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.

项目概要 dsDNA 病毒（包括有尾噬菌体、疱疹病毒和腺病毒）的组装是一种高度自动化的技术。 涉及一系列蛋白质相互作用的协调过程，导致感染性病毒颗粒的形成。 这项研究的中心目标是对具体的机制有一个详细的理解 蛋白质：控制该组装过程的蛋白质相互作用。 暂时地，由它们的构象可塑性驱动，这是必不可少的。 对于有尾 dsDNA 病毒组装，噬菌体 P22（一种 dsDNA 病毒支架）证明了这一点。 蛋白质驱动门户蛋白单体寡聚成环，随后并入 衣壳原 (PC) 会优先结合门户的 PC 构象，并包装 DNA。 直到头部充满为止，此时，门户发生构象转变为其成熟病毒体（MV）形式。 触发终止酶蛋白的释放和阻断门通道的蛋白的结合 在完整的 MV 中，Portal 呈现出与病毒体之后的 PC 和 MV 形式不同的另一种构象。 尽管门户复合体对于成功的病毒粒子组装至关重要，但它仍将其 DNA 注入宿主细胞。 它是 DNA 易位的管道，它在 DNA 包装和信号传导过程中发挥作用，以完成 包装尚未被机械地定义。核心假设是包装的构象变化。 需要门户复合体来调节病毒成功成熟的过程，我将对此进行测试。 假设使用噬菌体 P22，它提供了一个优秀的 dsDNA 模型组装系统。 简单的遗传学和完善的生物化学比更复杂的 dsDNA 病毒具有优势。 目标 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 蛋白质变体能够制造具有适当形态的感染性噬菌体，从而包装正确的噬菌体 在目标 2 中，我将获得与终止酶结合的门户复合物的冷冻电镜结构。 总的来说，这些数据将代表我们的重大进步。 通过揭示门户网站的构象变化来了解 dsDNA 病毒组装途径 总体而言，阐明 P22 组装所涉及的细节可以导致识别。 抑制 dsDNA 人类病原体组装的潜在抗病毒药物靶位点。