Structural Biology Of Virus Assembly

病毒组装的结构生物学

• 批准号: 8559281
• 负责人: ALASDAIR C. STEVEN
• 金额: $ 141.17万
• 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/8559281
• 关键词: ATP phosphohydrolase; Accounting; Adenoviruses; Allosteric Regulation; Antibodies; Antigenic Diversity; Antigens; Antiviral Agents; Bacteriophages; Binding; Binding Sites; Biological Models; Biological Process; Capsid; Capsid Proteins; Cells; Complex; Computing Methodologies; Cryoelectron Microscopy; DNA; DNA Packaging; Dengue Virus; Dimerization; Disease Progression; Dose; Double Stranded RNA Virus; Electrons; Endocytosis; Endosomes; Epitopes; Exhibits; Genetic Transcription; Genome; Goals; Heating; Hemagglutinin; Hepatitis; Hepatitis B Virus; Hepatitis B e Antigens; Herpesviridae; Herpesvirus 1; Human; Image; In Vitro; Individual; Infection; Investigation; Left; Lipoprotein (a); Liver; Location; Macromolecular Complexes; Maps; Mass Spectrum Analysis; Mediating; Membrane; Minor; Modeling; Molecular; Molecular Models; Monoclonal Antibodies; Morphology; Mus; Newcastle disease virus; Nucleocapsid; Pathway interactions; Patients; Play; Polymerase; Positioning Attribute; Process; Property; Proteins; Pseudomonas aeruginosa; Publications; Publishing; RNA; Recruitment Activity; Regulation; Reporting; Research; Resolution; Ribonucleoproteins; Role; Rotation; Rubella virus; Shapes; Side; Site; Sodium Chloride; Specificity; Staging; Structure; Surface; System; Tail; Time; Viral; Viral Genome; Virion; Virus; Virus Assembly; Work; base; density; dimer; electron tomography; flexibility; image reconstruction; influenzavirus; insight; interest; irradiation; molecular modeling; monomer; novel; particle; pathogen; pathogenic bacteria; response; scaffold; structural biology; three dimensional structure

During FY12, we focussed mainly on the following four subprojects: (1) Hepatitis B Virus Capsid Assembly. We study the HBV capsid protein which presents two of the three clinically important antigens - core antigen (cAg, capsids) and e-antigen (eAg, unassembled protein) - of this major human pathogen. After first showing that capsid protein self-assembles from dimers into shells of two different sizes, we obtained, in 1997, a cryo-EM density map in which we visualized the 4-helix bundle that forms the dimerization motif. This was the first time that such detailed information had been achieved by cryo-EM. We went on to investigate the antigenic diversity of HBV by using cryo-electron microscopy (EM) to characterize the conformational epitopes of seven monoclonal antibodies by cryo-EM and molecular modeling. In FY12, we completed and published two projects mentioned in last year's report. In one (2), we sought to relate our previous work with murine monoclonals to the immunological response of infected humans by investigating the binding of polyclonal anti-cAg antibodies from a patient. The observed Fab-related density could be reproduced by modeling with just five Fabs whose locations match those of the murine monoclonals previously studied. These results validate the mouse as a model system. We also investigated the reactivity of an antibody found in copious quantities in the livers of patients suffering from fulminant hepatitis and found that this antibody binds tangentially to a novel site on the side of the spike. These results support the idea that antibodies with particular specificities may correlate with different stages of disease progression. We also investigated the properties of eAg), which differs from cAg in having at its N-terminus an additional ten residues, a remnant of its propeptide. eAg and cAg are antigenically distinct but are cross-reactive (1). One eAg-specific Fab forms a stable complex with eAg that yielded crystals that diffract to 0.33 nm resolution. In the resulting structure, the eAg monomer is seen to have a similar fold to the cAg monomer but an entirely different mode of dimerization, related by a rotation of 140 degrees. This switch accounts for the profound differences in assembly properties and antigenicity between the two proteins. These results have been submitted for publication. (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, reflecting common evolutionary origins. In FY12, we pursued the following investigations. (2a) Regulation of genome packaging. The capsids of double-stranded RNA viruses serve as 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 reported the location of the P2 polymerase inside the viral procapsid. In FY10, we completed a study using