Uncoating of the Herpes Simplex Virus Genome

单纯疱疹病毒基因组的脱壳

• 批准号: 9504499
• 负责人: James F. Conway
• 金额: $ 23.46万
• 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 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9504499
• 关键词: 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起着至关重要的作用。感染的早期步骤包括：病毒和宿主膜的融合，以及将衣壳释放到细胞质中；沿微管沿着核包络的capsid的依赖性依赖的贩运；在核孔中对接衣壳；并将病毒基因组释放到细胞核中。衣壳与核孔复合物（NPC）的结合似乎是通过与NPC蛋白NUP214和NUP358的相互作用来介导的。另外，NPC结合需要importin beta和功能性RANGTP/GDP周期。感染早期阶段的一个关键知识差距是衣壳如何与NPC接合以及触发病毒基因组从衣壳中释放的原因。包装的HSV DNA在衣壳内产生数十个大气的压力，这种压力可能会将病毒基因组易位到核中。已经发现PUL25和PUL36中温度敏感的突变体在NPC处停靠，但两个突变体都无法释放DNA，这表明这两种蛋白在NPC对接和不涂层中的作用可以分开。该提案的目的是了解NPC上的衣壳在停靠后包装基因组的触发释放。我们最近报道了完整病毒体内成像的疱疹病毒衣壳的高质量冷冻重建，该疱疹病毒capsid允许使用PUL25的亚基和域组织的详细模型，并确定了PUL25与capsid和pul36相关的亚基触点。基于我们对新型PUL25 HSV突变体的初步研究，该研究结合了核膜，但不脱落，我们假设PUL25通过与PUL36和/或与NPC蛋白之一的相互作用的相互作用来启动DNA释放。我们建议在两个具体的目的中检验我们的假设，即i）定义pul25在CAPSID与NPC结合及其在触发Capsid中DNA释放中的作用的作用； ii）确定触发HSV基因组不涂层的衣壳/NPC相互作用的结构变化。这些研究将研究基因组脱膜的生化和结构基础，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断病毒复制的靶标。