Structural Basis for Long-Range Genetic Interactions in Retroviral CA Assembly

逆转录病毒 CA 组装中长程遗传相互作用的结构基础

• 批准号: 7684590
• 负责人: REBECCA C. CRAVEN
• 金额: $ 19.23万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7684590
• 关键词: Amides; Amino Acid Substitution; Amino Acids; Anti-Retroviral Agents; Area; Biochemical; Biochemistry; C-terminal; Capsid; Capsid Proteins; Cleaved cell; Collaborations; Collection; Complex; Correlation Studies; Deuterium; Development; Distant; Frequencies; Gagging; Genetic; Goals; HIV; Hot Spot; Human; Hydrogen; Laboratories; Life Cycle Stages; Maps; Modeling; Molecular; Molecular Conformation; Morphogenesis; Morphology; Mutation; NMR Spectroscopy; Pharmaceutical Preparations; Positioning Attribute; Process; Protein Conformation; Protein Region; Proteins; Research Personnel; Resolution; Retroviridae; Reverse Transcription; Role; Rous sarcoma virus; Series; Site; Staging; Structural Protein; Structure; Suppressor Mutations; System; T-Lymphocyte; Tertiary Protein Structure; Testing; Viral; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; Work; base; defined contribution; gag Gene Products; genetic analysis; in vivo; inhibitor/antagonist; intermolecular interaction; methyl group; molecular rearrangement; mutant; particle; public health relevance; research study; three dimensional structure; two-dimensional; virology

DESCRIPTION (provided by applicant): The retroviral CA (or capsid) protein has several essential roles in the virus life cycle organizing the packing of the Gag polyprotein during immature particle assembly, directing morphogenesis of the mature capsid in the virion core, and finally, acting in poorly defined role(s) during the early stages of replication. The ability of the CA protein to undergo complex molecular rearrangements and conformational changes is critical for its ability to support the different functions in the viral life cycle. The carboxy-terminal domain (CTD) of the protein is a structural module that participates in intermolecular interactions between Gag polyproteins in the immature particle and in CA-CA association after the maturation process cleaves Gag, rearranges the constituents and activates the virion core for subsequent reverse transcription. A role for highly conserved residues in the CTD in contributing to the intermolecular interactions both before and after maturation has been indicated by genetic analyses and structural studies involving several retroviruses. The work of the Craven laboratory in analyzing a large set of mutations in the Rous sarcoma virus CA protein has documented the dual role of this major homology region in both immature and mature virion function and demonstrated the ability of secondary substitutions at distant and diverse sites to compensate for lethal mutations in this area. In particular, a hot spot for suppression of these mutations has been mapped to the second 1-helix of the CTD. The findings suggest that this region of the protein works together with the conserved motif to control a critical conformational switch needed for correct maturation of the capsid. The proposed experiments will combine the genetic and biophysical expertise of two investigators to directly test the role of the 12 helix of the CTD in achieving a protein conformation that is competent for assembly of functional capsids. Specifically, these studies will use NMR spectroscopy to assess changes in the three-dimensional structure of the CTD upon introduction of particular lethal and suppressor mutations. In addition a series of different amino acids will be substituted at one position in the CTD which has been shown to be a particular hot spot for suppression of lethal MHR mutations. The structural effects caused by different substitutions of the CTD will be correlated with their in vivo effects on virus replication, morphology, and core stability. It is expected that this work will enable us define the contribution of the 12 helix to a conformational switch that is critical for retrovirus maturation and which is likely to have similar counterparts in other viral systems. PUBLIC HEALTH RELEVANCE: In retroviruses such as HIV, the human T-cell lymphotropic virus and the Rous sarcoma virus, a stage of the virus life cycle known as maturation involves very dramatic structural changes in the interior of the virus particle, leading to the activation of its infectious potential. The studies described in this proposal will use a combination of genetic and protein structural approaches to examine the molecular mechanisms that control this process. A detailed understanding of this essential step of virus infection will allow development of better maturation inhibitors for use as antiretroviral drugs.

描述（由申请人提供）：逆转录病毒Ca（或衣壳）蛋白在病毒生命周期中有多个重要作用，该病毒生命周期在未成熟颗粒组装过程中组织了GAG多蛋白的堆积，指导了病毒素内核中成熟的衣壳的形态发生，最后在重复的早期阶段起作用。 CA蛋白经历复杂分子重排和构象变化的能力对于支持病毒生命周期中不同功能的能力至关重要。蛋白质的羧基末端结构域（CTD）是一个结构模块，参与未成熟粒子中的GAG多蛋白之间的分子间相互作用和成熟过程后CA-CA关联中的cabobular相互作用，使组成部分重新安排并激活Virion核心，以进行随后的反向转录。 CTD中高度保守的残基在成熟之前和之后有助于分子间相互作用的作用。 Craven实验室在分析Rous肉瘤病毒CA蛋白中的大量突变方面的工作已经证明了该主要同源性区域在未成熟和成熟的病毒率功能中的双重作用，并证明了在远处和多种位点次级取代以补偿该区域中致死突变的能力。特别是，抑制这些突变的热点已被映射到CTD的第二个1螺旋。研究结果表明，该蛋白质的该区域与保守的基序一起工作，以控制正确成熟的衣壳所需的临界构象开关。提出的实验将结合两个研究者的遗传和生物物理专业知识，以直接测试CTD 12螺旋在实现蛋白质构象中的作用，该蛋白质构型有能力用于组装功能性衣壳的组装。具体而言，这些研究将使用NMR光谱法来评估CTD的三维结构的变化，并引入特定的致命和抑制突变。另外，在CTD中的一个位置将取代一系列不同的氨基酸，这已被证明是抑制致命MHR突变的特殊热点。由CTD的不同取代引起的结构效应将与它们的体内对病毒复制，形态和核心稳定性的影响相关。预计这项工作将使我们能够定义12架构象开关的贡献，该构象开关对于逆转录病毒至关重要，并且在其他病毒系统中可能具有相似的对应物。公共卫生相关性：在逆转录病毒中，人类T细胞淋巴病毒和ROUS肉瘤病毒（称为成熟的病毒生命周期的一个阶段）涉及病毒颗粒内部的非常巨大的结构变化，导致其感染潜力的激活。该提案中描述的研究将使用遗传和蛋白质结构方法的组合来检查控制该过程的分子机制。对病毒感染的这一基本步骤的详细理解将允许开发更好的成熟抑制剂作为抗逆转录病毒药物。