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

HIV1 RNA 运输、病毒组装和病毒粒子结构的定义机制

• 批准号: 10014388
• 负责人: WEI-SHAU HU
• 金额: $ 74.64万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014388
• 关键词: Address; Affect; Biological; Capsid Proteins; Cell Nucleus; Cell membrane; Cells; Cleaved cell; Complex; Cytoplasm; Diffuse; Diffusion; Elements; Engineering; Ensure; Epidemic; Event; Evolution; Family; Fluorescence; Fluorescence Microscopy; Frequencies; Future; Gene Expression; Genome; Goals; HIV; HIV Genome; HIV-1; Human; Infection; Kinetics; Knowledge; Label; Lead; Length; Location; Mediating; Monitor; Movement; Parents; Peptide Hydrolases; Play; Population; Process; Production; Property; Proteins; Proviruses; RNA; Regulation; Research; Retroviridae; Role; Shapes; Signal Transduction; Site; Structure; T-Lymphocyte; Testing; Time; Translating; Travel; Treatment Protocols; Ultraviolet Rays; Vaccines; Viral; Viral Genome; Viral Packaging; Viral Proteins; Virion; Virus; Virus Assembly; Virus Replication; Walking; dimer; gag Gene Products; genetic information; in vivo; insight; live cell imaging; live cell microscopy; macromolecule; particle; pathogen; pol Gene Products; protein complex; single molecule; stem; trafficking; viral RNA; virological synapse; virus genetics

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.

我们正在研究 HIV-1 大分子的贩运和组装。一旦离开细胞核，HIV-1 RNA 需要前往各个亚细胞位置来发挥其功能，包括与另一种病毒 RNA 形成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，以及确定病毒 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，并确定塑造病毒粒子中 RNA 结构的因素。 _背景：为了产生传染性颗粒，HIV-1 RNA 和蛋白质运输到质膜（主要的病毒组装位点）。 Gag 蛋白驱动 HIV-1 组装并与病毒 RNA 和蛋白质相互作用，以确保病毒基因组和复制机制的包装。此外，Gag 与宿主蛋白相互作用以促进病毒排出。人们经常认为，导致组装的 HIV-1 RNA 和 Gag 的相互作用是在细胞质中启动的。为了更好地了解病毒组装的调节，我们正在研究细胞质 HIV-1 Gag:RNA 和 RNA:RNA 相互作用。我们还在研究 T 细胞中的 HIV-1 RNA 运输，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一个成熟的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) 被激活并裂解 Gag/Gag-Pol 多蛋白以释放成熟蛋白。这个过程允许病毒体结构重新排列，包括形成圆锥形核心的衣壳蛋白。我们正在通过活细胞成像研究蛋白水解裂解和病毒成熟的时间。 _该项目的研究旨在解决有关 HIV-1 大分子贩运和病毒组装的几个尚未解答的问题，这是病毒复制的重要过程。 _成就：HIV-1 RNA 必须进入特定的亚细胞区室才能翻译或包装成病毒颗粒。 RNA 及其编码蛋白质的功能需要适当的 RNA 运输。然而，人们对 HIV-1 RNA 如何在细胞质中转运知之甚少。我们对 HIV-1 RNA 进行可视化，并通过单分子追踪监测其在细胞质中的运动。我们观察到大多数 HIV-1 RNA 分子以无方向、随机游走的方式移动，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，HIV-1 RNA 的很大一部分可能以 Gag-RNA 复合物的形式运输，其特性可能与不含 Gag 的 RNA 有很大不同。因此，我们还分析了在有足够的Gag用于病毒粒子组装的情况下细胞质HIV-1 RNA的运动，发现HIV-1 RNA仍然通过扩散运输，其流动性类似于不能表达功能性Gag的RNA。这些研究明确了对 HIV-1 基因表达很重要的主要机制。极化T细胞不仅构成体内HIV-1靶细胞的大部分，而且在HIV-1通过细胞间感染传播的过程中发挥着关键作用。为了确定极化 T 细胞中 HIV-1 RNA 的分布，我们使用活细胞显微镜和 Bgl-YFP 构建体对 RNA 进行可视化，该构建体特异性识别工程化到 HIV-1 基因组中的茎环序列。我们发现 HIV-1 RNA 以 Gag 依赖性方式富集在尾肢质膜附近。这些结果表明HIV-1 RNA在病毒组装过程中得到富集。由于富含 Gag 的尾足动物更有可能形成病毒突触，因此这种靶向有利于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 是否可以影响彼此的亚细胞定位，如果可以，则需要哪些元件来实现这种效果。为了深入了解 RNA 包装和病毒组装机制，我们通过全内反射荧光 (TIRF) 显微镜检查了质膜附近病毒 RNA 和 Gag-RNA 相互作用的动态。 HIV-1 RNA 通过 BglG 蛋白用可光转换的 Eos 蛋白进行标记，该蛋白可识别病毒基因组中工程化的茎环序列。紫外线照射会导致 Eos 发生不可逆的结构变化，并将其发射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换 Eos 以及随着时间的推移跟踪光转换红色 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 会短暂停留在质膜附近。 Gag 的存在显着增加了 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物的 HIV-1 RNA 的比例。我们观察到 HIV-1 RNA 包装的频率取决于 Gag 的表达水平。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）被整合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 衣壳化的 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，这种相互作用的生物学后果是什么。 __我们研究了 HIV-1 RNA 在病毒组装过程中的作用。研究表明，在没有病毒 RNA 的情况下，HIV-1 颗粒含有细胞 RNA；因此，病毒RNA不需要形成HIV-1颗粒。我们假设 HIV-1 全长 RNA 促进病毒颗粒的形成。为了检验我们的假设，我们检查了有或没有含有 HIV-1 包装信号的 RNA 的颗粒形成效率。我们发现，虽然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基因组的作用取决于细胞中Gag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。