Structural Biology Of Virus Assembly

病毒组装的结构生物学

• 批准号: 10265846
• 负责人: PAUL T WINGFIELD
• 金额: $ 84.62万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10265846
• 关键词: Adopted; Algorithms; Antigens; Antiviral Agents; Architecture; Bacteriophage T4; Bacteriophage T7; Bacteriophages; Binding; Capsid; Cell Nucleus; Cells; Cessation of life; Complex; Core Protein; Cryoelectron Microscopy; Crystallization; Cytoplasm; DNA; DNA Packaging; Data; Double Stranded DNA Virus; Electron Microscopy; Electrostatics; Environment; Floor; Genome; Genomics; Geometry; Glycoproteins; Goals; Grouping; HIV; HIV-1; Head; Hepatitis B; Hepatitis B Virus; Herpesviridae; Herpesvirus 1; Housing; Human; Image; In Vitro; Individual; Intervention; Isometric Exercise; Joints; Liver diseases; Location; Macromolecular Complexes; Modeling; Modification; Molecular; Molecular Conformation; Motor; Myxococcus xanthus; Nuclear; Nuclear Envelope; Nuclear Inner Membrane; Nuclear Outer Membrane; Nucleic Acids; Pathway interactions; Peripheral; Phosphotransferases; Play; Population; Potential Energy; Prolate; Proteins; Reaction; Reporting; Research; Resolution; Retroviridae; Running; Science; Side; Structure; System; Techniques; Thermus thermophilus; Trust; United States National Institutes of Health; Universities; Vaccines; Viral; Viral Genome; Virion; Virus; Virus Assembly; Work; density; dimer; ds-DNA; extracellular; human pathogen; interest; macromolecular assembly; mutant; particle; polypeptide; programs; protein complex; reconstruction; structural biology; terminase; thermostability; tomography; virus core

Summary: We seek to identify features with potential for antiviral drug and vaccine interventions, and to elucidate mechanisms at play in the assembly, maturation, and activation of large macromolecular complexes in general. In this context, we work on several viral systems with particular focus on viruses with genomes of double-stranded DNA. These viruses have the largest viral genomes and correspondingly elaborate virion structures. We also have a major interest in hepatitis B virus, a major human pathogen, and in retroviruses, primarily HIV-1. The latter (HIV-related studies) is the subject of a separate report (AR041166-11). During FY18, we made progress on several subprojects: Herpesvirus assembly. Over the past 25 years, we have studied many aspects of herpesvirus assembly, mostly on herpes simplex virus type 1(HSV-1). In recent years, our focus shifted to nuclear egress. The HSV-1 capsid assembles in the nucleus, then migrates into the cytoplasm for subsequent steps on the pathway. The Primary Enveloped Virion (PEV) is a transient particle formed in the perinuclear space as the DNA-filled capsid traverses the nuclear envelope. As the capsid buds through the inner nuclear membrane, it becomes coated with nuclear egress complex (NEC) protein. This yields a PEV whose envelope fuses with the outer nuclear membrane, releasing the capsid into the cytoplasm. To obtain enough PEVs for structural analysis, we isolated them from US3-null-infected cells (pUS3 is a virally encoded kinase). We found that PEVs differ from mature extracellular virions (MEVs) in several respects. PEVs have very few glycoprotein spikes whereas MEVs are densely coated with them. PEVs are 20% smaller than MEVs and there is little space between the capsid and the NEC layer, whereas in an MEV, this space is more capacious and is occupied by the tegument. To further characterize the portal vertex we worked with A-capsids (empty capsids in a mature conformational state), using a UL25-null strain which yields a high percentage of A-capsids. Tomographic data were recorded and, using a processing algorithm developed in the LSBR, it was possible to identify the portal vertex on these capsids. The portal protein pUL6 is seen to be embedded in the capsid floor of the capsid. In these