Defining Mechanisms of HIV-1 Gag:RNA Interactions and Virus Assembly

我们正在研究HIV-1大分子的运输和组装。一旦它离开细胞核，HIV-1 RNA需要前往不同的亚细胞位置来执行其功能，包括与另一种病毒RNA二聚体并组装成病毒颗粒。我们当前和未来的研究重点是探索细胞中Gag:RNA相互作用的启动，检查T细胞中的RNA运输，以及定义病毒RNA基因组在颗粒组装中的作用。我们还将通过活细胞成像确定病毒成熟的动力学，并确定在病毒粒子中形成RNA结构的因素。【背景】为了产生感染性颗粒，HIV-1 RNA和蛋白质运输到质膜，这是病毒的主要组装位点。Gag蛋白驱动HIV-1组装，并与病毒RNA和蛋白质相互作用，以确保病毒基因组的包装和复制机制。此外，Gag与宿主蛋白相互作用使病毒排出体外。通常认为HIV-1 RNA和Gag的相互作用导致组装是在细胞质中开始的。为了更好地理解病毒组装的调控，我们正在检查细胞质HIV-1 Gag:RNA和RNA:RNA的相互作用。我们也在研究HIV-1 RNA在T细胞中的转运，并探索RNA基因组在HIV组装中的作用。本项目的研究试图解决关于HIV-1大分子贩运和病毒组装的几个悬而未决的问题，这是病毒复制的基本过程。我们可视化HIV-1 RNA，并通过单分子跟踪监测其在细胞质中的运动，并确定大多数HIV-1 RNA分子具有弥漫性运动。此外，我们发现，在Gag存在的情况下，HIV-1 RNA通过扩散运输，其移动性与无法表达功能性Gag的RNA相似。这些研究已经确定了HIV-1基因表达的重要机制。极化T细胞不仅在体内构成HIV-1靶细胞的大多数，而且在HIV-1通过细胞间感染的传播中起着关键作用。为了确定HIV-1 RNA在极化T细胞中的分布，我们可视化了RNA，发现HIV-1 RNA在尾足动物质膜附近以gag依赖的方式富集。这些结果表明HIV-1 RNA在病毒组装过程中富集。由于gag富集的尾足动物更容易形成病毒学突触，这种靶向性促进了细胞介导的感染和病毒在体内的传播。我们用全内反射荧光（TIRF）显微镜研究了病毒RNA和Gag-RNA在质膜附近相互作用的动力学。我们发现，在没有Gag的情况下，大多数HIV-1 rna暂时停留在质膜附近。Gag的存在显著增加了rna在质膜附近停留的时间。我们观察到HIV-1 RNA包装的频率依赖于Gag表达水平。我们的结果表明，只有一小部分到达质膜的HIV-1 rna（大约十分之一到三分之一）被合并到病毒蛋白复合物中。这些研究确定了HIV-1 RNA在质膜上的动态，并获得了导致RNA包衣的RNA- gag相互作用的时间信息。我们已经研究了HIV-1 RNA在病毒组装过程中的作用。我们假设HIV-1全长RNA促进了病毒颗粒的形成。为了验证我们的假设，我们检测了携带和不携带含有HIV-1包装信号的RNA的颗粒形成效率。我们发现，尽管病毒颗粒可以在没有RNA基因组存在的情况下产生，但HIV-1 RNA基因组促进了HIV-1颗粒的产生。此外，RNA基因组的作用依赖于Gag在细胞中的表达水平。这些观察结果与我们的假设一致，即包装二聚体RNA是HIV-1组装的成核过程。成果：虽然在HIV-1 RNA的5' UTR中已经发现了几个核衣壳（NC）结合位点，但这些位点是否直接指导HIV-1 RNA基因组包装还没有得到充分的研究。已经证明HIV-1 NC与暴露的鸟苷结合。因此，我们在nc结合位点的暴露鸟苷上引入了G-to-A替代，并检测了这些突变RNA与野生型RNA竞争包装的能力。我们观察到多个nc结合位点影响RNA包装；在测试的位点中，位于5' UTR茎环1的位点具有最显著的影响。这些位点以前被报道为主要的nc结合位点，使用化学探针反向足迹测定和主要的Gag结合位点，使用体外测定。我们发现替换3到4个鸟苷导致包装缺陷不到两倍。然而，当突变组合时，观察到严重的缺陷。此外，组合突变对RNA包装缺陷具有协同作用，这表明Gag:RNA相互作用存在冗余性，并且需要多个Gag蛋白结合RNA以封装HIV-1基因组。此外，我们研究了暴露在5'非翻译区的鸟苷对HIV-2基因组包装是否重要。我们发现，即使只有三个鸟苷发生突变，也会显著降低RNA包装效率。然而，并不是所有的鸟苷都有同样的效果；相反，存在一个等级顺序，其中一个主站点、一个次要站点和三个三级站点被识别。此外，这些位点存在功能重叠，多个位点的突变可以协同作用，导致基因组包装缺陷。HIV-1 Gag:RNA相互作用介导基因组包装，但机制尚不清楚。我们假设，除了RNA结合外，Gag的其他特性也有助于基因组包装。为了研究Gag对基因组包装的重要特征，我们建立了互补系统，分离Gag的颗粒组装和RNA结合功能：我们使用一组Gag蛋白来驱动颗粒组装，使用一个RNA结合的Gag来包装HIV-1 RNA。我们已经开发了两种类型的rna结合Gag，其中包装由真实的核衣壳（NC）结构域或非病毒rna结合结构域介导。我们发现，在这两种情况下，影响Gag多聚或质膜锚定特性的突变会减少或取消RNA包装。这些突变Gag可以聚集成颗粒，但不能有效地包装RNA基因组。我们的研究结果表明，HIV-1 RNA包装发生在质膜上，RNA结合Gag需要在RNA上聚合以封装病毒基因组。

We are studying the trafficking of HIV-1 macromolecules and assembly. Once it has exited the nucleus, HIV-1 RNA needs to travel to various subcellular locations to carry out its functions, including dimerizing with another viral RNA and assembling into a viral particle. Our current and future studies are focused on exploring the initiation of Gag:RNA interaction in the cells, examining RNA trafficking in T cells, and defining the role of the viral RNA genome in particle assembly. We will also determine the kinetics of virus maturation by live-cell imaging and determine the factors that shape RNA structures in the virions. ___BACKGROUND: To generate infectious particles, HIV-1 RNA and proteins traffic to the plasma membrane, the major virus assembly site. The Gag protein drives HIV-1 assembly and interacts with viral RNA and proteins to ensure the packaging of the viral genome and replication machinery. Additionally, Gag interacts with host proteins for virus egress. It has often been suggested that the interactions of HIV-1 RNA and Gag leading to assembly are initiated in the cytoplasm. To better understand the regulation of virus assembly, we are examining cytoplasmic HIV-1 Gag:RNA and RNA:RNA interactions. We are also studying HIV-1 RNA trafficking in T cells and exploring the role of the RNA genome in HIV assembly. ___The studies in this project sought to address several unanswered questions on the trafficking of HIV-1 macromolecules and virus assembly, which are essential processes in viral replication. We visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking and determined that most of the HIV-1 RNA molecules have diffusive movement. Additionally, we showed that, in the presence of Gag, HIV-1 RNA is transported by diffusion with mobility similar to that of RNAs unable to express functional Gag. These studies have defined a major mechanism important to HIV-1 gene expression. Polarized T cells not only constitute a majority of HIV-1 target cells in vivo but also play a critical role in the spread of HIV-1 via cell-to-cell infection. To determine the distribution of HIV-1 RNA in polarized T cells, we visualized the RNA and found that HIV-1 RNAs were enriched near the uropod plasma membrane in a Gag-dependent manner. These results indicated that HIV-1 RNA is enriched during the process of virus assembly. As the Gag-enriched uropod is more likely to form a virological synapse, such targeting facilitates cell-mediated infection and virus spread in vivo. : We have examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by total internal reflection fluorescence (TIRF) microscopy. