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

该提案旨在填补二十面体病毒组装方面的关键知识空白;机制和 病毒基因组以高精度和保真度流入和流出病毒衣壳的控制。加尾 dsDNA噬菌体T4是我们的模型病毒。该提案还将把这些基本知识转化为基因 治疗肌肉萎缩症，一种使人衰弱的退行性肌肉疾病，导致过早死亡。 我们提出了一个创新的实验设计，提出了一个新的“分子阀”的假设。的 一个假说指出，在独特的门户顶点复杂的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衣壳中排出。 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 在不同的结构和构象状态的纳米机器的不对称重建。这些机器 将基因组转移到病毒衣壳中，产生加压冷凝物（向内的基因组流）， 流动和定位以进行递送，并允许基因组在遇到新宿主时排出（向外的基因组流动）。 在具体目标1中，我们将生成DNA包装机的原子级结构，其包括 门静脉顶点绑定包装电机和其中间状态在主动易位。详细 包装机制将制定将有广泛的影响，以生物，真核生物和古细菌 包括疱疹病毒和腺病毒的病毒。为准备和数据建立了强有力的工作流程 收集包装复合物使用一个新构建的超级充电“酸性衣壳”突变体。在特定 目的2，我们将阐明颈连接器瓣膜复合体与门静脉结合的动力学机制， 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停滞并将其定位以在尾部之后递送的阀复合物 对接我们提出了一个新的发现，涉及参与宿主核酸结合蛋白Hfq， 这些动态交易。在具体目标3中，我们将探索基因组喷射的机制， 在感染前、感染期间和感染后对基因组喷射机进行冷冻-EM重建。一 初步的冷冻电镜重建首次揭示了螺旋卷尺测量蛋白质-DNA的密度 在喷射管的最里面的核心复杂。在具体目标4中，我们将纳入基本知识 从具体目标1-3中获得，以建立一种新的、大容量的T4基因治疗载体， 长度~11-kb肌营养不良蛋白基因导入人肌肉细胞以及肌营养不良小鼠模型。 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， 研究小组将揭示病毒基因组流动的基本机制，以及它们翻译成潜在的 基于噬菌体的基因疗法这些将对病毒学和人类免疫学产生广泛的影响。 疾病治疗。

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.