reconstructions, we also detected a sizeable density overlying the portal vertex that may represent the viral terminase complex. Packing of DNA and internal proteins in bacteriophage T4. In earlier work, our cryo-EM studies of bacteriophage T7 provided strong evidence for the "concentric spool model whereby the DNA is coiled around the portal axis in nested shells (Cerritelli et al., Cell 91, 271-290 1997). We have now investigated whether a similar arrangement pertains in T4, which has a 4-fold larger genome, a prolate capsid, and lacks the cylindrical protein core of T7. Cryo-EM yielded side-views and axial views (defined as such relative to the axis running through the portal vertex) of prolate (wild-type) heads and of isometric (mutant) heads. Giant heads were imaged in sideview. We conclude that in isometric heads, the DNA is spooled around the portal axis, essentially as in T7; in giant heads, the spool is rotated by 90o. The innermost regions of DNA-filled heads are less ordered. We infer that the conformations of encapsidated genomes represent an energy-minimized compromise between electrostatic repulsion effects involving DNA duplexes and the strain associated with bending the DNA. Although T4 does not emulate T7 in having a highly ordered core of internal proteins, it does have substantial amounts of internal proteins. In both systems, these proteins are destined for delivery into a host cell whose locations in the head have been unclear. To investigate the internal proteins ofT4, of which there are two, gpAlt and gpIPIII, we have used bubblegram imaging, a technique invented in the LSBR (Wu et al., Science 335, 182 2012). In this way we found that both gpAlt and gpIPII are excluded from a highly ordered peripheral zone but otherwise are distributed seemingly at random throughout the capsid interior. The peripheral zone coincides with that occupied by shells of coiled DNA. DNA packaging into supersized T=7 capsids of a thermophilic virus. This project has been a joint undertaking with F. Antson (University of York) as part of a Wellcome Trust/NIH collaborative program. Double-stranded DNA viruses including bacteriophages and herpesviruses package their genomes into preformed capsids, using ATP-driven motors. Seeking to advance structural and mechanistic understanding, we established in vitro packaging for a thermostable bacteriophage, P23-45 of Thermus thermophilus. Both the unexpanded procapsid and the expanded capsid can package DNA in the presence of large terminase and ATP. Cryo- EM reconstructions were determined to 0.4 nm resolution for both the expanded capsid and the procapsid. Unexpectedly, the capsid was found to observe T=7 quasi-symmetry, despite the P23-45 genome being twice as large as those of T=7 and other phages (e.g. HK97, P22) with the same T-number (7) in which DNA packing density is thought to approach the maximum physically possible. Our reconstructions explain this anomaly, whereby modifications to the canonical HK97 fold permit a capsid volume twice as large. We also obtained a 1.95 crystal structure for the portal protein and performed symmetry-free reconstructions for the procapsid and expanded capsid to define its setting in the portal vertex. We found that capsid expansion elicits a substantial change in the conformation of the portal protein, while still allowing DNA to be packaged. Hepatitis B virus core-antigen. Hepatitis B virus has infected approximately one third of the human population and causes almost 1 million deaths from liver disease annually. The capsid is a defining feature of a virus, distinct from host components, and therefore a target for intervention. Unusually for a virus, Hepatitis B assembles two capsids, with different geometries, from the same dimeric protein. Geometric principles dictate that the subunits in this system occupy seven different