Structural Biology Of Virus Assembly

病毒组装的结构生物学

• 批准号: 7964881
• 负责人: ALASDAIR C. STEVEN
• 金额: $ 132.69万
• 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/7964881
• 关键词: ATP phosphohydrolase; Affinity; Allosteric Regulation; Annual Reports; Antibodies; Antigenic Diversity; Antigens; Antiviral Agents; Atomic Force Microscopy; Bacteriophage T7; Bacteriophages; Binding; Calorimetry; Capsid; Capsid Proteins; Cells; Cereals; Charge; Chromatin; Chromatin Structure; Coupling; Cryoelectron Microscopy; DNA; DNA Packaging; Data; Dependence; Dimerization; Double Stranded RNA Virus; Elasticity; Enzymes; Epitopes; Filament; Genetic Transcription; Genome; Goals; Heating; Hepatitis B Virus; Hepatitis B e Antigens; Herpesviridae; Histones; Human; Immune system; Immunodominant Epitopes; Immunoglobulin G; Incubated; Individual; Investigation; Knowledge; Label; Location; Macromolecular Complexes; Malignant neoplasm of cervix uteri; Maps; Mass Spectrum Analysis; Measures; Mechanics; Methods; Modeling; Molecular; Monoclonal Antibodies; Morphogenesis; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Nucleosomes; Paper; Papillomavirus; Patients; Physical condensation; Plasma; Play; Polymerase; Polyomavirus; Positioning Attribute; Property; Proteins; Publishing; RNA; Recombinants; Reporting; Research; Resolution; Role; Rupture; Scaffolding Protein; Scanning; Signal Transduction; Simian virus 40; Site; Structure; Surface Plasmon Resonance; System; Tail; Time; Variant; Viral; Viral Genome; Virion; Virus; Virus Assembly; base; cold temperature; density; dimer; early onset; electron tomography; expectation; follow-up; insight; interest; molecular dynamics; mutant; nanoindentation; particle; pathogen; polypeptide; pressure; reconstruction; research study; scaffold; structural biology; tomography

During FY08, we focussed on three subprojects. (1) Hepatitis B Virus Capsid Assembly. We study the HBV capsid protein which presents two of the three clinically important antigens - core antigen (capsids) and e-antigen (unassembled protein) - of this major human pathogen. After first showing that capsid protein self-assembles from dimers into capsids of two different sizes, we obtained, in 1997, a cryo-EM density map in which much of the secondary structure was visible, including the 4-helix bundle that forms the dimerization motif. This was the first time that such detailed information had been achieved by cryo-EM. Our subsequent research helped delineate the path of the polypeptide chain. We went on to investigate the antigenic diversity of HBV by using cryo-EM to characterize the conformational epitopes of seven different monoclonal antibodies raised against capsids. In FY09, we followed three lines of investigation. (i) We completed a project reported in FY08 in which surface plasmon resonance was used to measure the binding affinities of a set of murine monoclonal antibodies commonly used to discriminate between core- and e-antigen, including several that we previously characterized by cryo-EM. Unexpectedly, most antibodies bind to both antigens with high affinity. The exceptions are antibody e6 which detects an epitope accessible only on dimers and occluded on capsids, and antibody 3120 which detects an epitope presented only on capsids because its epitope spans an inter-dimer interface. (ii) To investigate the immunodominant epitopes recognized by the human immune system in infected patients, we isolated antibodies from plasma obtained from a HBV-positive patient (provided by Drs T. Heller and J. Liang, NIDDK), prepared Fabs from them, and studied their decoration of purified recombinant capsids by cryo-electron microscopy and 3D-reconstruction. Contrary to expectation, these polyclonal Fabs did not form a continuous canopy over the capsid but instead bound preferentially to two discreet sites; one