Uncoating of the Herpes Simplex Virus Genome

单纯疱疹病毒基因组的脱壳

• 批准号: 9372274
• 负责人: James F. Conway
• 金额: $ 19.42万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-10 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9372274
• 关键词: Alpha Cell; Antiviral Agents; Binding; Biochemical; Capsid; Capsid Proteins; Cell Nucleus; Cells; Chickenpox; Complex; Cryoelectron Microscopy; Cytoplasm; DNA; Defect; Development; Disease; Docking; Dynein ATPase; Encephalitis; Genetic Transcription; Genetic study; Genome; Herpes Labialis; Herpes zoster disease; Herpesviridae; Herpesvirus 1; Image; Infection; Intervention; Knowledge; Lead; Life Cycle Stages; Maps; Mediating; Membrane; Methodology; Microtubules; Modeling; Molecular; Nuclear Envelope; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Pharmaceutical Preparations; Play; Process; Proteins; Reporting; Resolution; Role; Simplexvirus; Structure; System; Testing; Viral; Viral Genome; Virion; Virus; Virus Replication; base; beta Karyopherins; improved; insight; mutant; novel; planetary Atmosphere; pressure; reconstitution; reconstruction; temperature sensitive mutant; trafficking; viral DNA

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.

单纯疱疹病毒 (HSV) 衣壳在病毒生命周期的多个步骤中发挥着关键作用。感染的早期步骤包括：病毒和宿主膜融合以及衣壳释放到细胞质中；衣壳沿微管向核膜的动力蛋白依赖性运输；将衣壳对接在核孔处；并将病毒基因组释放到细胞核中。衣壳与核孔复合物 (NPC) 的结合似乎是由衣壳蛋白 pUL25 和衣壳栓系的外皮蛋白 pUL36 通过它们与 NPC 蛋白 Nup214 和 Nup358 的相互作用介导的。此外，NPC 结合需要导入 beta 和功能性 RanGTP/GDP 循环。感染早期阶段的一个关键知识缺口是衣壳如何与 NPC 结合以及触发病毒基因组从衣壳中释放的因素。包装的 HSV DNA 在衣壳内产生数十个大气压的压力，这种压力可能会驱动病毒基因组易位到细胞核中。已发现 pUL25 和 pUL36 中的温度敏感突变体与 NPC 对接，但两种突变体均无法释放 DNA，表明这两种蛋白在 NPC 对接和脱壳中的作用可以分开。该提案的目的是了解衣壳与 NPC 对接后触发包装基因组释放的因素。我们最近报道了对完整病毒粒子内成像的疱疹病毒衣壳的高质量冷冻电镜重建，这允许建立 pUL25 亚基和结构域组织的详细模型，并确定 pUL25 与衣壳和 pUL36 形成的亚基接触。基于我们对一种新型 pUL25 HSV 突变体的初步研究，该突变体的衣壳结合核膜但不脱壳，我们假设 pUL25 通过与 pUL36 和/或一种 NPC 蛋白相互作用来启动 DNA 释放。我们建议在两个具体目标中检验我们的假设：i) 定义 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) 确定触发 HSV 基因组脱壳的衣壳/NPC 相互作用的结构变化。这些研究将检查基因组脱壳的生化和结构基础，提供有关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。