In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters
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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DOI：
10.1073/pnas.1211759109
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发表时间：
2012-10-16
影响因子：
11.1
通讯作者：
Hurley, James H.
中科院分区：
文献类型：
--
作者：
Carlson, Lars-Anders;Hurley, James H.
大多数膜包膜病毒的释放依赖于转运所需的内体分选复合物（ESCRT）机制的宿主蛋白。HIV-1是典型的ESCRT依赖性病毒。HIV-1和早期ESCRT因子TSG 101和阿利克斯之间的直接相互作用已经被详细绘制。然而，ESCRT募集到HIV-1出芽位点的完整途径，最终与晚期作用的CHMP 4，CHMP 3，CHMP 2和CHMP 1亚基的组装，是不完全理解的。在这里，我们报告的生化重建ESCRT招聘病毒装配网站，使用纯化的蛋白质和巨大的单层囊泡。将HIV-1的豆蔻酰化全长Gag蛋白纯化至单分散性。Myr-Gag在含有质膜脂质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，并招募早期ESCRT复合物ESCRT-I或阿利克斯，其适当依赖于Gag PTAP和LYP（X）（n）L基序。阿利克斯直接募集关键的ESCRT-III亚基CHMP 4。只有当ESCRT-II和CHMP 6作为中介因素存在时，ESCRT-I才能招募CHMP 4。在CHMP 4的下游，CHMP 3和CHMP 2协同组装，存在有效募集所需的两种亚基。除非通过CHMP 3和CHMP 2完成该途径，否则不会招募非常迟效因子CHMP 1。这些发现定义了在HIV-1出芽位点完成ESCRT组装所需的最小组件集，并为系统的体外结构和生物物理解剖提供了起点。
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)(n)L motifs. ALIX directly recruits the key ESCRT-III subunit CHMP4. ESCRT-I can only recruit CHMP4 when ESCRT-II and CHMP6 are present as intermediary factors. Downstream of CHMP4, CHMP3 and CHMP2 assemble synergistically, 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.