In situ structures of three components essential to human cytomegalovirus pathogenesis: genome-packaging machinery, capsid-associated tegument and prefusion glycoprotein complexes

人类巨细胞病毒发病机制所必需的三个成分的原位结构：基因组包装机制、衣壳相关的外皮和融合前糖蛋白复合物

• 批准号: 10597018
• 负责人: Z Hong ZHOU
• 金额: $ 47.19万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10597018
• 关键词: 3-Dimensional; Acquired Immunodeficiency Syndrome; Antibodies; Antiviral Agents; Architecture; Bacterial Artificial Chromosomes; Bacteriophages; Binding; Capsid; Capsid Proteins; Cell fusion; Cell physiology; Cell-Matrix Junction; Cells; Chickenpox; Collaborations; Complex; Congenital Abnormality; Coupled; Cryo-electron tomography; Cryoelectron Microscopy; Cytomegalovirus; Cytomegalovirus Infections; DNA; DNA Packaging; Data Collection; Developed Countries; Developing Countries; Development; Diameter; Drug Targeting; Elderly; Electrons; Epitopes; Genome; Glycoproteins; Herpesviridae; Herpesvirus Type 3; Human; Immune; Immunocompetent; Immunocompromised Host; In Situ; Individual; Infection; Life; Maps; Mediating; Membrane Proteins; Methods; Modeling; Mutagenesis; Mutation; Nature; Pathogenesis; Persons; Phase; Pilot Projects; Population; Primates; Process; Proteins; Publications; Relaxation; Reporting; Research; Resolution; Resource-limited setting; Science; Seroprevalences; Structure; Surface; Technology; Testing; Therapeutic; Vaccines; Viral; Virion; Virus; Work; chemical bond; congenital infection; density; design; ds-DNA; gain of function; gammaherpesvirus; glycoprotein structure; high resolution imaging; human data; human imaging; human model; image processing; immunosuppressed; improved; inhibitor; insight; member; neonatal patient; neutralizing monoclonal antibodies; new therapeutic target; novel; novel therapeutics; novel vaccines; organ transplant recipient; particle; pressure; protein complex; protein protein interaction; rational design; three dimensional structure; vaccine candidate; vaccine development; viral resistance

Though latently infecting most of us asymptomatically, human cytomegalovirus (HCMV) is a leading viral cause of birth defects and can be life-threatening to immune-compromised individuals. As a member of the β- herpesvirus subfamily of the Herpesviridae and the most structurally and genetically complex herpesvirus (e.g., its genome is twice that of chickenpox-causing varicella zoster virus, an α-herpesvirus), HCMV is one of the largest of all viruses and presents a major challenge to structure determination. Architecturally similar to other herpesviruses, HCMV is composed of a glycoprotein-containing envelope, a tegument layer, and a bacteriophage-like icosahedral capsid enclosing a genome of a single dsDNA molecule. Distinctive from members of the α- and γ-herpesvirus subfamilies are two processes central to HCMV infection: 1) its large genome needs to be packaged through a portal complex and then stabilized by a unique tegument protein pp150; 2) the process of cell fusion by gB involves a unique pentameric glycoprotein complex gH/gL/UL128/UL130/UL131. These processes thus can be targeted for structure-guided design for novel vaccines and anti-virals against HCMV infections. By cryo electron microscopy (cryoEM), the PI’s group obtained the first three-dimensional structure of HCMV capsid at 18Å resolution in 1999, which was followed by progressive improvement in resolution, culminating at the recent 3.9Å resolution structure reported in Science. Our pilot studies resolved the portal complex and pp150-capsid interactions absent from α- and γ-herpesvirus subfamilies. Furthermore, we have demonstrated atomic resolution structure determination for membrane protein complexes and—in collaboration with Merck—obtained preliminary cryoEM data for HCMV pentameric glycoprotein complexes. We hypothesize that our state-of-the-art technologies in electron-counting cryoEM, symmetry relaxation and local refinement methods, and HCMV BAC technologies together would now allow us to determine in situ structures of genome packaging/ejection portal complex, pp150 and glycoprotein complexes, and when combined with structure-guided mutagenesis, to identify essential hot-spot residues critical to the interactions among these proteins. Harnessing technology breakthroughs in cryoEM and structure-guided mutagenesis, the proposed research aims to: (1) obtain in situ structure of the portal of DNA genome packaging and ejection machinery at near-atomic resolution and identify residues critical to capsid assembly and stabilization; (2) determine the in situ structure of pp150 at about 2Å resolution and identify the chemical bonds between capsid and capsid-interacting pp150 residues, particularly the cys tetrad conserved among primate cytomegaloviruses; (3) obtain atomic structures of purified pentameric glycoprotein complex in complex with three neutralizing monoclonal antibodies, as well as their in situ pre-fusion glycoprotein structures on viral envelope by cryo electron tomography for comparison. The expected results should inform efforts in designing inhibitors and vaccines against HCMV infections.