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently. The presence of Gag significantly increased the time RNAs stay near the plasma membrane. We observed that the frequency of HIV-1 RNA packaging was dependent on the Gag expression level. Our results showed that only a small proportion of the HIV-1 RNAs (approximately one tenth to one third) that reached the plasma membrane was incorporated into viral protein complexes. These studies determined the dynamics of HIV-1 RNA on the plasma membrane and obtained the temporal information of RNA-Gag interactions that lead to RNA encapsidation. ___We have studied the role of HIV-1 RNA during virus assembly. We hypothesize that HIV-1 full-length RNA facilitates the formation of viral particles. To test our hypothesis, we examined the efficiencies of particle formation with and without RNA containing HIV-1 packaging signal. We found that, although viral particles can be generated without the presence of RNA genome, the HIV-1 RNA genome facilitates the production of HIV-1 particles. Furthermore, the effects of the RNA genome are dependent on the level of Gag expressed in the cells. These observations are consistent with our hypothesis that packaging a dimeric RNA is the nucleation process of HIV-1 assembly. ACCOMPLISHMENT: Although several nucleocapsid (NC)-binding sites have been identified in the 5' UTR of HIV-1 RNA, whether these sites direct HIV-1 RNA genome packaging has not been fully investigated. It has been shown that HIV-1 NC binds exposed guanosines. Thus, we introduced G-to-A substitution on exposed guanosines in the NC-binding sites and examined the ability of these mutant RNAs to compete for packaging with wild-type RNA. We observed that multiple NC-binding sites affect RNA packaging; of the sites tested, those located at stem-loop 1 of the 5' UTR had the most significant effects. These sites were previously reported as the primary NC-binding sites using a chemical probing reverse-footprinting assay and the major Gag binding sites using an in vitro assay. We found that substituting 3 to 4 guanosines resulted in less than twofold defects in packaging. However, when mutations were combined, severe defects were observed. Furthermore, combining mutations had synergistic effects on RNA packaging defects, suggesting redundancy in Gag:RNA interactions and a requirement for multiple Gag proteins to bind RNA to encapsidate the HIV-1 genome. Additionally, we examined whether exposed guanosines in the 5' untranslated region are important for HIV-2 genome packaging. We found that mutating as few as three guanosines significantly reduce RNA packaging efficiency. However, not all guanosines examined have the same effect; instead, a hierarchical order exists wherein a primary site, a secondary site, and three tertiary sites are identified. Furthermore, there are functional overlaps in these sites and mutations of more than one site can act synergistically to cause genome packaging defects. HIV-1 Gag:RNA interactions mediate genome packaging, but the mechanism remains unclear. We posited that, besides RNA binding, other properties of Gag contribute to genome packaging. To examine features of Gag that are important for genome packaging, we established complementation systems that separate the particle-assembling and RNA-binding functions of Gag: we used a set of Gag proteins to drive particle assembly and an RNA-binding Gag to package HIV-1 RNA. We have developed two types of RNA-binding Gag in which packaging is mediated by the authentic nucleocapsid (NC) domain or by a nonviral RNA-binding domain. We found that in both cases, mutations that affect the multimerization or plasma membrane anchoring properties of Gag reduce or abolish RNA packaging. These mutant Gag can coassemble into particles but cannot package the RNA genome efficiently. Our findings indicate that HIV-1 RNA packaging occurs at the plasma membrane and RNA-binding Gag needs to multimerize on RNA to encapsidate the viral genome.