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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters
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In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

机译：在膜结合的HIV-1 Gag簇上运输所需的内体分选复合体的有序组装体的体外重建

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摘要

Most membrane-enveloped viruses depend on host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release. HIV-1 is the prototypic ESCRT-dependent virus. The direct interactions between HIV-1 and the early ESCRT factors TSG101 and ALIX have been mapped in detail. However, the full pathway of ESCRT recruitment to HIV-1 budding sites, which culminates with the assembly of the late-acting CHMP4, CHMP3, CHMP2, and CHMP1 subunits, is less completely understood. Here, we report the biochemical reconstitution of ESCRT recruitment to viral assembly sites, using purified proteins and giant unilamellar vesicles. The myristylated full-length Gag protein of HIV-1 was purified to monodispersity. Myr-Gag forms clusters on giant unilamellar vesicle membranes containing the plasma membrane lipid PI(4,5)P_2. These Gag clusters package a fluorescent oligonucleotide, and recruit early ESCRT complexes ESCRT-I or ALIX with the appropriate dependence on the Gag PTAP and LYP(X)_nL motifs. ALIX directly recruits the key ESCRT-III subunit CHMP4. ESCRT-I can only recruit CHMP4 when ESCRT-II arid CHMP6 are present as intermediary factors. Downstream of CHMP4, CHMP3 and CHMP2 assemble syner-gistically, with the presence of both subunits required for efficient recruitment. The very late-acting factor CHMP1 is not recruited unless the pathway is completed through CHMP3 and CHMP2. These findings define the minimal sets of components needed to complete ESCRT assembly at HIV-1 budding sites, and provide a starting point for in vitro structural and biophysical dissection of the system.
机译：大多数膜包膜病毒依赖于运输（ESCRT）机器释放所需的内体分选复合物的宿主蛋白。 HIV-1是原型ESCRT依赖病毒。 HIV-1与早期ESCRT因子TSG101和ALIX之间的直接相互作用已被详细绘制。但是，对ESCRT募集到HIV-1出芽位点的完整途径的了解还不完全，该途径最终与后期作用的CHMP4，CHMP3，CHMP2和CHMP1亚基的组装有关。在这里，我们报告使用纯化的蛋白质和巨大的单层囊泡将ESCRT募集到生化重组到病毒装配位点。 HIV-1的肉豆蔻酰化的全长Gag蛋白被纯化至单分散性。 Myr-Gag在包含质膜脂质PI（4,5）P_2的巨大单层囊泡膜上形成簇。这些Gag簇包装荧光寡核苷酸，并募集早期的ESCRT复合物ESCRT-1或ALIX，并适当依赖于Gag PTAP和LYP（X）_nL基序。 ALIX直接募集关键的ESCRT-III亚基CHMP4。当ESCRT-II和CHMP6作为中介因子存在时，ESCRT-I只能募集CHMP4。 CHMP4，CHMP3和CHMP2的下游协同组装，同时存在有效募集所需的两个亚基。除非通过CHMP3和CHMP2完成该途径，否则不会募集作用非常晚的因子CHMP1。这些发现确定了在HIV-1出芽位点完成ESCRT组装所需的最少组件，并为系统的体外结构和生物物理解剖提供了起点。

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