APOBEC PROTEINS AND RIBOVIRAL RESTRICTION

APOBEC 蛋白质和核病毒限制

基本信息

批准号：
10190817
负责人：
Jordan T Becker
金额：
$ 6.64万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10190817
关键词：
Active Sites Address Affinity Antiviral Agents Antiviral Therapy Area Binding Biochemical Biological Assay CD4 Positive T Lymphocytes Career Transition Award Cell Line Cells Cellular biology Collaborations Complex Conflict (Psychology)Crystallization Cytosine DNA Data Data Set Deamination Development Dimerization Enzymes Epitopes Event Fluorescent in Situ Hybridization Foundations Future Genetic Genome Goals HIV-1 Human Human poliovirus Image Imaging Techniques Immunoprecipitation Infection Influenza A virus Knock-out Lentivirus Letters Macaca nemestrina Mediating Microscopy Modeling Molecular Nucleic Acids Pan Genus Pathogenicity Positioning Attribute Proteins Protocols documentation Protomer Publications RNA RNA Binding RNA Editing RNA I RNA Sequences RNA Viruses RNA immunoprecipitation sequencing Reporting Research Retroviridae Series Site Structure Surface Surveys System T-Lymphocyte Technical Expertise Testing Vaccines Viral Viral Physiology Virion Virus Virus Replication Work antiretroviral therapy base comparative deep sequencing dimer experimental study genomic RNA high resolution imaging improved innovative technologies monomer novel overexpression pathogen single molecule spatiotemporal therapy design transcriptome sequencing translational study vector viral RNA virology

项目摘要

Human immunodeficiency virus type 1 (HIV-1) is engaged in an ongoing evolutionary conflict with APOBEC3 (A3) proteins. Retrovirus-restricting A3’s (A3D, A3F, A3G, and A3H) gain access to progeny virions produced in infected cells primarily by binding RNAs to be packaged into virions. This positions the A3 enzymes for restricting virus replication in target cells by DNA deamination-dependent and -independent mechanisms. However, lentiviruses like HIV-1 encode an A3-counteraction protein called Vif (virion infectivity factor) responsible for degradation of these A3 enzymes. There are currently no antiretroviral therapies designed to promote A3 packaging or disrupt Vif-dependent A3 degradation. Our lab recently solved the crystal structure of human A3H and these high-resolution images revealed a novel and unanticipated RNA binding mechanism in which two A3H monomers become dimeric by complexing with duplex RNA. Moreover, the active site required for cytosine deamination occurs adjacent to the RNA-binding surface raising the possibility of RNA cytosine deamination (i.e. RNA editing). I will test the hypothesis that A3H binds to specific RNA structures in cellular and viral RNAs to restrict RNA-based viruses through deamination-dependent (RNA editing) and deamination- independent mechanisms through two specific aims. In Aim 1, I will identify the HIV-1 and cellular duplex RNA species bound by endogenous A3H through a series of A3H-RNA immunoprecipitation and deep sequencing experiments (RIP-seq). A3H-RNA interactions will be further interrogated by advanced imaging techniques including an in cellulo interaction protocol and single molecule fluorescence in situ hybridization. In Aim 2, I propose RIP-seq of A3H in infected with RNA-only viruses, including influenza A virus and poliovirus, to identify viral RNA structures bound and sites of RNA editing. I will determine if A3H changes the infectious titer of these viruses in single and multiple round infections. Finally, I will use fluorescent microscopy and RNA imaging to validate these A3H-RNA interactions. In summary, these proposed experiments use innovative technologies to improve our understanding of A3H-mediated restriction of HIV-1 as well as have the potential to open an entirely new research area of APOBEC-mediated riboviral restriction.

人类免疫缺陷病毒1型（HIV-1）与正在进行的进化冲突与 APOBEC3（A3）蛋白质。逆转录病毒限制A3（A3D，A3F，A3G和A3H）访问进度病毒在感染细胞中主要是通过结合RNA包装到病毒中产生的。这可以定位A3酶通过DNA脱氨基依赖性和非依赖性机制限制靶细胞中病毒复制。但是，诸如HIV-1之类的慢病毒编码一种称为VIF（视觉感染因子）的A3-Counteraction蛋白负责这些A3酶降解。目前尚无抗逆转录病毒疗法促进A3包装或破坏依赖VIF的A3降解。我们的实验室最近解决了人A3H和这些高分辨率图像揭示了一种新颖而意外的RNA结合机制通过与双链RNA复合，两个A3H单体变成了二聚体。此外，需要主动网站胞嘧啶死亡发生在RNA结合表面附近，从而增加了RNA胞嘧啶的可能性脱氨（即RNA编辑）。我将检验以下假设：A3H与细胞和细胞中特定的RNA结构结合病毒RNA通过脱氨基依赖性（RNA编辑）和脱氨酸来限制基于RNA的病毒在AIM 1中，我将识别HIV-1和细胞双链体RNA 通过一系列A3H-RNA免疫沉淀和深度测序，由内源A3H绑定的物种实验（RIP-SEQ）。高级成像技术将进一步询问A3H-RNA相互作用包括纤维素相互作用方案和单分子荧光原位杂交。在AIM 2中，我在感染仅RNA病毒的A3H的提案RIP-Seq，包括影响力病毒和脊髓灰质炎病毒，以鉴定病毒RNA结构与RNA编辑的位点结合。我将确定A3H是否会改变其中的传染性滴度单一和多圆感染中的病毒。最后，我将使用荧光显微镜和RNA成像验证这些A3H-RNA相互作用。总而言之，这些提出的实验使用创新技术来提高我们对A3H介导的HIV-1限制的理解，并有可能完全开放 APOBEC介导的核武器限制的新研究领域。