Investigating the conformational changes of the portal protein that drive DNA packaging in a dsDNA virus

研究双链 DNA 病毒中驱动 DNA 包装的门户蛋白的构象变化

• 批准号: 10677356
• 负责人: Makayla Leroux
• 金额: $ 4.52万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-23 至 2025-11-24
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677356
• 关键词: Adenoviruses; Affect; Antiviral Agents; Bacteriophage P22; Bacteriophages; Binding; Binding Proteins; Biochemistry; Biological Assay; Capsid Proteins; Cells; Charge; Complex; Cryoelectron Microscopy; DNA; DNA Packaging; Data; Development; Double Stranded DNA Virus; Drug Targeting; Fluorescence; Genetic; Genome; Goals; Head; Health; Herpesviridae; Human; Light; Methods; Modeling; Molecular Conformation; Morphology; Mutate; Pathway interactions; Potassium; Process; Production; Proteins; Research; Resolution; Role; Salmonella typhimurium; Scaffolding Protein; Series; Signal Transduction; Site; Structure; System; Tail; Techniques; Testing; Time; Transmission Electron Microscopy; Variant; Viral; Viral Proteins; Virion; Virus; Virus Assembly; Wing; Work; drug development; ds-DNA; experimental study; human pathogen; in vivo; innovation; monomer; novel; protein complex; protein protein interaction; terminase

PROJECT SUMMARY Assembly of dsDNA viruses, including the tailed bacteriophages, herpesviruses, and adenoviruses, is a highly coordinated process involving a series of protein interactions that lead to the formation of an infectious virion. The central goal of this research is to achieve a detailed mechanistic understanding of the specific protein:protein interactions that govern this assembly process. During assembly, proteins interact temporally, driven by their conformational plasticity. The dodecameric portal protein complex, which is essential for tailed dsDNA virus assembly, exemplifies this. In bacteriophage P22, a model for dsDNA viruses, scaffolding protein drives the oligomerization of portal protein monomers into rings which are subsequently incorporated into procapsids (PCs). Terminase proteins then preferentially bind the PC conformation of portal, and package DNA until the head is full. At this point, portal undergoes a conformational switch to its mature virion (MV) form that triggers the release of the terminase proteins and the binding of proteins that stopper the portal channel resulting in the complete MV. Portal takes on another distinct conformation from its PC and MV forms after the virion injects its DNA into host cells. Despite the portal complex being critical for successful virion assembly, owing to it being the conduit for DNA translocation, its roles during DNA packaging and signaling for the completion of packaging have not been mechanistically defined. The central hypothesis is that conformational changes of the portal complex are required to regulate the processes involved in successful viral maturation. I will test this hypothesis using bacteriophage P22 which provides an excellent dsDNA model assembly system. Overall, its simple genetics and well-established biochemistry offer advantages over more complex dsDNA viruses. The experiments proposed in Aim 1 will elucidate the signal for DNA packaging completion by interrogating portal protein variants’ ability to make infectious phages with the appropriate morphology that package the correct amount of DNA. In Aim 2, I will obtain the cryo-EM structure of the portal complex bound to terminase, which has not been solved at high-resolution. Collectively, these data will represent a significant advancement of our understanding of the dsDNA viral assembly pathway by shedding light on how conformational changes of portal drive DNA packaging. Overall, elucidation of the details involved in P22 assembly can lead to the identification of potential anti-viral drug target sites to inhibit assembly of dsDNA human pathogens.

项目摘要 DSDNA病毒的组装，包括尾细菌，疱疹病毒和腺病毒，是一种高度 涉及一系列蛋白质相互作用的协调过程，这些蛋白质相互作用导致形成感染性病毒体。 这项研究的核心目标是对特定的特定理解实现详细的机械理解 蛋白质：控制这种组装过程的蛋白质相互作用。在组装过程中，蛋白质相互作用 暂时，由它们的构象可塑性驱动。十二体门户蛋白质复合物，这是必不可少的 对于尾部的dsDNA病毒组装，这是这样的。在细菌p22中，DSDNA病毒的模型，脚手架 蛋白质将门户蛋白质单体的寡聚化驱动到环中，随后将其纳入 procapsids（PC）。然后，末端酶蛋白优先结合门户的PC构象，然后包装DNA 直到头满。此时，门户经历了会议切换到其成熟的病毒粒子（MV）形式，该形式 触发末端酶蛋白的释放和蛋白的结合，该蛋白会阻止门户的通道产生 在完整的MV中。 Portal在Virion之后与PC和MV形式进行了另一个不同的会议 将其DNA注射到宿主细胞中。尽管门户综合体对于成功的病毒装配至关重要 它是DNA易位的导管，其在DNA包装过程中的作用和完成的信号 包装尚未机械定义。中心假设是 需要门户复合物来调节成功的病毒成熟过程中涉及的过程。我会测试这个 使用拟噬菌p22的假设，该假设提供了出色的DSDNA模型组装系统。总体而言，它 简单的遗传学和良好的生物化学提供了比更复杂的DSDNA病毒的优点。这 AIM 1中提出的实验将通过询问门户来阐明DNA包装完成的信号 蛋白质变体具有适当的形态，可以包装正确 DNA的量。在AIM 2中，我将获得与末端酶结合的门户配合物的冷冻EM结构，该结构 尚未以高分辨率解决。总的来说，这些数据将代表我们的重大进步 通过阐明门户的构象变化，了解DSDNA病毒组装途径 驱动DNA包装。总体而言，阐明P22组装中涉及的细节可以导致识别 潜在的抗病毒药物靶位部位抑制DSDNA人类病原体的组装。