located on the spike-like protrusions (sites occupied by the so-called immunodominant loops), and one in the region between these protrusions. These data indicate that the human IgG repertoire actually detects a rather limited set of epitopes very similar to those that we previously observed with murine monoclonal antibodies. (iii) Following up on the "native" high resolution mass spectrometry experiments reported in FY08 in which the masses of both size variants of the capsid were determined to within 0.1%, we pursued a collaborative project to measure the mechanical properties of both capsids by nanoindentation methods carried out by atomic force microscopy. The two capsids have similar overall stability and elasticity (Young's modulus of 0.4 GPa), but differ in their subunit exchange rates, as determined by mass spectrometry of capsids incubated with isotopically labeled subunits. The directional dependence of capsid elasticity was predicted from coarse-grained molecular dynamics simulation and compared with the nano-indentation data. (2) Assembly and Maturation of Bacteriophage Capsids. Our interest in capsid assembly lies in the massive conformational changes that accompany their maturation. These transitions afford unique insights into allosteric regulation. We study maturation of several phages to exploit expedient aspects of each system. The tailed phages afford an excellent model for herpesvirus capsids (see (2) above), reflecting common evolutionary origins. In FY08, we focussed on thre projects. (i) We used a combination of scanning calorimetry and cryo-EM to investigate the encapsidation of phage DNA, which represents an extreme case of genome condensation. Phage HK97 is well suited to study this phenomenon in view of detailed knowledge of its capsid structure. We found that, as filled capsids are heated, their DNA is released at relatively low temperatures (40 to 50 degrees). Heating increases the internal pressure, causing the capsid to rupture, releasing the DNA. DNA packaging also induces a change in the capsid structure that is reflected both in an earlier onset of thermal denaturation than empty capsids and in subtle morphological differences. (We also detected a similar effect in herpesvirus capsids - see (2) above). We envisage that this transition in the capsid shell is transmitted to the portal, altering its interactions with the packaging enzyme and thus signaling that packaging is complete. This project, outlined in annual report FY08, was completed and published in FY09. (ii) The capsids of double-stranded RNA viruses serve as specialized compartments for the replication and transcription of the viral genomes. We investigate the structural basis of this remarkable phenomenon in the phage phi6 system, which has a tripartite genome. In FY08, we published a paper describing the location of the P2 polymerase in the interior of the viral procapsid, as determined by cryo-EM of wild type and mutant particles. P2 is substoichiometric, occupying only 3 - 10 (depending on the mutant) of 20 potential sites. In FY09, we extended these studies by cryo-electron tomography with particular focus on the question of whether sites occupied by P2 correlate with external sites occupied by P4, the packaging ATPase. The observed distributions of P2 and P4 occupancy indicate that both proteins are randomly distributed and therefore there is no direct coupling between the activities of these two viral enzymes that respectively conduct RNA packaging, and replication and transcription. (iii) For tailed phages and herpesviruses, a scaffolding protein is essential for correct capsid assembly . The scaffolding protein coassembles with the capsid protein to form the precursor procapsid and is then expelled during DNA packaging. Little is known about how scaffolding proteins perform