Investigating the conformational changes of the portal protein that drive DNA packaging in a dsDNA virus

项目总结 Dsdna病毒的组装，包括尾部噬菌体、疱疹病毒和腺病毒，是一种高度 包括一系列蛋白质相互作用的协调过程，这些相互作用导致感染性病毒粒子的形成。这个 这项研究的中心目标是实现对具体机制的详细理解 蛋白质：支配这个组装过程的蛋白质相互作用。在组装过程中，蛋白质相互作用 在时间上，由它们的构象可塑性驱动。十二聚体门脉蛋白复合体，这是必不可少的 对于尾随dsDNA病毒组装，举例说明了这一点。在噬菌体P22中，dsDNA病毒的模型，支架 蛋白质驱动门户蛋白单体齐聚成环，这些环随后被结合到 Proapsids(PC)。然后，终端酶蛋白优先结合门户的PC构象，包装DNA 直到头满为止。在这一点上，门户经历了其成熟的病毒粒子(MV)形式的构象转换 触发终止酶蛋白的释放和蛋白的结合，从而阻断门静脉通道 在完整的MV中。在病毒粒子之后，门户呈现出与其PC和MV形式不同的另一种构象 将其DNA注入宿主细胞。尽管门户复合体对成功组装病毒粒子至关重要，但由于 它是DNA转运的管道，它在DNA包装中的作用以及完成 包装还没有被机械地定义。中心假设是分子的构象变化 门静脉复合体是调节病毒成功成熟所涉及的过程所必需的。我要测试一下这个 假设使用噬菌体P22，它提供了一个很好的dsDNA模型组装系统。总体而言，其 与更复杂的dsDNA病毒相比，简单的遗传学和成熟的生物化学提供了优势。这个 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 蛋白质变异体能够制造具有适当形态的传染性噬菌体，包装正确的 DNA的数量。在目标2中，我将获得与末端酶结合的门脉复合体的冷冻EM结构，该结构 没有在高分辨率下得到解决。总的来说，这些数据将代表着我们的 通过揭示门静脉的构象变化来理解dsDNA病毒组装途径 驱动DNA包装。总体而言，阐明P22组装所涉及的细节可以导致鉴定 潜在的抗病毒药物靶点以抑制dsDNA人类病原体的组装。

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.