Recognition of Viral DNA by APOBEC3 Proteins and their Antagonization by HIV Vif

APOBEC3 蛋白对病毒 DNA 的识别及其 HIV Vif 的拮抗作用

基本信息

批准号：
8992425
负责人：
Yong Xiong
金额：
$ 41.49万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-15 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8992425
关键词：
Acquired Immunodeficiency Syndrome Affinity Antiviral Agents Binding Biochemical Biochemistry Biological Assay Biophysics C-terminal Catalytic Domain Cells Chimeric Proteins Communities Comorbidity Complement Complex Cytidine Deaminase DNA DNA Sequence DNA-Protein Interaction Deaminase Electron Microscopy Fluorescence Genome Goals HIV HIV Infections HIV-1 Host Defense Host Defense Mechanism Human Immune Immune system Infection Knowledge Length Malignant Neoplasms Maps Mediating Methods Molecular Mutagenesis Mutate Pathway interactions Positioning Attribute Protein Engineering Protein Family Proteins Recruitment Activity Research Research Project Grants Reverse Transcription Solubility Spectrum Analysis Structure System Therapeutic Viral Viral Genome Viral Proteins Virion Virus Inhibitors Work X-Ray Crystallography antiretroviral therapy base combat design enzyme mechanism inhibitor/antagonist innovation multicatalytic endopeptidase complex novel pathogen preference prevent public health relevance structural biology success three dimensional structure tool ubiquitin-protein ligase viral DNA

项目摘要

DESCRIPTION (provided by applicant): The host immune system has diverse defenses that combat viral invasions, such as infection by human immunodeficiency virus type 1 (HIV-1), which causes acquired immunodeficiency syndrome (AIDS) and major comorbidities such as cancer. The human antiviral proteins APOBEC3G (A3G) and APOBEC3F (A3F) are cellular cytidine deaminases that suppress HIV infection by hypermutation of viral genome and physically blocking reverse transcription, which prevents the accumulation of HIV-1 DNA. To evade this host defense mechanism, HIV expresses the virion infectivity factor (Vif), which hijacks a cellular E3 ubiquitin ligase complex to target A3F/G for proteasome-mediated degradation. Despite intense research and progress in the structure elucidation of a Vif-containing E3 ubiquitin ligase complex, the mechanisms by which APOBEC3 (A3) proteins select specific viral DNA sequences and the molecular interactions employed by Vif to recognize A3 proteins remain unclear. Our overall goal is to establish the biochemical and structural principles by which A3 proteins mutate viral DNA and HIV-1 Vif sequesters the A3 proteins. To achieve this goal, we will use a combination of biochemistry, biophysics, structural biology, and cell-based functional assays to dissect the interactions between different A3 proteins and substrate DNAs, interrogate the assembly of the Vif/E3 ligase/A3 complex, and obtain detailed three-dimensional structural information of these interaction complexes. We have devised innovative strategies to capture structures of multiple A3 proteins in complex with their preferred DNA substrates. These results will provide essential information to help decipher the DNA recognition and sequence preference mechanisms of these enzymes. We will further delineate the structural details of Vif-A3 protein interactions by constructing rationally designed structural targets that represent the molecular interfaces. We will also investigate the substrate assembly pathway using spectroscopy methods and determine the structure of the completely assembled molecular complex containing A3, Vif and the E3 ubiquitin ligase. The success of the proposed work will significantly advance our understanding of an important host immune defense system against HIV and viral evasion strategy. Information obtained will facilitate the design of Vif inhibitors as a novel class of antiretroviral therapy to complement the existing armamentarium.

描述（由申请人提供）：宿主免疫系统具有多种防御机制来对抗病毒入侵，例如 1 型人类免疫缺陷病毒 (HIV-1) 感染，这种病毒会导致获得性免疫缺陷综合症 (AIDS) 和人类癌症等主要合并症。抗病毒蛋白 APOBEC3G (A3G) 和 APOBEC3F (A3F) 是细胞胞苷脱氨酶，通过病毒基因组的超突变和物理作用抑制 HIV 感染。阻断逆转录，从而防止 HIV-1 DNA 的积累。为了逃避这种宿主防御机制，HIV 表达病毒颗粒感染因子 (Vif)，该因子劫持细胞 E3 泛素连接酶复合物，以 A3F/G 为目标，进行蛋白酶体介导的降解。尽管在含 Vif 的 E3 泛素连接酶复合物的结构阐明方面进行了大量研究并取得进展，但 APOBEC3 (A3) 蛋白选择的机制特定的病毒 DNA 序列和 Vif 识别 A3 蛋白的分子相互作用仍不清楚，我们的总体目标是建立 A3 蛋白突变病毒 DNA 和 HIV-1 Vif 隔离 A3 蛋白的生化和结构原理。，我们将结合生物化学、生物物理学、结构生物学和基于细胞的功能分析来剖析不同 A3 蛋白和底物 DNA 之间的相互作用，探究 Vif/E3 的组装连接酶/A3 复合物，并获得这些相互作用复合物的详细三维结构信息，我们设计了创新策略来捕获多种 A3 蛋白与其首选 DNA 底物的复合物结构，这些结果将为帮助破译 DNA 识别提供重要信息。我们将通过构建合理设计的酶来进一步描述 Vif-A3 蛋白相互作用的结构细节。我们还将使用光谱方法研究底物组装途径，并确定包含 A3、Vif 和 E3 泛素连接酶的完全组装的分子复合物的结构。该工作的成功将显着增进我们的理解。获得的信息将促进 Vif 抑制剂作为一种新型抗逆转录病毒疗法的设计，以补充现有的治疗方案。