environments. We compared the two capsids by cryo-electron microscopy at 3.5 -resolution under identical conditions and found that the polypeptide chains adopt seven different conformations. These structures were used to calculate potential energies (analogous to elastic deformation or strain) for the individual chains, dimers, and several higher-order groupings discernible in the two lattices. We also calculated the binding energies between chains. We found that some groupings have substantially lower energy and are therefore potentially more stable, allowing us to predict likely intermediates on the two assembly pathways. We also observed such intermediates by electron microscopy of in vitro capsid assembly reactions. This is the first structural characterization of the early assembly intermediates of this important human pathogen. 5) Encapsulins are protein shells that resemble viral capsids in many respects, including the fold of their constituent subunits and the icosahedral symmetry of their molecular architecture. This fold was first observed in capsids of bacteriophage HK97. However, instead of housing genomic nucleic acid as in viral capsids, encapsulins accommodate other kinds of cargo. We obtained cryo-EM structure of M. xanthus encapsuling A structure at 3.18 Angstrom resolution and 3.45 Angstrom resolution for T=3 and T=1 particles. In addition, obtained the first crystal structure of encapsuling cargo protein EncB.

概括： 我们寻求识别具有抗病毒药物和疫苗干预潜力的特征，并阐明大分子复合物组装、成熟和激活中发挥作用的机制。在此背景下，我们研究了几种病毒系统，特别关注具有双链 DNA 基因组的病毒。这些病毒具有最大的病毒基因组和相应复杂的病毒体结构。我们还对乙型肝炎病毒（一种主要的人类病原体）和逆转录病毒（主要是 HIV-1）非常感兴趣。后者（HIV 相关研究）是另一份报告 (AR041166-11) 的主题。 2018 财年，我们在多个子项目上取得了进展： 疱疹病毒组装。在过去的 25 年里，我们研究了疱疹病毒组装的许多方面，主要是针对 1 型单纯疱疹病毒 (HSV-1)。近年来，我们的重点转向了核泄漏。 HSV-1 衣壳在细胞核中组装，然后迁移到细胞质中进行后续步骤。初级包膜病毒颗粒 (PEV) 是当充满 DNA 的衣壳穿过核膜时在核周空间中形成的瞬时颗粒。当衣壳穿过内核膜时，它就会被核出口复合物 (NEC) 蛋白包被。这产生了 PEV，其包膜与外核膜融合，将衣壳释放到细胞质中。为了获得足够的 PEV 进行结构分析，我们从 US3-null 感染的细胞中分离出它们（pUS3 是一种病毒编码的激酶）。我们发现 PEV 在几个方面与成熟的细胞外病毒体 (MEV) 不同。 PEV 的糖蛋白刺突很少，而 MEV 的糖蛋白刺突很密集。 PEV 比 MEV 小 20%，并且衣壳和 NEC 层之间的空间很小，而在 MEV 中，这个空间更宽敞并且被外皮占据。 为了进一步表征门顶点，我们使用 A-衣壳（处于成熟构象状态的空衣壳），使用产生高百分比 A-衣壳的 UL25 无效菌株。记录断层扫描数据，并使用 LSBR 中开发的处理算法，可以识别这些衣壳上的入口顶点。可以看出门蛋白 pUL6 嵌入衣壳的衣壳底中。在这些重建中，我们还检测到门静脉顶点上有相当大的密度，这可能代表病毒终止酶复合物。 噬菌体 T4 中 DNA 和内部蛋白质的包装。在早期工作中，我们对噬菌体 T7 的冷冻电镜研究为“同心线轴模型”提供了强有力的证据，即 DNA 在嵌套壳中围绕门轴盘绕（Cerritelli 等人，Cell 91, 271-290 1997）。现在研究了 T4 中是否存在类似的排列，T4 的基因组大 4 倍，具有长长的衣壳，并且缺乏 T7 的圆柱形蛋白质核心。冷冻电镜产生了长形（野生型）头部和等距（突变型）头部的侧视图和轴向视图（定义为相对于穿过门顶点的轴），我们得出结论：在等距头部中，DNA 绕着门轴缠绕，基本上与 T7 中一样；在巨型头部中，线轴旋转 90°。衣壳基因组的构象代表了涉及 DNA 双链体的静电排斥效应和与 DNA 弯曲相关的应变之间的能量最小化折衷。 尽管 T4 不像 T7 那样具有高度有序的内部蛋白质核心，但它确实具有大量的内部蛋白质。在这两个系统中，这些蛋白质都注定要被输送到宿主细胞中，而宿主细胞在头部的位置尚不清楚。为了研究 T4 的内部蛋白（其中有两种蛋白：gpAlt 和 gpIPIII），我们使用了气泡图成像，这是 LSBR 中发明的技术（Wu 等人，Science 335, 182 2012）。通过这种方式，我们发现 gpAlt 和 gpIPII 都被排除在高度有序的外围区域之外，但在整个衣壳内部似乎随机分布。外围区域与卷曲 DNA 壳所占据的区域一致。 DNA 包装到嗜热病毒的超大 T=7 衣壳中。该项目是与 F. Antson（约克大学）联合开展的项目，是 Wellcome Trust/NIH 合作项目的一部分。 包括噬菌体和疱疹病毒在内的双链 DNA 病毒使用 ATP 驱动的马达将其基因组包装到预先形成的衣壳中。为了加深对结构和机制的理解，我们建立了耐热噬菌体 P23-45 的体外包装。未扩增的衣壳原和扩增的衣壳都可以在大终止酶和 ATP 存在的情况下包装 DNA。冷冻电镜重建的扩展衣壳和原衣壳的分辨率均确定为 0.4 nm。出乎意料的是，尽管 P23-45 基因组是 T=7 和具有相同 T 数 (7) 的其他噬菌体 (例如 HK97、P22) 的两倍，但衣壳被发现观察到 T=7 准对称性。 DNA堆积密度被认为接近物理上可能的最大密度。我们的重建解释了这种异常现象，即对规范 HK97 折叠的修改允许衣壳体积增加两倍。我们还获得了门蛋白的 1.95 晶体结构，并对原衣壳和扩展衣壳进行了无对称重建，以定义其在门顶点的设置。我们发现衣壳扩张引起门户蛋白构象发生实质性变化，同时仍然允许 DNA 被包装。 乙型肝炎病毒核心抗原。乙型肝炎病毒感染了大约三分之一的人口，每年导致近 100 万人死于肝病。衣壳是病毒的一个决定性特征，与宿主成分不同，因此是干预的目标。对于病毒来说不同寻常的是，乙型肝炎由相同的二聚蛋白组装出两个具有不同几何形状的衣壳。几何原理规定该系统中的子单元占据七个不同的环境。我们在相同条件下通过冷冻电子显微镜在 3.5 分辨率下比较了两个衣壳，发现多肽链采用七种不同的构象。这些结构用于计算两个晶格中单个链、二聚体和几个高阶分组的势能（类似于弹性变形或应变）。我们还计算了链之间的结合能。我们发现某些分组的能量要低得多，因此可能更稳定，使我们能够预测两条组装途径上可能的中间体。我们还通过体外衣壳组装反应的电子显微镜观察了此类中间体。这是这种重要人类病原体早期组装中间体的首次结构表征。 5) 封装蛋白是蛋白质外壳，在许多方面类似于病毒衣壳，包括其组成亚基的折叠和分子结构的二十面体对称性。这种折叠首先在噬菌体 HK97 的衣壳中观察到。然而，封装蛋白并不像病毒衣壳那样容纳基因组核酸，而是容纳其他种类的货物。我们获得了 M. xanthus 封装 A 结构的冷冻电镜结构，T=3 和 T=1 颗粒的分辨率为 3.18 埃，分辨率为 3.45 埃。此外，获得了第一个封装货物蛋白EncB的晶体结构。