨10.3390/v15122289⟩. ⟨hal-04384226⟩