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

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

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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（由适用提供）：在病毒生命周期的晚期，HIV-1将病毒基因组的两个副本包装到每个组装HIV-1病毒颗粒中。尽管此过程对于产生传染性病毒至关重要，但在细胞中包装的位置以及如何在何处仍不清楚。因为鲜为人知，因此通常假定当GAG和HIV-1基因组RNA（GRNA）随机在细胞质中的任何地方互相找到彼此时，开始包装。相比之下，我们的发现主张了一个非常不同的模型，在该模型中，通过高度调节的过程，在特定的亚细胞复合物中启动了GRNA包装。我们的初步数据表明，在组装之前和组装过程中，在称为RNA颗粒的细胞复合物中，非翻译GRNA在很大程度上发现。此外，我们发现可以使用对细胞ATPase ABCE1的抗体分离含有GRNA和GAG的RNA颗粒的子类。那是ABCE1是包装发生的RNA颗粒的标记。此外，我们已经确定了三类含ABCE1的包装颗粒：涉及包装的早期，中间和晚期的颗粒。在这里，我们将利用我们的能力分离代表HIV-1包装中不同阶段的RNA颗粒，以定义参与GRNA封装启动和完成的分子事件。在AIM 1中，诱导的Jurkat细胞同步表达潜在的HIV-1基因组将使我们能够遵循我们已经鉴定出的每个包装颗粒，并进入释放的病毒。我们还将使用HIV-1感染的原代人T细胞确认HIV-1包装颗粒的物理相关性。在AIM 2中，我们将确定HIV-1包装颗粒的组件。这将使用假设驱动的方法进行，其中我们将在包装期间测试预期存在的病毒成分（例如赖氨酸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，中间和晚包装花岗岩的细胞蛋白质组和RNAome。最后，在AIM 3中，我们将使用创新的交联免疫沉淀（夹）技术来了解包装过程中发生的构象变化。其他人最近使用夹子在膜上检查了细胞质与在膜上的所有插科打g，发现包装过程中Gag-Grna接触位点的数量急剧增加。在这里，我们建议使用夹子来确定HIV-1包装颗粒是否是GAG蛋白使那些变化的GRNA接触的部位。我们还将使用夹确定与GRNA接触的宿主蛋白，因此很可能促进封装。 AIM 3中的研究还将确定接触堵嘴的宿主蛋白质，从而有助于定义包装过程中插科打gag的会议变化。总之，提出的目标将大大提高我们对细胞包装分子基础的理解，并将洞悉最近发现的小分子，这些分子抑制了病毒特异性的RNA颗粒。