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.

项目总结/摘要 疱疹病毒是双链DNA病毒，感染几乎所有哺乳动物，包括人类，使它们 高效病原体疱疹病毒复制周期中的一个标志性事件需要不成熟的衣壳， 在一个称为核出口的过程中，芽通过内核膜（INM）到达核周空间。 病毒排出的第一步是由保守的病毒核出口复合物（NEC）介导的， 这一提案的重点。NEC在膜上寡聚化的能力对于衣壳出芽是重要的，然而 进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驱动出芽的构象变化（目的1） 并开发基于肽的筛选平台，用于鉴定新型NEC抑制剂（目标2）。这项工作源于 从一个中心假设，从大量的初步数据制定，即NEC可以被抑制， 特别是通过干扰NEC寡聚化。这项工作的科学前提是制定一个详细的 疱疹病毒核出口的机制，为创新治疗化合物的设计提供信息。一 结合尖端生物物理技术，包括冷冻电子显微镜/断层扫描（cryoEM/T）， 沿着突变和功能的方法将被用来确定所经历的分子相互作用， 单纯疱疹NEC在萌芽期间，并确定抑制这一进程的途径。K99阶段 一项提案的结构不仅要回答围绕疱疹病毒核出口的这些基本问题， 还提供了一个平台，用于过渡到以识别为中心的独立研究生涯， 靶向在不同阶段发生的特异性蛋白质-蛋白质相互作用的小分子和肽抑制剂 疱疹病毒的复制这种蛋白质靶向技术将为确定特异性蛋白质提供一种新的手段 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库。 疾病这一阶段的重点将是通过研究NEC突变体来微调cryoEM/T的专业知识 旨在干扰寡聚化（与HHMI Janelia的cryoEM/T专家Zhiheng Yu博士合作 研究校园），获得病毒学技术的指导，对NEC突变体进行功能研究（在 与疱疹生物学专家爱荷华州大学的理查德·罗尔博士和哈佛大学的大卫·佩普博士合作， 哈佛医学院）和建立肽筛选平台（在约书亚克里泽博士的指导下 在塔夫茨大学-治疗肽设计的专家）。在一位非常有资格的 导师（Katya Heldwein博士-结构病毒学专家）和共同导师（Ralph Isberg博士- 博士后培训成功的学术生涯），在塔夫茨大学的支持性制度环境 大学，这一建议确保及时过渡到领导一个成功的独立的学术生涯。