PDF(Pubmed)
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.
摘要：
HIV-1出芽以及许多其他细胞过程需要运输所需的内体分选复合物(ESCRT)机制。由于空间分辨率和瞬时ESCRT-III募集，对HIV-1出芽位点的天然ESCRT-III复合物的理解受到限制。这里,我们开发了一种药物诱导的瞬时HIV-1出芽抑制工具,以提高ESCRT-III在出芽位点的寿命.我们生成了可自动切割的CHMP2A，CHMP3和CHMP4B与丙型肝炎病毒NS3蛋白酶的融合蛋白。我们在不存在和存在蛋白酶抑制剂Glecaprevir的情况下表征了CHMP-NS3融合蛋白的表达，稳定性,本地化,和HIV-1GagVLP萌芽。免疫印迹实验揭示了CHMP-NS3融合蛋白的快速稳定积累。值得注意的是,在药物管理后，CHMP2A-NS3和CHMP4B-NS3融合蛋白显著降低VLP释放,而CHMP3-NS3没有作用,但与CHMP2A-NS3协同作用。定位研究表明CHMP-NS3融合蛋白在质膜上的重新定位,内体，和GagVLP萌芽位点。通过结合使用透射电子显微镜和视频显微镜，我们揭示了CHMP2A-NS3和CHMP4B-NS3的药物依赖性积累,导致HIV-1Gag-VLP释放延迟.我们的发现为在HIV-1出芽过程中抑制ESCRT-III的功能后果提供了新的见解，并建立了新的工具来破译ESCRT-III在HIV-1出芽位点和其他ESCRT催化的细胞过程中的作用。
