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: 10395617
• 负责人: Z Hong ZHOU
• 金额: $ 34.76万
• 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 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10395617
• 关键词: 3-Dimensional; Acquired Immunodeficiency Syndrome; Antibodies; Antiviral Agents; Architecture; Bacterial Artificial Chromosomes; Bacteriophages; Binding; Caliber; Capsid; Capsid Proteins; Cell fusion; Cell physiology; Cell-Matrix Junction; Cells; Cercopithecine Herpesvirus 1; Chickenpox; Collaborations; Complex; Congenital Abnormality; Coupled; Cryo-electron tomography; Cryoelectron Microscopy; Cytomegalovirus; DNA; DNA Packaging; Data; Data Collection; Developed Countries; Developing Countries; Development; Drug Targeting; Elderly; Electrons; Epitopes; Genome; Glycoproteins; Herpesviridae; Herpesvirus Type 3; Hot Spot; 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; Time; Vaccines; Viral; Virion; Virus; Work; base; chemical bond; congenital infection; density; design; ds-DNA; gain of function; gammaherpesvirus; glycoprotein structure; high resolution imaging; image processing; immunosuppressed; 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.

尽管我们大多数人不对称地感染了我们大多数人，但人类巨细胞病毒（HCMV）是出生缺陷的主要病毒原因，可以威胁生命对免疫功能低下的个体。作为疱疹病毒科的β-疱疹病毒的成员，结构上最复杂的疱疹病毒（例如，其基因组是chapox引起的水viricella带状疱疹病毒的两倍，是α-herpesvirus）的两倍，α-herpesvirus），α-herpecmmv是所有病毒质量质疑的最大挑战的最重要的挑战。与其他疱疹病毒在结构上相似，HCMV由含糖蛋白的包膜，技术层和细菌样的二十面体capsid组成，封闭了单个dsDNA分子的基因组。与α-和γ-疱疹病毒亚家族的成员不同的是HCMV感染的两个过程：1）需要通过门户复合物包装大型基因组，然后通过独特的Tegument蛋白PP150稳定； 2）GB的细胞融合过程涉及独特的五聚糖糖蛋白复合蛋白GH/GL/UL128/UL130/UL131。因此，这些过程可以针对针对HCMV感染的新型疫苗和抗病毒的结构引导设计。通过冷冻电子显微镜（Cryoem），PI组在1999年在18Å分辨率下获得了HCMV帽的第一个三维结构，随后是分辨率的逐步改进，最终在科学中报道的最近的3.9Å分辨率结构上达到了最终。我们的试点研究解决了门户复合物，而α-和γ-疱疹病毒亚家族不存在PP150-CAPSID相互作用。此外，我们已经证明了膜蛋白复合物的原子分辨率结构测定，并与默克公司合作（用于HCMV五型五聚糖糖蛋白复合物）的初步冷冻数据。我们假设我们在电子计算冷冻，对称性放松和局部改进方法中的最先进技术，而HCMV BAC技术现在可以使我们能够确定基因组包装/弹性门户复合物的原位结构，PP150，PP150，PP150，PP150和糖蛋白蛋白复合物中的结合量相互作用，以识别这些杂种构成的构成，并识别构成的构成，并识别出来的确定性，并识别出来的构成。拟议的研究利用了冷冻和结构引导的诱变的技术突破，目的是：（1）在接近原子的分辨率下获得DNA基因组包装和射血机构门户的原位结构，并确定对Capsid组装和稳定至关重要的残留物； （2）在约2Å分辨率下确定pp150的原位结构，并确定衣壳和衣壳相互作用的PP150保留的化学键，尤其是在灵长类的巨细胞病毒中保守的Cys Tetrad； （3）获得与三种中和单克隆抗体的纯化的五聚蛋白糖蛋白复合物的原子结构，并通过冷冻电子层析成像在病毒包膜上进行的原位融合前糖蛋白结构进行比较。预期的结果应为针对HCMV感染设计抑制剂和疫苗而努力。