Uncoating of the Herpes Simplex Virus Genome

单纯疱疹病毒(HSV)衣壳在病毒生命周期的多个步骤中起着关键作用。感染的早期步骤包括：病毒和宿主膜的融合和衣壳进入细胞质；依赖动力蛋白的衣壳沿着微管运输到核膜；衣壳停靠在核孔；以及将病毒基因组释放到核中。衣壳与核孔复合体(NPC)的结合似乎是由衣壳蛋白pUL25和衣壳拴系被膜蛋白pUL36通过与NPC蛋白Nup214和Nup358相互作用而介导的。此外，NPC的结合需要Importin beta和一个功能正常的RanGTP/GDP循环。在感染的早期阶段，一个关键的知识缺口是衣壳如何与鼻咽癌接触，以及是什么触发了病毒基因组从衣壳中释放。包装好的单纯疱疹病毒DNA在衣壳内产生了数十个大气压的压力，这种压力可能会推动病毒基因组移位到细胞核中。PUL25和pUL36中的温度敏感突变体已被发现对接在NPC上，但这两个突变体都不能释放DNA，这表明这两种蛋白质在NPC对接和脱壳中的作用可以分离。这项建议的目的是了解是什么触发了在NPC对接衣壳后包装的基因组的释放。我们最近报道了在完整病毒粒子内成像的疱疹病毒衣壳的高质量冷冻EM重建，这使得pUL25的亚基和结构域组织的详细模型，并确定了pUL25与衣壳和pUL36的亚基接触。根据我们对一个新的pUL25 HSV突变体的初步研究，该突变体的衣壳与核膜结合，但不脱壳，我们假设pUL25通过与pUL36和/或与一种鼻咽癌蛋白相互作用而启动DNA释放。我们建议从两个特定的目标来验证我们的假设：i)确定pUL25在衣壳与NPC结合中的作用及其在触发衣壳DNA释放中的作用；ii)确定衣壳/NPC相互作用中触发HSV基因组脱壳的结构变化。这些研究将检查基因组脱壳的生化和结构基础，提供关于衣壳结构如何控制与NPC相互作用的独特信息，并可能揭示脆弱的结构中间体作为阻止病毒复制的目标。

The herpes simplex virus (HSV) capsid plays a critical role in multiple steps of the virus life cycle. Early steps in infection include: fusion of viral and host membranes and release of the capsid into the cytoplasm; dynein- dependent trafficking of the capsid along microtubules to the nuclear envelope; docking of the capsid at a nuclear pore; and release of the viral genome into the nucleus. Binding of the capsid to the nuclear pore complex (NPC) appears to be mediated by the capsid protein pUL25 and the capsid-tethered tegument protein pUL36 through their interaction with the NPC proteins Nup214 and Nup358. In addition, NPC binding requires importin beta and a functional RanGTP/GDP cycle. A key knowledge gap in the early stages of infection is how the capsid engages the NPC and what triggers release of the viral genome from the capsid. The packaged HSV DNA creates a pressure of tens of atmospheres within the capsid and this pressure likely drives the translocation of the viral genome into the nucleus. Temperature-sensitive mutants in pUL25 and pUL36 have been found to dock at the NPC but both mutants fail to release DNA indicating that the role of these two proteins in NPC docking and uncoating can be separated. The objective of this proposal is to understand what triggers release of the packaged genome after docking of the capsid at the NPC. We have recently reported high-quality cryoEM reconstructions of the herpesvirus capsid imaged inside intact virions which allowed for a detailed model of subunit and domain organization of pUL25 and identified subunit contacts that pUL25 makes with capsid and with pUL36. Based on our preliminary studies of a novel pUL25 HSV mutant whose capsid binds the nuclear membrane but does not uncoat, we hypothesize that pUL25 initiates DNA release through its interaction with pUL36 and/or with one of the NPC proteins. We propose to test our hypothesis in two specific aims that i) define the role of pUL25 in capsid binding to the NPC and its role in triggering DNA release from the capsid; and ii) determine the structural changes in the capsid/NPC interaction that trigger HSV genome uncoating. These studies will examine the biochemical and structural basis of genome uncoating, provide unique information on how the capsid structure controls interactions with the NPC, and may reveal vulnerable structural intermediates as targets for blocking virus replication.