Laboratory of Molecular Microbiology, NIAID, NIH, Bethesda, MD 20892, USA.
J Virol. 2011 Mar;85(5):2304-15. doi: 10.1128/JVI.01562-10. Epub 2010 Dec 15.
The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPXnL, that bind the cellular proteins Tsg101 and Alix, respectively. These interactions are thought to recruit members of the host fission machinery (ESCRT) to facilitate HIV-1 release. Here we report a new role for the p6-adjacent nucleocapsid (NC) domain in HIV-1 release. The mutation of basic residues in NC caused a pronounced decrease in virus release from 293T cells, although NC mutant Gag proteins retained the ability to interact with cellular membranes and RNAs. Remarkably, electron microscopy analyses of these mutants revealed arrested budding particles at the plasma membrane, analogous to those seen following the disruption of the PTAP motif. This result indicated that the basic residues in NC are important for virus budding. When analyzed in physiologically more relevant T-cell lines (Jurkat and CEM), NC mutant viruses remained tethered to the plasma membrane or to each other by a membranous stalk, suggesting membrane fission impairment. Remarkably, NC mutant release defects were alleviated by the coexpression of a Gag protein carrying a wild-type (WT) NC domain but devoid of all L domain motifs and by providing alternative access to the ESCRT pathway, through the in trans expression of the ubiquitin ligase Nedd4.2s. Since NC mutant Gag proteins retained the interaction with Tsg101, we concluded that NC mutant budding arrests might have resulted from the inability of Gag to recruit or utilize members of the host ESCRT machinery that act downstream of Tsg101. Together, these data support a model in which NC plays a critical role in HIV-1 budding.
HIV-1 Gag 的 p6 区域包含两个晚期（L）结构域，PTAP 和 LYPXnL，分别与细胞蛋白 Tsg101 和 Alix 结合。这些相互作用被认为招募了宿主分裂机制（ESCRT）的成员，以促进 HIV-1 的释放。在这里，我们报告了 p6 邻近核衣壳（NC）结构域在 HIV-1 释放中的新作用。NC 中碱性残基的突变导致 293T 细胞中病毒释放明显减少，尽管 NC 突变 Gag 蛋白仍保留与细胞膜和 RNA 相互作用的能力。值得注意的是，这些突变体的电子显微镜分析显示出在质膜处停滞的出芽颗粒，类似于在 PTAP 基序破坏后观察到的情况。这一结果表明 NC 中的碱性残基对于病毒出芽很重要。在生理上更相关的 T 细胞系（Jurkat 和 CEM）中分析时，NC 突变病毒仍然通过质膜或彼此之间的膜突连接在一起，这表明膜分裂受损。值得注意的是，通过共表达携带野生型（WT）NC 结构域但缺乏所有 L 结构域基序的 Gag 蛋白以及通过转染泛素连接酶 Nedd4.2s 提供替代途径进入 ESCRT 途径，可以缓解 NC 突变体的释放缺陷。由于 NC 突变体 Gag 蛋白仍与 Tsg101 相互作用，我们得出结论，Gag 不能招募或利用在 Tsg101 下游起作用的宿主 ESCRT 机制成员可能导致 NC 突变体出芽停滞。总之，这些数据支持了 NC 在 HIV-1 出芽中起关键作用的模型。
