HIV packaging occurs in RNA granules: implications for cell biology and anti-retroviral drugs

HIV 包装发生在 RNA 颗粒中：对细胞生物学和抗逆转录病毒药物的影响

• 批准号: 9353851
• 负责人: JAISRI R LINGAPPA
• 金额: $ 34.76万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-22 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9353851
• 关键词: ABCE1 gene; ATP phosphohydrolase; Anti-Retroviral Agents; Antibodies; Antiviral Agents; Appearance; Binding Proteins; Capsid; Categories; Cell Fraction; Cell Line; Cell membrane; Cells; Cellular biology; Complex; Cytoplasm; Cytoplasmic Granules; Data; Development; Dimerization; Event; Genetic Transcription; Genome; HIV; HIV-1; Human; Immunoprecipitation; Jurkat Cells; Kinetics; Life Cycle Stages; Ligase; Membrane; Modeling; Molecular; Molecular Conformation; Pharmaceutical Preparations; Physiological; Polyribosomes; Population; Process; Production; Proteins; Proteome; Proviruses; RNA; RNA marker; Reverse Transcription; Ribosomes; Series; Site; T-Lymphocyte; Techniques; Technology; Testing; Time; Translating; Viral; Viral Genome; Viral Packaging; Virion; Virus; base; crosslink; dimer; gag Gene Products; genomic RNA; innovation; innovative technologies; insight; lysine-tRNA; new therapeutic target; novel; protein crosslink; public health relevance; small molecule; trafficking

 DESCRIPTION (provided by applicant): During the late stages of the virus life cycle, HIV-1 packages two copies of the viral genome into each assembling HIV-1 virus particle. Although this process is critical for production of infectious virus, where and how packaging occurs in cells remains unclear. Because so little is known, it is generally assumed that packaging is initiated when Gag and HIV-1 genomic RNA (gRNA) randomly find each other anywhere in the cytoplasm. In contrast, our findings argue for a very different model, in which gRNA packaging is initiated within a specific subcellular complex through a highly regulated process. Our preliminary data reveal that prior to and during assembly, non-translating gRNA is largely found in cellular complexes termed RNA granules. Moreover, we find that a subclass of RNA granules containing gRNA and Gag can be isolated using an antibody to the cellular ATPase ABCE1. Thus, ABCE1 is a marker for the RNA granules in which packaging takes place. Additionally, we have identified three categories of ABCE1-containing packaging granules: those involved in early, intermediate, and late stages of packaging. Here we will use our ability to isolate RNA granules representing different stages in HIV-1 packaging to define molecular events involved in initiation and completion of gRNA encapsidation. In Aim 1, inducing Jurkat cells to synchronously express latent HIV-1 genomes will allow us to follow the kinetics of gRNA trafficking through each of the packaging granules we have identified and into released virus. We will also confirm the physiological relevance of HIV-1 packaging granules using HIV-1 infected primary human T cells. In Aim 2, we will identify components of HIV-1 packaging granules. This will be done using a hypothesis-driven approach, in which we will test for viral components expected to be present during packaging (e.g. lysyl tRNA primer and a dimeric form of gRNA), and using a discovery- based approach to define the cellular proteome and RNAome of early, intermediate, and late packaging granules. Finally, in Aim 3, we will use innovative crosslinking-immunoprecipitation (CLIP) technology to understand conformational changes that occur during packaging. Others recently examined all Gag in the cytoplasm vs. at membranes using CLIP and found that the number of Gag-gRNA contact sites increase dramatically during packaging. Here, we propose to use CLIP to determine whether HIV-1 packaging granules are the sites in which Gag proteins make those changing gRNA contacts. We will also use CLIP to identify host proteins that make contact with gRNA and are therefore likely to promote encapsidation. Studies in Aim 3 will also identify host proteins that contact Gag, thereby helping to define conformational changes that Gag undergoes during packaging. Together, the proposed aims will greatly advance our understanding of the molecular basis of packaging in cells, and will provide insight into recently discovered small molecules that inhibit virus-specific RNA granules.

 描述（由申请人提供）：在病毒生命周期的后期阶段，HIV-1将两个病毒基因组拷贝包装到每个组装的HIV-1病毒颗粒中。虽然这个过程对于感染性病毒的产生至关重要，但包装在细胞中的位置和方式仍不清楚。由于所知甚少，通常认为包装是在Gag和HIV-1基因组RNA（gRNA）在细胞质中随机发现彼此时开始的。相比之下，我们的研究结果支持一种非常不同的模型，其中gRNA包装是通过高度调控的过程在特定的亚细胞复合物内启动的。我们的初步数据显示，在组装之前和组装期间，非翻译gRNA主要存在于称为RNA颗粒的细胞复合物中。此外，我们发现含有gRNA和Gag的RNA颗粒的亚类可以使用细胞ATP酶ABCE 1的抗体分离。因此，ABCE 1是RNA颗粒的标记物，其中包装发生。此外，我们还确定了三种含ABCE 1的包装颗粒：涉及包装早期、中期和后期阶段的颗粒。在这里，我们将利用我们的能力，分离RNA颗粒代表不同阶段的HIV-1包装，以确定分子事件参与的启动和完成gRNA折叠。在目标1中，诱导Jurkat细胞同步表达潜伏的HIV-1基因组将使我们能够跟踪gRNA通过我们已经鉴定的每个包装颗粒并进入释放的病毒的动力学。我们还将使用HIV-1感染的原代人T细胞证实HIV-1包装颗粒的生理相关性。在目标2中，我们将鉴定HIV-1包装颗粒的组分。这将使用假设驱动的方法来完成，其中我们将测试预期在包装期间存在的病毒组分（例如赖氨酰tRNA引物和gRNA的二聚体形式），并使用基于发现的方法来定义早期、中期和晚期包装颗粒的细胞蛋白质组和RNA组。最后，在目标3中，我们将使用创新的交联免疫沉淀（CLIP）技术来了解包装过程中发生的构象变化。其他人最近使用CLIP检查了细胞质中相对于膜处的所有Gag，并发现在包装期间Gag-gRNA接触位点的数量显著增加。在这里，我们建议使用CLIP来确定HIV-1包装颗粒是否是Gag蛋白使那些改变gRNA接触的位点。我们还将使用CLIP来鉴定与gRNA接触的宿主蛋白，因此可能促进腺苷酸化。目标3中的研究还将鉴定与Gag接触的宿主蛋白，从而帮助定义Gag在包装过程中经历的构象变化。总之，提出的目标将大大推进我们对细胞包装分子基础的理解，并将为最近发现的抑制病毒特异性RNA颗粒的小分子提供深入了解。