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

虽然人类巨细胞病毒(HCMV)在无症状的情况下潜伏感染我们大多数人，但它是导致出生缺陷的主要病毒原因，并可能危及免疫受损的人的生命。作为疱疹病毒科β疱疹病毒亚家族的一员，也是结构和基因最复杂的疱疹病毒(例如，其基因组是引起水痘的水痘带状疱疹病毒的两倍)，是所有病毒中最大的病毒之一，对结构确定提出了重大挑战。在结构上与其他疱疹病毒相似，HCMV由含有糖蛋白的包膜、被层和包裹单个dsDNA分子基因组的类似噬菌体的二十面体衣壳组成。与α和γ疱疹病毒亚家族成员不同的是，巨细胞病毒感染有两个核心过程：1)其大基因组需要通过一个门户复合体包装，然后由独特的被膜蛋白pp150稳定；2)GB的细胞融合过程涉及一个独特的五聚体糖蛋白复合体Gh/Gl/UL128/UL130/UL131。因此，这些过程可以作为针对HCMV感染的新型疫苗和抗病毒药物的结构指导设计的目标。1999年，利用冷冻电子显微镜(CryoEM)，Pi的小组获得了第一个分辨率为18？的人巨细胞病毒衣壳的三维结构，随后分辨率逐渐提高，最终达到了最近在《科学》杂志上报道的3.9？分辨率结构。我们的初步研究解决了α-和γ-疱疹病毒亚家族所缺乏的门脉复合体和pp150-衣壳相互作用。此外，我们还展示了膜蛋白复合体的原子分辨结构测定，并与默克公司合作，获得了HCMV五聚体糖蛋白复合体的初步低温电子显微镜数据。我们假设，我们在电子计数冷冻EM、对称性松弛和局部精化方法方面的最先进技术，以及HCMV BAC技术，现在将使我们能够原位确定基因组包装/喷射门户复合体、pp150和糖蛋白复合体的结构，并与结构导向突变相结合，以确定对这些蛋白质之间相互作用至关重要的必要热点残基。利用低温电子显微镜和结构导向突变技术的突破，这项拟议的研究旨在：(1)以近原子分辨率获得DNA基因组包装和喷射机械入口的原位结构，并鉴定对衣壳组装和稳定至关重要的残基；(2)以约2？分辨率测定pp150的原位结构，并鉴定衣壳与衣壳相互作用的pp150残基之间的化学键，特别是灵长类巨细胞病毒之间保守的四聚体；(3)获得纯化的五聚体糖蛋白复合体的原子结构，以及它们在病毒被膜上原位融合前的糖蛋白结构用于比较。预期的结果应该为设计针对HCMV感染的抑制剂和疫苗的努力提供参考。

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.