Abstract：
Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.
摘要：
人类免疫缺陷病毒（HIV）-1的组装是通过Gag与质膜（PM）的内部小叶结合而启动的。Gag靶向由其N-末端肉豆蔻酰化基质(MA)结构域和PM磷脂酰肌醇4,5-二磷酸[PI(4,5)P2]介导。在Gag集会上，包膜（Env）糖蛋白被募集到组装位点；该过程取决于Gag的MA结构域和Env细胞质尾。为了调查Env招聘的动态，我们应用化学二聚体系统通过可逆PI（4,5）P2消耗结合超分辨率和活细胞显微镜来操纵HIV-1组装。这种方法使我们能够控制和同步HIV-1组装，并实时跟踪Env招募到各个新生组装地点。单病毒体追踪显示Gag和Env在HIV-1组装位点以相似的动力学积累。PI(4,5)P2耗尽阻止GagPM靶向和Env簇形成，确认Env招聘的Gag依赖性。在显示预组装Gag晶格的单元格中，PI(4,5)P2耗尽导致完整组装域的解体，因为不仅Gag而且Env集群都从PM中迅速丢失。这些结果证明了Gag诱导和维持的膜微环境的存在，这吸引了Env。PI（4,5）P2耗尽引起的Gag团簇解离显然破坏了这种微环境，导致从以前的组装域中丢失Env。IMPORTANCEHuman免疫缺陷病毒（HIV）-1在感染细胞的质膜上组装，导致膜包裹的病毒体出芽。HIV-1组装是由HIV-1的主要结构蛋白Gag启动的复杂过程。有趣的是,HIV-1仅将少数包膜（Env）糖蛋白掺入出芽的病毒体中，尽管在表达HIV的细胞的质膜上检测到新生Gag组件周围的大量Env积累。Gag的基质结构域和Env细胞质尾巴在Env募集到HIV-1组装位点及其掺入新生病毒体中起作用。然而,这些过程的调节还没有完全理解。通过结合化学二聚化系统来操纵具有超分辨率和活细胞显微镜的HIV-1组装，我们的研究为Gag之间的相互作用提供了新的见解，Env,在病毒组装过程中和宿主细胞膜，并将Env掺入HIV-1病毒体中。