this function. We study this phenomenon in the phage T7 system. Mass analysis of T7 procapsids indicates that the gp9 copy number averages 150 but can vary by as much as 30% from particle to particle. Examination of native T7 procapsids by cryoelectron tomography revealed a loosely ordered network of scaffolding filaments, with as many as ten filaments per particle. These filaments emanate from the portal vertex. The extended structure and positively charged C-terminus of gp9 likely play an important role in these interactions. The implications of these observations for T7 morphogenesis are under assessment. (3) Papillomaviruses and polyomaviruses. These viruses, which include human pathogens that induce cervical cancer, have the unique property of appropriating histone proteins from the host cell when assembling the viral minichromosome. In FY08, we started a project to analyze the structure of encapsidated SV40 minichromosomes by cryo-electron tomography. In a set of 100 reconstructed full virions extracted from several high-quality reconstructions, we find that the number of nucleosomes that they contain varies from 17 to 23 and that their spatial arrangement does not match the icosahedral symmetry of the capsid. Furthermore, analysis of extracted chromatin core densities suggests that overall chromatin structure is variable from particle to particle. Ongoing efforts are directed towards computationally modeling of the positions and orientations of nucleosomes within individual particle reconstructions.

2008 财年，我们重点关注三个子项目。 (1)乙型肝炎病毒衣壳组装。我们研究了 HBV 衣壳蛋白，它代表了这种主要人类病原体的三种临床重要抗原中的两种 - 核心抗原（衣壳）和 e 抗原（未组装蛋白）。在首次表明衣壳蛋白从二聚体自组装成两种不同大小的衣壳后，我们于 1997 年获得了冷冻电镜密度图，其中大部分二级结构是可见的，包括形成二聚体的 4 螺旋束主题。这是第一次通过冷冻电镜获得如此详细的信息。我们随后的研究帮助描绘了多肽链的路径。我们通过使用冷冻电镜来表征针对衣壳的七种不同单克隆抗体的构象表位，继续研究 HBV 的抗原多样性。 2009 财年，我们进行了三项调查。 (i) 我们完成了 2008 财年报告的一个项目，其中使用表面等离振子共振来测量一组通常用于区分核心抗原和 e 抗原的鼠单克隆抗体的结合亲和力，包括我们之前通过冷冻抗原表征的几种抗体。 EM。 出乎意料的是，大多数抗体以高亲和力与两种抗原结合。例外的是抗体 e6，它检测仅在二聚体上可接近并封闭在衣壳上的表位，以及抗体 3120，它检测仅在衣壳上存在的表位，因为其表位跨越二聚体间界面。 (ii) 为了研究感染患者中人类免疫系统识别的免疫显性表位，我们从 HBV 阳性患者的血浆中分离出抗体（由 T. Heller 和 J. Liang 博士提供，NIDDK），从中制备 Fab，并通过冷冻电子显微镜和 3D 重建研究了纯化重组衣壳的装饰。与预期相反，这些多克隆 Fab 并未在衣壳上形成连续的冠层，而是优先结合到两个离散位点；一个位于尖峰状突起上（被所谓的免疫显性环占据的位置），另一个位于这些突起之间的区域。这些数据表明，人类 IgG 库实际上检测到一组相当有限的表位，与我们之前在鼠单克隆抗体中观察到的表位非常相似。 (iii) 继 2008 财年报告的“天然”高分辨率质谱实验（其中衣壳的两种尺寸变体的质量确定在 0.1% 以内）之后，我们开展了一个合作项目，通过以下方法测量两种衣壳的机械特性：通过原子力显微镜进行纳米压痕方法。 两种衣壳具有相似的整体稳定性和弹性（杨氏模量为 0.4 GPa），但其亚基交换率不同，这是通过与同位素标记亚基一起孵育的衣壳的质谱法测定的。通过粗粒度分子动力学模拟预测衣壳弹性的方向依赖性，并与纳米压痕数据进行比较。 (2)噬菌体衣壳的组装和成熟。我们对衣壳组装的兴趣在于其成熟过程中发生的巨大构象变化。这些转变为变构调节提供了独特的见解。我们研究了几种噬菌体的成熟，以利用每个系统的有利方面。带尾噬菌体为疱疹病毒衣壳提供了一个极好的模型（参见上面的（2）），反映了共同的进化起源。 2008 财年，我们重点开展了三个项目。 (i) 我们结合使用扫描量热法和冷冻电镜来研究噬菌体 DNA 的衣壳化，这代表了基因组浓缩的极端情况。鉴于其衣壳结构的详细知识，噬菌体 HK97 非常适合研究这种现象。 我们发现，当填充的衣壳被加热时，它们的 DNA 在相对较低的温度（40 至 50 度）下释放。加热会增加内部压力，导致衣壳破裂，释放 DNA。 DNA 包装还会引起衣壳结构的变化，这反映在比空衣壳更早发生热变性以及细微的形态差异上。 （我们还在疱疹病毒衣壳中检测到类似的效果 - 参见上面的 (2)）。我们设想衣壳中的这种转变被传递到门户，改变其与包装酶的相互作用，从而发出包装完成的信号。该项目于 2008 财年年度报告中概述，并于 2009 财年完成并发布。 (ii)双链RNA病毒的衣壳充当病毒基因组复制和转录的专门区室。我们研究了噬菌体 phi6 系统中这一显着现象的结构基础，该系统具有三部分基因组。在 2008 财年，我们发表了一篇论文，描述了通过野生型和突变体颗粒的冷冻电镜测定的 P2 聚合酶在病毒衣壳内部的位置。 P2 是亚化学计量的，仅占据 20 个潜在位点中的 3 - 10 个（取决于突变体）。 2009 财年，我们通过冷冻电子断层扫描扩展了这些研究，特别关注 P2 占据的位点是否与 P4（包装 ATP 酶）占据的外部位点相关的问题。观察到的 P2 和 P4 占据分布表明这两种蛋白质是随机分布的，因此这两种分别进行 RNA 包装、复制和转录的病毒酶的活性之间没有直接耦合。 (iii) 对于有尾噬菌体和疱疹病毒，支架蛋白对于衣壳的正确组装至关重要。支架蛋白与衣壳蛋白共同组装形成前体衣壳，然后在 DNA 包装过程中被排出。人们对支架蛋白如何发挥这一功能知之甚少。 我们在噬菌体 T7 系统中研究了这种现象。 T7 原衣壳的质量分析表明，gp9 拷贝数平均为 150，但不同颗粒之间的差异可能高达 30%。 通过冷冻电子断层扫描对天然 T7 衣壳的检查显示出一个松散有序的支架丝网络，每个颗粒有多达 10 个丝。这些细丝从门顶点发出。 gp9 的延伸结构和带正电荷的 C 末端可能在这些相互作用中发挥重要作用。 这些观察结果对 T7 形态发生的影响正在评估中。 (3)乳头瘤病毒和多瘤病毒。这些病毒（包括诱发宫颈癌的人类病原体）具有在组装病毒微型染色体时从宿主细胞中获取组蛋白的独特特性。 2008 财年，我们启动了一个项目，通过冷冻电子断层扫描分析衣壳化的 SV40 微型染色体的结构。在从几次高质量重建中提取的一组 100 个重建的完整病毒粒子中，我们发现它们包含的核小体数量从 17 到 23 不等，并且它们的空间排列与衣壳的二十面体对称性不匹配。 此外，对提取的染色质核心密度的分析表明，整体染色质结构因颗粒而异。 正在进行的工作旨在对单个粒子重建中核小体的位置和方向进行计算建模。