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

 描述(由申请人提供)：宿主免疫系统有多种防御系统来对抗病毒入侵，例如感染人类免疫缺陷病毒1型(HIV-1)，它会导致获得性免疫缺陷综合症(AIDS)和主要共病，如癌症。人类抗病毒蛋白APOBEC3G(A3G)和APOBEC3F(A3F)是细胞胞苷脱氨酶，通过病毒基因组的高度突变和物理阻断逆转录来抑制HIV感染，从而阻止HIV-1DNA的积聚。为了逃避这种宿主防御机制，HIV表达病毒粒子传染性因子(Vif)，该因子劫持细胞内的E3泛素连接酶复合体，靶向A3F/G进行蛋白酶体介导的降解。尽管人们对含有Vif的E3泛素连接酶复合体的结构进行了深入的研究并取得了进展，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分析来剖析不同A3蛋白与底物DNA之间的相互作用，询问Vif/E3连接酶/A3复合体的组装，并获得这些相互作用复合体的详细三维结构信息。我们设计了创新的策略来捕获多个A3蛋白与其首选DNA底物形成的复合体的结构。这些结果将为破译这些酶的DNA识别和序列偏好机制提供必要的信息。我们将通过构建合理设计的结构来进一步描绘Vif-A3蛋白相互作用的结构细节 代表分子界面的结构靶标。我们还将利用光谱学方法研究底物组装途径，并确定完全组装的包含A3、Vif和E3泛素连接酶的分子复合体的结构。这项拟议工作的成功将极大地促进我们对对抗艾滋病毒和病毒逃避策略的重要宿主免疫防御系统的理解。所获得的信息将有助于将Vif抑制剂设计为一种新的抗逆转录病毒疗法，以补充现有的设施。

 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.