Mechanisms of Conformational Dynamics and Inhibition of the HSV-1 Nuclear Egress Complex

项目摘要/摘要 疱疹病毒是双链dna病毒，感染几乎所有的哺乳动物，包括人类，使它们 高效的病原体。疱疹病毒复制周期中的标志性事件需要未成熟的衣壳 芽通过内核膜(INM)进入核周空间，这一过程称为核出口。 病毒退出的这一关键的第一步是由保守的病毒核出口复合体(NEC)介导的，是 这项提案的重点。NEC在膜上寡聚的能力对衣壳萌发很重要，但 执行这一过程所经历的构象变化是未知的。这项研究的长期目标是 确定NEC寡聚化的破坏如何扰乱推动萌发的构象变化(目标1) 并开发基于多肽的筛选平台，以确定新的NEC抑制剂(目标2)。这项工作源于 根据大量初步数据形成的中心假设，NEC可以被抑制， 特别是通过干扰NEC的齐聚反应。这项工作的科学前提是制定详细的 疱疹病毒核外泄的机制为创新治疗化合物的设计提供信息。一个 结合尖端生物物理技术，包括低温电子显微镜/断层扫描(低温EM/T)， 以及突变和功能方法将被用来识别经历过的分子相互作用 单纯性疱疹NEC在萌芽过程中的作用，并找出抑制这一过程的途径。这方面的K99阶段 提案的结构不仅是为了回答围绕疱疹病毒核出口的这些基本问题 还提供了一个向以识别为中心的独立研究职业过渡的平台 针对不同阶段发生的特定蛋白质-蛋白质相互作用的小分子和多肽抑制剂 疱疹病毒复制。这种类型的蛋白质靶向将提供一种确定特定蛋白质的新方法 在病毒内发挥作用，并扩大可用于治疗这种疾病的治疗靶点的曲目 疾病。这一阶段的重点将是通过研究NEC突变体来微调低温EM/T方面的专业知识 旨在扰乱齐聚(与HHMI Janelia的低温EM/T专家余志恒博士合作 研究校园)，获得病毒学技术指导，以执行NEC突变体的功能研究(在 与爱荷华大学的疱疹生物学专家理查德·罗勒博士和大卫·尼普博士合作 哈佛医学院)，并建立肽筛选平台(在Joshua Kritzer博士的建议下 塔夫茨大学的治疗性多肽设计专家)。在一个非常合格的人的指导下 导师(Katya Heldwein博士--结构病毒学专家)和共同导师(Ralph Isberg博士-- 成功学术生涯的博士后培训)，在塔夫茨大学的支持性机构环境中 作为一所大学，这项提议确保了及时过渡到领导一个成功的独立学术生涯。

Project Summary/Abstract Herpesviruses are double-stranded DNA viruses that infect almost all mammals, including humans, making them highly effective pathogens. A hallmark event in the herpesvirus replication cycle requires immature capsids to bud through the inner nuclear membrane (INM) to the perinuclear space in a process termed nuclear egress. This essential first step in viral exit is mediated by the conserved viral nuclear egress complex (NEC) and is the focus of this proposal. The ability of the NEC to oligomerize on membranes is important for capsid budding, yet the conformational changes undergone to perform this are unknown. The long-term goal of this research is to determine how disruption of NEC oligomerization perturbs conformational changes that drive budding (Aim 1) and develop peptide-based screening platforms for identifying novel NEC inhibitors (Aim 2). This work stems from a central hypothesis, formulated from substantial preliminary data, that the NEC can be inhibited, specifically by perturbing NEC oligomerization. The scientific premise of this work is to formulate a detailed mechanism of herpesvirus nuclear egress to inform the design of innovative therapeutic compounds. A combination of cutting-edge biophysical techniques, including cryoelectron microscopy/tomography (cryoEM/T), along with mutational and functional approaches will be used to identify the molecular interactions undergone by Herpes Simplex NEC during budding and to identify routes for inhibiting this process. The K99 phase of this proposal is structured to not only answer these fundamental questions surrounding herpesvirus nuclear egress but also provide a platform for transitioning to an independent research career centered around identifying small molecule and peptide inhibitors targeting specific protein-protein interactions occurring at various stages of herpesviral replication. This type of protein targeting will provide a novel means for determining specific protein function within the virus and expand the repertoire of therapeutic targets available for the treatment of this disease. Focus during this phase will be on fine-tuning expertise in cryoEM/T by investigating NEC mutants designed to perturb oligomerization (in collaboration with cryoEM/T expert Dr. Zhiheng Yu at the HHMI Janelia Research Campus), gain instruction in virological techniques to perform functional studies on NEC mutants (in collaboration with herpes biology experts Dr. Richard Roller at the University of Iowa and Dr. David Knipe at Harvard Medical School) and establish peptide screening platforms (under advisement from Dr. Joshua Kritzer at Tufts University – an expert in therapeutic peptide design). Under the guidance of an extremely qualified mentor (Dr. Katya Heldwein – an expert in structural virology) and co-mentor (Dr. Ralph Isberg – an expert in postdoctoral training for successful academic careers), within the supportive institutional environment of Tufts University, this proposal ensures a timely transition to leading a successful independent academic career.