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

这项建议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进出病毒衣壳的控制。尾巴 双链DNA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 治疗肌营养不良症，这是一种会导致早逝的衰弱退行性肌肉疾病。 通过提出一个新颖的“分子阀”假说，我们提出了一种创新的实验设计。这个 假说认为，位于独特入口顶端的复杂分子阀控制着基因组流入 并通过动态的结构和构象变化走出病毒衣壳。 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 纳米机器在不同结构和构象状态下的不对称重构。这些机器 将基因组转移到病毒衣壳中，产生加压凝聚物(向内基因组流)，阻止基因组 流动和定位以供传递，并允许在遇到新宿主时排出基因组(向外基因组流动)。 在特定目标1中，我们将生成DNA包装机的原子级结构，包括 门脉顶端绑定的包装马达及其在主动移位过程中的中间状态。一个详细的 将制定对噬菌体、真核生物和古生物具有广泛影响的包装机制 病毒，包括疱疹病毒和腺病毒。已经建立了强大的准备和数据工作流程 包装复合体的收集使用了一个新构建的超级带电的“酸性衣壳”突变体。具体而言 目的2，我们将阐明颈-连接瓣膜复合体与门脉结合的动力学机制。 在不同的装配状态下生成结构。这些结构将说明构象变化 导致基因组流停止的瓣膜复合体在包装后并将其定位为在尾部之后交付 对接。我们提出了一项新的发现，涉及宿主核酸结合蛋白Hfq参与 这些动态交易。在具体目标3中，我们将探索不对称抛射基因组的机制。 感染前、感染期间和感染后基因组喷射机的冷冻-EM重建。一个 低温电子显微镜的初步重建首次揭示了螺旋卷尺测量蛋白质-DNA的密度 喷出管最内层的复合体。在具体目标4中，我们将纳入基本知识 从特定的目标1-3获得建立一个新的，大容量，T4基因治疗载体，以提供完整的 将长度为~11kb的dystrophin基因导入人肌肉细胞，以及导入肌营养不良症小鼠模型。 凭借我们42年来在T4噬菌体组装和基因组包装方面的专业知识，我们的协同 研究小组将揭示病毒基因组流动的基本机制，并将其翻译成潜在的 基于噬菌体的变革性基因疗法。这些都将对病毒学和人类产生广泛的影响。 疾病疗法。

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.