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

• 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
• 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

 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，例如人类免疫缺陷病毒 1 型 (HIV-1) 感染，这种病毒会导致获得性免疫缺陷综合症 (AIDS) 和癌症等主要合并症。人类抗病毒蛋白 APOBEC3G (A3G) 和 APOBEC3F (A3F) 是细胞胞苷脱氨酶，可通过病毒基因组的超突变和物理阻断逆转录来抑制 HIV 感染，从而防止 HIV-1 DNA 的积累。为了逃避这种宿主防御机制，HIV 表达病毒粒子感染因子 (Vif)，该因子劫持细胞 E3 泛素连接酶复合物以靶向 A3F/G 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了进展，但 APOBEC3 (A3) 蛋白选择特定病毒 DNA 序列的机制以及 Vif 识别 A3 蛋白所采用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。为了实现这一目标，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测定来剖析不同A3蛋白和底物DNA之间的相互作用，询问Vif/E3连接酶/A3复合物的组装，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 底物复合物的结构。这些结果将提供重要信息，帮助破译这些酶的 DNA 识别和序列偏好机制。我们将通过构建合理设计的结构进一步描绘 Vif-A3 蛋白相互作用的结构细节 代表分子界面的结构目标。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分子复合物的结构。这项工作的成功将极大地增进我们对针对艾滋病毒的重要宿主免疫防御系统和病毒逃避策略的理解。获得的信息将有助于 Vif 抑制剂的设计，作为一类新型抗逆转录病毒疗法，以补充现有的治疗方案。