Structural Mechanisms Of Genome Flow In Bacteriophage T4 And Their Biomedical Applications

噬菌体T4基因组流动的结构机制及其生物医学应用

• 批准号: 10635661
• 负责人: Venigalla B. Rao
• 金额: $ 48.48万
• 依托单位: CATHOLIC UNIVERSITY OF AMERICA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-15 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10635661
• 关键词: Archaeal Viruses; Bacteriophage T4; Bacteriophages; Binding; Biochemistry; Capsid; Categories; Cells; Cessation of life; Complex; Cryoelectron Microscopy; DNA; DNA Packaging; DNA Structure; Data; Data Collection; Defect; Degenerative Disorder; Disease; Docking; Duchenne muscular dystrophy; Dystrophin; Experimental Designs; Future; Gene Delivery; Genes; Genetic; Genome; Head; Human; In Vitro; Individual; Infection; Interdisciplinary Study; Investigation; Knowledge; Length; Lentivirus; Life Cycle Stages; Ligands; Measures; Modeling; Molecular; Molecular Conformation; Motor; Muscle; Muscle Cells; Muscular Dystrophies; Myopathy; Nanovirus; Neck; Organism; Phase; Physical condensation; Planet Earth; Positioning Attribute; Preparation; Process; Proteins; Regulation; Reporter Genes; Research; Research Proposals; Resolution; Series; Structure; Surface; System; Tail; Therapeutic; Time; Translating; Translational Research; Translations; Tube; Viral; Viral Genome; Virus; conformational conversion; delivery vehicle; density; design; ds-DNA; effective therapy; experimental study; extracellular; gene therapy; genetic payload; human disease; improved; in vivo; innovation; mouse model; muscular dystrophy mouse model; mutant; nanocapsid; nanomachine; nanoparticle; nanoparticle delivery; novel; nucleic acid binding protein; pressure; protein complex; reconstruction; seal; targeted delivery; therapeutic gene; vector; virology

This proposal aims to fill a critical knowledge gap in the assembly of icosahedral viruses; mechanisms and controls by which a viral genome flows into and out of a virus capsid with high precision and fidelity. The tailed dsDNA bacteriophage T4 is our model virus. The proposal will also translate this basic knowledge into a gene therapy for muscular dystrophy, a debilitating degenerative muscular disease that causes early death. We propose an innovative experimental design by advancing a novel “molecular valve” hypothesis. The hypothesis states that a sophisticated molecular valve at the unique portal vertex controls genome flow into and out of a virus capsid through dynamic structural and conformational changes. By integrating genetics, biochemistry, and cryo-electron microscopy, we will generate a series of asymmetric reconstructions of nanomachines in different structural and conformational states. These machines translocate genome into virus capsid creating a pressurized condensate (inward genome flow), arrest genome flow and position for delivery, and allow genome ejection upon encountering new host (outward genome flow). In specific aim 1, we will generate atomic level structures of the DNA packaging machine consisting of the portal vertex-bound packaging motor and its intermediate states during active translocation. A detailed packaging mechanism will be formulated that will have broad implications to phages, eukaryotic and archaeal viruses including herpes and adeno viruses. A strong work-flow has been established for preparation and data collection of packaging complexes using a newly constructed super-charged “acidic capsid” mutant. In specific aim 2, we will elucidate the dynamic mechanism of the neck-connector valve complex bound to portal by generating structures in different assembly states. These structures would illustrate conformational changes in the valve complex that lead to arrest of genome flow post-packaging and position it for delivery following tail docking. We present a novel discovery involving the participation of a host nucleic acid binding protein Hfq in these dynamic transactions. In specific aim 3, we will probe the mechanism of genome ejection by asymmetric cryo-EM reconstructions of the genome ejection machine pre-infection, during-infection, and post-infection. A preliminary cryo-EM reconstruction revealed, for the first time, density for a helical tape measure protein-DNA complex in the innermost core of the ejection tube. In specific aim 4, we will incorporate the basic knowledge gained from specific aims 1-3 to establish a novel, large capacity, T4 gene therapy vehicle to deliver the full- length ~11-kb dystrophin gene into human muscle cells as well as into a muscular dystrophy mouse model. Relying on our 42-years of expertise on T4 phage assembly and genome packaging, our synergistic research team will uncover the basic mechanisms of genome flow in viruses and their translation into potentially transformative phage-based gene therapeutics. These will have broad implications to virology and human disease therapies.

该建议旨在填补二十面体病毒组装的关键知识差距。机制和 病毒基因组以高精度和忠诚度流入和流出病毒capsid的对照。尾巴 dsDNA噬菌体T4是我们的模型病毒。该提案还将这些基本知识转化为基因 肌肉营养不良的疗法，这是一种令人衰弱的退化性肌肉疾病，会导致早期死亡。 我们通过推进一种新颖的“分子瓣膜”假设来提出创新的实验设计。这 假设指出，独特的门户顶点处的复杂分子阀控制基因组流入 并通过动态结构和构象变化从病毒capsid中。 通过整合遗传学，生物化学和冷冻电子显微镜，我们将生成一系列 纳米机器在不同结构和构象状态下的不对称重建。这些机器 将基因组转化为病毒衣壳，形成加压冷凝物（内基因组流），停滞基因组 流量和输送位置，并允许遇到新宿主（向外基因组流）时进行基因组估计。 在特定目标1中，我们将生成由DNA包装机的原子水平结构 在活动易位期间，门户网站结合的包装电机及其中间状态。详细的 包装机制将制定对噬菌体，真核生物和古细菌具有广泛影响 包括疱疹和腺病毒在内的病毒。为准备和数据建立了强大的工作流程 采用新建的超级充电“酸性衣壳”突变体的包装配合物收集。具体 AIM 2，我们将阐明颈部连接阀复合物的动态机理，该复合物由门户结合到门户 在不同的组装状态下产生结构。这些结构将说明 导致基因组流量后包装并将其放置在尾巴后交付的阀复合体 停靠。我们提出了一个新的发现，涉及宿主核酸结合蛋白HFQ参与 这些动态交易。在特定的目标3中，我们将通过不对称探测基因组射血的机理 基因组射血机的冷冻EM重建，感染期间和感染后。一个 初步的冷冻EM重建，首次揭示了螺旋胶带测量蛋白-DNA的密度 在弹出管的最内核中的复合物。在特定目标4中，我们将纳入基本知识 从特定的目标1-3中获得的，以建立一种新型，大容量的T4基因治疗工具，以提供全部 长度〜11-kb肌营养不良蛋白基因进入人类肌肉细胞以及肌肉营养不良小鼠模型。 依靠我们在T4噬菌体组件和基因组包装方面的42年专业知识，我们的协同作用 研究团队将发现病毒中基因组流的基本机制，并将其转化为潜在 基于变革噬菌体的基因疗法。这些将对病毒学和人类具有广泛的影响 疾病疗法。