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bioRxiv · 10.1101/2023.10.16.562494

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

Wang, H.·Gallet, B.·Moriscot, C.·Pezet, M.·Chatellard, C.·Kleman, J.-P.·Gottlinger, H.·Weissenhorn, W.·Boscheron, C.
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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 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.

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Wang, H., Gallet, B., Moriscot, C., Pezet, M., Chatellard, C., Kleman, J.-P., Gottlinger, H., Weissenhorn, W., Boscheron, C.. 2023-10-16. An inducible ESCRT-III inhibition tool to control HIV-1 budding. https://doi.org/10.1101/2023.10.16.562494

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