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.

在08财年期间，我们专注于三个副标题。 （1）丙型肝炎病毒衣壳组件。我们研究了这种主要人类病原体的三种临床上重要抗原（Capsids）和E-抗原（未组装蛋白）中的三种HBV衣壳蛋白。在首次表明从二聚体变成两个不同尺寸的衣壳的衣壳蛋白的自组合后，我们于1997年获得了一个冷冻EM密度图，其中大部分二级结构都是可见的，其中包括形成二聚体基序的4个螺旋束。这是Cryo-Em首次获得此类详细信息。我们随后的研究有助于描绘多肽链的路径。我们继续使用冷冻EM来表征HBV的抗原多样性，以表征针对衣壳的七种不同单克隆抗体的构象表位。在09财年，我们遵循了三条调查。 （i）我们完成了一个在08财年报告的项目，其中使用表面等离子体共振来测量一组通常用于区分核心和e-antigen的鼠单克隆抗体的结合亲和力，其中包括几种以前以冷冻em为特征的。 出乎意料的是，大多数抗体与两种具有高亲和力的抗原结合。例外是抗体E6，它检测仅在二聚体上可访问的表位并在衣壳上闭塞，而抗体3120检测仅在帽壳上显示的表位，因为其表位横跨二聚体之间的二聚体界面。 (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.与期望相反，这些多克隆晶圆厂没有在衣壳上形成连续的树冠，而是优先与两个谨慎的位点结合。一个位于尖峰状的突起（由所谓的免疫循环占据的位置），在这些突起之间的区域中一个。这些数据表明，人IgG曲目实际上检测到一组相当有限的表位，与我们先前观察到的鼠单克隆抗体相似的表位。 （iii）跟随08财年报告的“天然”高分辨率质谱实验，其中确定CapSID的两个大小变体的质量在0.1％以内，我们追求了一个协作项目，以测量通过原子力显微镜通过纳米构造方法携带的两种Capsids的机械性能。 这两个衣壳具有相似的总体稳定性和弹性（Young的模量为0.4 GPA），但其亚基汇率率有所不同，这取决于用同位素标记的亚基孵育的衣壳的质谱法确定。通过粗粒分子动力学模拟预测衣壳弹性的定向依赖性，并与纳米注册数据进行了比较。 （2）噬菌体衣壳的组装和成熟。我们对Capsid组装的兴趣在于它们成熟的巨大构象变化。这些过渡提供了对变构调节的独特见解。我们研究了几个噬菌体的成熟，以利用每个系统的权宜方面。尾巴噬菌体为疱疹病毒衣壳提供了出色的模型（请参见上面的（2）），反映了常见的进化起源。在08财年，我们专注于三个项目。 （i）我们使用扫描量热法和冷冻EM的组合来研究噬菌体DNA的封装，这代表了基因组凝结的极端情况。据详细了解其衣壳结构，噬菌体HK97非常适合研究这种现象。 我们发现，由于填充的衣壳被加热，其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包装还诱导了衣壳结构的变化，而胶囊结构既反映在热变性的早期发作中，而不是空的衣壳和微妙的形态差异。 （我们还检测到在疱疹病毒衣壳中也有类似的作用 - 请参见上面的（2））。我们设想，Capsid壳中的这种过渡已传输到门户，从而改变了其与包装酶的相互作用，从而发出了包装已完成的信号。该项目在08财年的年度报告中概述并于2009财年发布。 （ii）双链RNA病毒的衣壳充当病毒基因组复制和转录的专门室。我们研究了具有三方基因组的噬菌体Phi6系统中这种非凡现象的结构基础。在08财年，我们发表了一篇论文，描述了P2聚合酶在病毒procapsid内部的位置，该论文由野生型和突变颗粒的冷冻EM确定。 P2是取代的，仅占有20个潜在位点的3-10（取决于突变体）。在09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占用的位点是否与包装ATPase P4占据的外部位点相关的问题。观察到的P2和P4占用率的分布表明，这两种蛋白都是随机分布的，因此这两种病毒酶的活性分别进行RNA包装，复制和转录之间没有直接的耦合。 （iii）对于尾巴和疱疹病毒，脚手架蛋白对于正确的衣壳组件至关重要。脚手架蛋白与衣壳蛋白结合成形，形成前体procapsid，然后在DNA包装期间排出。关于脚手架蛋白如何执行此功能，知之甚少。 我们在噬菌体T7系统中研究了这种现象。 T7 procapsids的质量分析表明，GP9拷贝数平均150，但在粒子之间可以变化30％。 对天然T7 procapsid的检查通过冷冻层析成像显示出一个松散有序的脚手架细丝网络，每个颗粒的细丝多达十个细丝。这些细丝源自门户顶点。 GP9的扩展结构和带正电荷的C末端可能在这些相互作用中起重要作用。 这些观察结果对T7形态发生的含义正在评估中。 （3）乳头瘤病毒和多瘤病毒。这些病毒包括诱导宫颈癌的人类病原体，在组装病毒小浓度小体时，具有从宿主细胞中使用组蛋白蛋白的独特特性。在08财年，我们启动了一个项目，以通过低温电子断层扫描分析封装的SV40微型浓度体的结构。在从几个高质量重建中提取的一组100个重建的全病毒体中，我们发现它们所包含的核小体数量从17到23不等，并且它们的空间排列与capsid的二十面体对称性不符。 此外，对提取的染色质核密度的分析表明，总体染色质结构从粒子到粒子都是可变的。 持续的努力是针对单个粒子重建中核小体的位置和方向的计算建模。