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

基本信息

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
项目类别：
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

项目摘要

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（由申请人证明）：在病毒生命周期的后期，尽管该过程是在传染病的生产过程中，但在细胞中的包装却尚不清楚。它通常假设包装是在细胞质中随机找到彼此时彼此在彼此中随机找到的。揭示在组装过程中，非翻译GRNA是在称为RNA颗粒的细胞复合物中的大finda。三类含ABCE1包装的颗粒：与包装的早期阶段相关的颗粒，HRE将隔离RNA颗粒，以隔离RNA颗粒niv-1 niv-1包装，以定义参与AIM 1中GRNA的启动和压缩的分子事件，诱导Jurkat细胞同步潜在的HIV-1基因组，使我们的grna troffick槽的动力学动力学，我们已经确定并释放了intedo释放的病毒。人类的T细胞在AIM 2中，我们将确定HIV-1包装颗粒的成分，其中我们将在包装期间测试预期的病毒式编译（例如，赖赛TRNA底漆和二聚体形式的GRNA）包装颗粒。在AIM 3中，我们将使用创新的交联 - 免疫沉淀（剪辑）技术来了解包装期间发生的构象变化，并发现hiv-1包装颗粒的gag-grna接触数量也会增加。使用夹子识别hosteins用GRNA进行的作用，因此可能会促进AIM 3中的研究。