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（由适用提供）：宿主免疫缺陷系统具有抵抗病毒感染的潜水防御能力，例如通过人类免疫缺陷病毒1型感染（HIV-1），导致获得性免疫缺陷综合征（AIDS）和主要的合并症，例如癌症。人类抗病毒蛋白APOBEC3G（A3G）和APOBEC3F（A3F）是细胞胞化死亡，可通过病毒基因组的过度抑制HIV感染并在物理上阻断逆转录，从而阻止HIV-1 DNA的积累。为了逃避这种宿主防御机制，HIV表达了病毒体感染因子（VIF），该因子劫持了细胞E3泛素连接酶复合酶，以靶向A3F/g，以用于蛋白酶体介导的降解。尽管对含有VIF的E3泛素连接酶复合物的结构进行了激烈的研究和进展，但APOBEC3（A3）蛋白选择特定的病毒DNA序列以及VIF进行的分子相互作用以识别A3蛋白的机制仍然不清楚。我们的总体目标是建立生化和结构原理，A3蛋白突变病毒DNA和HIV-1 VIF隔离A3蛋白。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法，以剖析不同A3蛋白和底物DNA之间的相互作用，询问VIF/E3 Ligase/A3复合物的组装，并获得这些互动复合物的详细三维结构信息。我们已经设计了创新的策略，以捕获与其首选的DNA底物复杂的多种A3蛋白的结构。这些结果将提供基本信息，以帮助解读这些酶的DNA识别和序列偏好机制。我们将通过理性设计的构建VIF-A3蛋白相互作用的结构细节进一步描述 代表分子界面的结构靶标。我们还将使用光谱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络合物的结构。拟议工作的成功将大大提高我们对针对艾滋病毒和病毒进化策略的重要宿主免疫防御系统的理解。获得的信息将促进VIF抑制剂作为一种新型的抗逆转录病毒疗法的设计，以完成现有的武器群。