尽管人类巨细胞病毒（HCMV）潜伏感染我们大多数人而无症状，但它是导致出生缺陷的主要病毒原因，并且可能危及免疫功能低下个体的生命。作为疱疹病毒科β-疱疹病毒亚科的成员，也是结构和遗传最复杂的疱疹病毒（例如，其基因组是引起水痘的水痘带状疱疹病毒（一种α-疱疹病毒）的两倍），HCMV是所有病毒中最大的一种，对结构确定提出了重大挑战。 HCMV 在结构上与其他疱疹病毒相似，由含糖蛋白的包膜、外皮层和包围单个 dsDNA 分子基因组的噬菌体样二十面体衣壳组成。与α-和γ-疱疹病毒亚科成员不同的是HCMV感染的两个核心过程：1）其大基因组需要通过门复合物包装，然后通过独特的外皮蛋白pp150来稳定； 2）gB的细胞融合过程涉及独特的五聚体糖蛋白复合物gH/gL/UL128/UL130/UL131。因此，这些过程可用于针对 HCMV 感染的新型疫苗和抗病毒药物的结构指导设计。通过冷冻电子显微镜 (cryoEM)，PI 团队于 1999 年获得了第一个分辨率为 18Å 的 HCMV 衣壳三维结构，随后分辨率不断提高，最终在《科学》杂志上报道了最近的 3.9Å 分辨率结构。我们的初步研究解决了 α-和 γ-疱疹病毒亚科中不存在的门静脉复合体和 pp150-衣壳相互作用。此外，我们还与默克公司合作，展示了膜蛋白复合物的原子分辨率结构测定，并获得了 HCMV 五聚体糖蛋白复合物的初步冷冻电镜数据。我们假设，我们在电子计数冷冻电镜、对称弛豫和局部精化方法以及 HCMV BAC 技术方面最先进的技术现在将使我们能够确定基因组包装/喷射门户复合物、pp150 和糖蛋白复合物的原位结构，并与结构引导诱变相结合，以确定对这些蛋白质之间的相互作用至关重要的重要热点残基。利用冷冻电镜和结构引导诱变方面的技术突破，该研究旨在：（1）以近原子分辨率获得DNA基因组包装和喷射机械门户的原位结构，并识别对衣壳组装和稳定至关重要的残基； (2) 以大约 2Å 的分辨率确定 pp150 的原位结构，并鉴定衣壳和衣壳相互作用的 pp150 残基之间的化学键，特别是灵长类巨细胞病毒中保守的 cys 四分体； (3)通过冷冻电子断层扫描获得纯化的五聚体糖蛋白复合物与三种中和单克隆抗体复合物的原子结构，以及它们在病毒包膜上的原位融合前糖蛋白结构，以进行比较。预期结果将为设计针对 HCMV 感染的抑制剂和疫苗的努力提供信息。