cryo-electron tomography to map the distributions of P2-occupied sites and of the external sites occupied by P4, the packaging ATPase. In FY11, we investigated the expansion transformation undergone by the procapsid during RNA packaging. To investigate procapsid transformation, we induced expansion in vitro by acidification, heating, and elevated salt concentration. The results identify two structural intermediates between the procapsid and the mature capsid (7). Phi6 has an additional protein component, P7, which functions as a packaging accessory protein with involvement also in assembly. However, its location has been unknown. In FY12, we used cryo-EM to localize P7 by difference mapping between procapsids with different compositions (8). We found that P7 resides on the interior surface of the procapsid at sites that overlap those of P2, indicating competition between these two proteins and implying that substoichiometric quantities of both are sufficient to fulfil their biological functions. The P7 binding sites are arranged around the three-fold axes, suggesting that the protein promotes assembly by stabilizing an initiation complex. (2b) PhiKZ is a large and complex virus that infects the pathogenic bacterium Pseudomonas aeruginosa. The virion has a large icosahedral capsid containing densely packed DNA (280kbp) as well as a proteinaceous "inner body" which is invisible in cryo-electron micrographs because of contrast-matching with the surrounding DNA. We found, serendipitously, that the inner body is exceptionally sensitive to electron irradiation and explodes into bubbles of gaseous products at doses that leave the surrounding capsid only slightly blurred. We developed a computational method of analyzing these bubblegrams to locate the inner body in individual nucleocapsids and then to determine its structure (5). The inner body is 24 nm wide, 105 nm long, and consists of multiple stacked tiers with 6-fold symmetry. Mass spectrometry and SDS-PAGE indicate that the inner body has five major proteins, present in 100-200 copies each, as well as a number of minor proteins (6). The shape and position of the inner body suggest that it plays a role of organizer in the DNA packaging process. A working hypothesis is that inner body proteins are injected into the host cell along with the DNA where they fulfil functions needed early in infection. (3) Herpesviruses have an icosahedral nucleocapsid surrounded by an amorphous tegument and a lipoprotein envelope (3). The tegument comprises at least 20 proteins destined for delivery into the host cell. As the tegument does not have a regular structure, the question arises of how its proteins are recruited. The HSV-1 tegument is known to contact the capsid at its vertices and two proteins, UL36 and UL37, had been identified as candidates for this interaction. We showed by cryo-EM that capsids with and without UL37 exhibit a vertex-associated density that represents the ordered portion of UL36 (336 kDa).These observations (4) support the hypothesis that UL36 provides a flexible scaffold to which other tegument proteins, including UL37, bind. They also indicate how sequential conformational changes in the maturing nucleocapsid control the ordered binding of UL36 and UL37. (4) While much of the research described above was performed with icoshedral capsids which may be imaged by high-resolution image reconstruction, many viruses do not conform to this symmetry. Nevertheless, their three-dimensional structures may be studied by cryo-electron tomography. We published the first such analysis of a pleiomorphic virus - herpes simplex virus type 1 - in 2003 and have gone on to make numerous other applications. In addition to completing studies of Rubella virus (13), Newcastle disease virus (12), immature adenovirus (15), and maturing Dengue virus (14), in FY12 we studied how influenza virus responds to acidic pH (9). Influenza virus enters host cells by endocytosis. The low pH of endosomes triggers conformational changes in hemagglutinin (HA) that mediate fusion of the viral and endosomal membranes. At pH 4.9, we observed dramatic changes in morphology: elongated particles were no longer observed; larger particles representing fused virions appeared; the HA spikes became conspicuously disorganized; a layer of M1 matrix protein was no longer resolved on most virions; and the ribonucleoprotein complexes (RNPs) coagulated on the interior surface of the virion. These observations have illuminated the cell entry pathway of influenza virus.

