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

HSV-1核出口复合物的构象动力学和抑制机制

• 批准号: 10776119
• 负责人: Elizabeth Bennett Draganova
• 金额: $ 24.9万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-03 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10776119
• 关键词: Affect; Automobile Driving; Binding; Biological Assay; Biology; Blindness; Capsid; Cell Nucleus; Cells; Cellular biology; Chemicals; Collaborations; Complex; Coupled; Coupling; Cryo-electron tomography; Cryoelectron Microscopy; Custom; Cytoplasm; Data; Data Analyses; Data Collection; Defect; Disease; Double Stranded DNA Virus; Drug Targeting; Encephalitis; Engineering; Ensure; Environment; Event; Experimental Designs; Future; Goals; Herpes Labialis; Herpes Simplex Infections; Herpesviridae; Herpesviridae Infections; Herpesvirus 1; Human; Immunocompromised Host; In Vitro; Individual; Infection; Institution; Instruction; Interferometry; Iowa; Knock-out; Malignant Neoplasms; Mammals; Mediating; Medical; Membrane; Mentors; Methodology; Methods; Molecular Conformation; Monitor; Mutation; Newborn Infant; Nuclear; Nuclear Inner Membrane; Outcome; Peptides; Pharmaceutical Preparations; Phase; Point Mutation; Population; Process; Protein Inhibition; Proteins; Qualifying; Research; Role; Route; Series; Structure; Surface; Techniques; Testing; Therapeutic; Universities; Viral; Viral Proteins; Virion; Virus; Virus Replication; Work; X-Ray Crystallography; biophysical techniques; career; design; driving force; inhibitor; innovation; latent infection; light scattering; medical schools; mutant; neonate; new therapeutic target; novel; particle; pathogen; peptide drug; post-doctoral training; prevent; protein function; protein protein interaction; screening; small molecule; stem; therapeutic target; tomography; virology

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.

项目摘要/摘要 疱疹病毒是双链的DNA病毒，几乎感染了包括人类在内的所有哺乳动物 高效的病原体。疱疹病毒复制周期中的一个标志性事件需要未成熟的衣壳 在称为核出口的过程中，穿过内部核膜（INM）到达核周空间。 病毒出口的第一步是由配置的病毒核出口复合物（NEC）介导的，是 该提议的重点。 NEC在膜上寡聚的能力对于衣壳萌芽很重要，但是 会议的变化经历了执行此操作的情况。这项研究的长期目标是 确定NEC寡聚化的破坏是如何驱动萌芽的构象变化（AIM 1） 并开发基于肽的筛选平台来识别新型NEC抑制剂（AIM 2）。这项工作步骤 从通过大量初步数据提出的中心假设可以抑制NEC， 特别是通过扰动NEC寡聚化。这项工作的科学前提是形成详细的 疱疹病毒核出口机制，以告知创新治疗化合物的设计。一个 尖端生物物理技术的结合，包括冷冻光学显微镜/层析成像（冷冻/T）， 除突变和功能方法外，还将使用 在萌芽过程中疱疹单纯疱疹，并确定抑制此过程的途径。 K99阶段 提案的结构不仅是为了回答有关疱疹病毒核出口的这些基本问题 但也提供了一个平台，以过渡到以识别为中心的独立研究职业 针对特定蛋白质 - 蛋白质相互作用的小分子和肽抑制剂发生在各个阶段 疱疹病毒复制。这种类型的蛋白质靶向将为确定特定蛋白质提供新颖的手段 病毒内部功能并扩大可用于治疗的治疗靶标的曲目 疾病。在此阶段的重点将通过研究NEC突变体来进行冷冻/T的微调专业知识 旨在扰动低聚（与Cryoem/T专家Zhiheng Yu博士合作 研究校园），在病毒学技术方面获得教学，以对NEC突变体进行功能研究（在 与爱荷华大学的理查德·罗勒（Richard Roller）博士与疱疹生物学专家和David Knipe博士合作 哈佛医学院）并建立肽筛查平台（在Joshua Kritzer博士的建议下 塔夫茨大学（Tufts University） - 肽设计专家）。在非常合格的指导下 导师（结构性病毒学专家Katya Heldwein博士）和同事（Ralph Isberg博士 - 专家 在成功的学术职业的博士后培训），在簇的支持机构环境中 大学，该建议确保了及时过渡到成功的独立学术职业。