Defining Mechanisms of HIV1 RNA Trafficking, Virus Assembly and Virion Structure

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。_BACKGROUND：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。未成熟的颗粒需要经过成熟过程才能成为传染性病毒。在此过程中，Gag- pol多蛋白中的蛋白酶（PR）被激活，裂解Gag/Gag- pol多蛋白，释放成熟蛋白。这个过程允许病毒粒子结构的重排，包括衣壳蛋白，形成一个锥形核心。我们正在利用活细胞成像技术研究蛋白裂解和病毒成熟的时间。本项目的研究旨在解决关于HIV-1大分子贩运和病毒组装的几个悬而未决的问题，这是病毒复制的重要过程。HIV-1 RNA必须进入特定的亚细胞区室才能被翻译或包装成病毒颗粒。适当的RNA转运是RNA及其编码蛋白发挥功能所必需的。然而，人们对HIV-1 RNA如何在细胞质中运输知之甚少。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。我们观察到大多数HIV-1 RNA分子以非定向、随机行走的方式移动，并且RNA移动的均方距离随时间线性增加，表明扩散运动。当Gag表达时，很大一部分HIV-1 RNA可能以Gag-RNA复合物的形式运输，其性质可能与无Gag RNA有很大不同。因此，我们也分析了HIV-1 RNA在有足够Gag用于病毒粒子组装时的细胞质运动，发现HIV-1 RNA仍然通过扩散运输，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，我们使用活细胞显微镜和Bgl-YFP构建物来可视化RNA， Bgl-YFP构建物专门识别HIV-1基因组中的茎环序列。我们发现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。为了更好地了解HIV-1大分子的运输，我们目前正在确定HIV-1 RNA和Gag是否可以相互影响亚细胞定位，如果是的话，需要哪些元素来实现这种影响。为了深入了解RNA包装和病毒组装机制，我们使用全内反射荧光（TIRF）显微镜检查了病毒RNA和Gag-RNA在质膜附近相互作用的动力学。HIV-1 RNA通过BglG蛋白标记为光可转换的Eos蛋白，BglG蛋白可识别设计到病毒基因组中的茎环序列。紫外线照射使Eos发生不可逆的结构变化，使其发出的荧光从绿色变为红色。HIV-1 RNA的动态是通过质膜附近的光转化Eos和随时间跟踪光转化的红色Eos标记RNA信号的数量来确定的。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。然后，我们量化了质膜附近被包装成组装病毒复合体的HIV-1 rna的比例。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。我们目前正在研究HIV-1 RNA和Gag是否在细胞质中相互作用，如果是的话，这种相互作用的生物学后果是什么。我们已经研究了HIV-1 RNA在病毒组装中的作用。研究表明，在缺乏病毒RNA的情况下，HIV-1颗粒含有细胞RNA；因此，形成HIV-1颗粒并不需要病毒RNA。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生成，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。我们目前正在剖析RNA包装所需的Gag特性。

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. _Immature particles need to go through a maturation process to become infectious viruses. During this process, protease (PR) in the Gag-Pol polyprotein is activated and cleaves Gag/Gag-Pol polyproteins to release mature proteins. This process allows the rearrangement of the virion structure including the capsid proteins, which form a conical core. We are studying the timing of the proteolytic cleavage and virus maturation by using live-cell imaging. _The studies in this project seek to address several unanswered questions on the trafficking of HIV-1 macromolecules and virus assembly, which are essential processes in viral replication. _ACCOMPLISHMENTS: HIV-1 RNA must go to specific subcellular compartments to be translated or packaged into viral particles. Proper RNA trafficking is required for the functions of RNA and its encoded proteins. However, little was known about how HIV-1 RNA is transported in the cytoplasm. We visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules moved in a nondirectional, random-walk manner, and that the mean-squared distance traveled by the RNA increased linearly with time, indicative of diffusive movement. When Gag was expressed, a significant portion of HIV-1 RNA may be transported as Gag-RNA complexes, whose properties could differ greatly from Gag-free RNA. Therefore, we also analyzed the cytoplasmic HIV-1 RNA movement in the presence of sufficient Gag for virion assembly and found that HIV-1 RNA is still 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 by using live-cell microscopy and a Bgl-YFP construct that specifically recognizes stem-loop sequences engineered into the HIV-1 genome. We 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. To better understand the trafficking of HIV-1 macromolecules, we are currently determining whether HIV-1 RNA and Gag can affect each other's subcellular localization and, if so, which elements are required for such effects. _To gain insights into RNA packaging and virus assembly mechanisms, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by total internal reflection fluorescence (TIRF) microscopy. HIV-1 RNA was labeled with a photo-convertible Eos protein via a BglG protein that recognizes stem-loop sequences engineered into the viral genome. UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. The dynamics of HIV-1 RNA were determined by photoconverting Eos near the plasma membrane and by following the population of the photoconverted red-Eos-labeled RNA signals over time. 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 then quantified the proportion of HIV-1 RNAs near the plasma membrane that was packaged into assembling viral complexes. 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 are currently studying whether HIV-1 RNA and Gag interact in the cytoplasm, and if so, what the biological consequences of such interactions are. __We have studied the role of HIV-1 RNA during virus assembly. It has been shown that in the absence of the viral RNA, HIV-1 particles contain cellular RNAs; thus, viral RNA is not required to form HIV-1 particles. 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, HIV-1 RNA genome facilitates the production of HIV-1 particles. Furthermore, the effects of 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. We are currently dissecting the Gag properties required for RNA packaging.