通过诱导抑制内体分选转运复合体Ⅲ（ESCRT-III）控制HIV-1出芽的方案。

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

作者信息

Wang Haiyan, Weissenhorn Winfried, Boscheron Cécile

机构信息

University Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), Grenoble, France.

出版信息

STAR Protoc. 2025 May 13;6(2):103808. doi: 10.1016/j.xpro.2025.103808.

DOI:10.1016/j.xpro.2025.103808

PMID:40372921

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12142511/

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..

摘要

我们提出了一种使用与丙型肝炎病毒NS3蛋白酶融合的ESCRT-III蛋白对HIV-1病毒样颗粒（VLP）释放进行时间抑制的方案。这些融合蛋白的功能类似于野生型ESCRT-III，但在添加NS3抑制剂格卡瑞韦后会转化为显性负性抑制剂。该程序包括将Gag和CHMP-NS3-Green质粒共转染到HEK293或HeLa细胞中，然后进行药物处理。还包括蛋白质表达分析、通过免疫印迹进行VLP定量以及VLP释放动力学的活细胞成像步骤。有关此方案的使用和执行的完整详细信息，请参考Wang等人的研究。