HARC Center: HIV Accessory and Regulatory Complexes

描述(由申请人提供)：在整个艾滋病毒生命周期中，病毒-宿主复合体在病毒生物学中发挥着不可或缺的作用。组装范围从蛋白质-蛋白质和蛋白质-核酸二元复合体到更大的多组分复合体。这些复合体的高分辨率结构提供了为艾滋病治疗的干预策略提供模板的潜力，但到目前为止，结构只在少数情况下得到了解决。获得这样的结构信息受到限制，部分原因是几种艾滋病毒蛋白质在生化上表现不佳，而且经常是孤立的无结构的。这些特征可能反映了蛋白质在整个病毒生命周期中瞬时结合到不同的细胞伙伴时采用不同构象的固有属性。该项目名为HARC中心(HIV附件和调节复合体)，专注于执行重要调节和辅助功能的五种关键HIV蛋白-整合酶(IN)、TAT、REV、VIF和Nef-及其相互作用的病毒和细胞蛋白伙伴。我们的主要目标是获得在病毒生命周期的早期阶段形成的艾滋病毒-宿主细胞相互作用的全面结构图，特别是那些调节基因表达、细胞信号或细胞生物学的相互作用。其中一些病毒和宿主蛋白在与其核酸或蛋白质伴侣络合时会发生显著的构象变化，了解这种动态行为的分子细节是一个重要的结构目标。我们对调控复合体的关注将为艾滋病毒的生物学和发病机制提供新的见解，并定义新的治疗目标来对抗艾滋病毒感染。HARC中心将利用蛋白质表达和共表达策略，使用一系列全面的结构方法来表征功能复合体，包括质谱学、X射线结晶学、核磁共振和冷冻-EM。为了克服迄今限制进展的障碍，我们建议为技术发展作出实质性努力。将探索基本的新方法来优化表达到结晶，利用结晶学和核磁共振数据分析蛋白质动力学，并通过冷冻-EM确定小分子和非均相复合体的结构。强大的外展和协作部分将吸引来自该中心以外的研究人员，特别是那些在艾滋病毒机制、蛋白质生物化学和病毒学方面具有专业知识的研究人员。 总体来说

DESCRIPTION (provided by applicant): Throughout the HIV life cycle, viral-host complexes play an integral role in the biology of the virus. Assemblies range from binary protein-protein and protein-nucleic acid complexes to much larger multicomponent complexes. High-resolution structures of these complexes offer the potential to provide templates for intervention strategies in the treatment of AIDS, but structures have so far been solved in only a few cases. Obtaining such structural information has been limited in part because several of the HIV proteins are poorly behaved biochemically and often are unstructured in isolation. These characteristics may reflect an inherent property of the proteins to adopt different conformations when bound transiently to different cellular partners throughout the viral life cycle. This project, called the HARC Center (HIV Accessory and Regulatory Complexes), focuses on five key HIV proteins that perform important regulatory and accessory functions - Integrase (IN), Tat, Rev, Vif, and Nef- and their interacting viral and cellular protein partners. Our primary aim is to achieve a comprehensive structural picture of HIV-host cell interactions formed during early phases of the viral life cycle, particularly those that regulate gene expression, cell signaling, or cell biology. Several of these viral and host proteins undergo significant conformational changes when complexed to their nucleic acid or protein partners, and understanding the molecular details of this dynamic behavior is an important structural goal. Our focus on regulatory complexes will provide new insights into the biology and pathogenesis of HIV and define new therapeutic targets to combat HIV infection. The HARC Center will harness protein expression and co-expression strategies to characterize functional complexes using a comprehensive battery of structural methods, including mass spectrometry, x- ray crystallography, NMR, and cryo-EM. To overcome the obstacles that have limited progress to date, we propose substantial efforts for technological development. Essential new methods will be explored to optimize expression-to-crystallization, analyze protein dynamics using crystallographic and NMR data, and determine structures of small and heterogeneous complexes by cryo-EM. Strong outreach and collaborative components will draw investigators from outside the Center, particularly those with expertise in HIV mechanisms, protein biochemistry, and virology. OVERALL