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.

该提案旨在填补二十面体病毒组装机制和方面的关键知识空白； 控制病毒基因组以高精度和保真度流入和流出病毒衣壳。 双链DNA噬菌体T4是我们的模型病毒，该提案也将把这些基础知识转化为基因。 肌肉营养不良症的治疗，这是一种导致过早死亡的衰弱性退行性肌肉疾病。 我们通过提出一种新颖的“分子阀”假说，提出了一种创新的实验设计。 假设指出，独特的门户顶点处的复杂分子阀门控制着基因组流入 并通过动态结构和构象变化脱离病毒衣壳。 通过整合遗传学、生物化学和冷冻电子显微镜，我们将产生一系列 不同结构和构象状态下纳米机器的不对称重建。 将基因组转移到病毒衣壳中，产生加压冷凝物（向内基因组流），阻止基因组 传递的流动和位置，并允许在遇到新宿主时基因组弹出（外向基因组流动）。 在具体目标 1 中，我们将生成 DNA 包装机的原子级结构，其中包括 主动易位过程中门户顶点绑定包装电机及其中间状态。 将制定对噬菌体、真核生物和古细菌产生广泛影响的包装机制 病毒包括疱疹病毒和腺病毒已经建立了强大的工作流程用于准备和数据。 使用新构建的超电荷“酸性衣壳”突变体的包装复合物的集合。 目标 2，我们将通过以下方式阐明颈部连接器瓣膜复合体与门静脉结合的动态机制 生成不同组装状态的结构这些结构将说明构象变化。 导致包装后基因组流动停滞并将其定位为跟随尾部递送的阀门复合物 我们提出了一项新发现，涉及宿主核酸结合蛋白 Hfq 的参与。 这些动态事务在具体目标 3 中，我们将探讨不对称基因组喷射的机制。 感染前、感染期间和感染后基因组喷射机的冷冻电镜重建。 初步冷冻电镜重建首次揭示了螺旋卷尺蛋白质-DNA 的密度 在具体目标4中，我们将结合基本知识。 从具体目标 1-3 中获得的成果是建立一种新型、大容量、T4 基因治疗载体，以提供全面的治疗效果。 将长度约 11 kb 的肌营养不良蛋白基因导入人类肌肉细胞以及肌营养不良症小鼠模型中。 凭借我们在 T4 噬菌体组装和基因组包装方面 42 年的专业知识，我们的协同 研究小组将揭示病毒基因组流动的基本机制及其转化为潜在的 基于噬菌体的变革性基因疗法将对病毒学和人类产生广泛​​的影响。 疾病疗法。