2012 财年，我们主要关注以下四个子项目： (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（cAg，衣壳）和 e 抗原（eAg，未组装蛋白）。在首次证明衣壳蛋白从二聚体自组装成两种不同大小的壳后，我们于 1997 年获得了冷冻电镜密度图，其中我们可视化了形成二聚化基序的 4 螺旋束。这是第一次通过冷冻电镜获得如此详细的信息。我们继续使用冷冻电镜 (EM) 来研究 HBV 的抗原多样性，并通过冷冻电镜和分子建模来表征七种单克隆抗体的构象表位。 2012 财年，我们完成并发布了去年报告中提到的两个项目。在一 (2) 中，我们试图通过研究来自患者的多克隆抗 cAg 抗体的结合，将我们之前对鼠单克隆抗体的研究与受感染人类的​​免疫反应联系起来。观察到的 Fab 相关密度可以通过仅使用 5 个 Fab 进行建模来重现，这些 Fab 的位置与之前研究的鼠单克隆抗体的位置相匹配。这些结果验证了鼠标作为模型系统的有效性。我们还研究了在患有暴发性肝炎的患者肝脏中发现的大量抗体的反应性，发现这种抗体与尖峰一侧的新位点切向结合。这些结果支持这样的观点：具有特定特异性的抗体可能与疾病进展的不同阶段相关。 我们还研究了 eAg 的特性，它与 cAg 的不同之处在于其 N 末端有一个额外的 10 个残基，即其前肽的残余物。 eAg 和 cAg 具有不同的抗原性，但具有交叉反应性 (1)。一种 eAg 特异性 Fab 与 eAg 形成稳定的复合物，产生衍射分辨率为 0.33 nm 的晶体。在所得结构中，eAg 单体与 cAg 单体具有相似的折叠，但二聚化模式完全不同，通过 140 度的旋转相关。这种转变解释了两种蛋白质之间组装特性和抗原性的巨大差异。这些结果已提交发表。 (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。有尾噬菌体为疱疹病毒衣壳提供了一个极好的模型，反映了共同的进化起源。 2012 财年，我们进行了以下调查。 (2a)基因组包装的调节。双链RNA病毒的衣壳充当病毒基因组复制和转录的区室。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。 2008 财年，我们报道了 P2 聚合酶在病毒衣壳内的位置。 2010 财年，我们完成了一项研究，使用冷冻电子断层扫描来绘制 P2 占据位点和 P4（包装 ATP 酶）占据的外部位点的分布图。 2011 财年，我们研究了 RNA 包装过程中原衣壳所经历的扩增转化。 为了研究衣壳转化，我们通过酸化、加热和升高盐浓度在体外诱导扩增。 结果确定了衣壳和成熟衣壳之间的两个结构中间体 (7)。 Phi6 有一个额外的蛋白质成分 P7，它充当包装辅助蛋白，也参与组装。然而，它的位置一直未知。在 2012 财年，我们使用冷冻电镜通过不同成分的原衣壳之间的差异图谱来定位 P7 (8)。我们发现 P7 位于衣壳内表面上与 P2 重叠的位点上，表明这两种蛋白质之间存在竞争，并意味着两者的亚化学计量足以实现其生物学功能。 P7 结合位点围绕三重轴排列，表明该蛋白质通过稳定起始复合物来促进组装。 (2b) PhiKZ 是一种大型且复杂的病毒，可感染致病菌铜绿假单胞菌。该病毒粒子有一个巨大的二十面体衣壳，其中含有密集的 DNA (280kbp) 以及一个蛋白质“内部主体”，由于与周围 DNA 的对比度匹配，在冷冻电子显微照片中看不见该蛋白质“内部主体”。我们偶然发现，内部身体对电子辐射异常敏感，并在电子辐射剂量下爆炸成气态产物气泡，使周围的衣壳仅略微模糊。我们开发了一种计算方法来分析这些气泡图，以定位单个核衣壳的内部主体，然后确定其结构 (5)。内部主体宽 24 nm，长 105 nm，由多个六重对称的堆叠层组成。质谱分析和 SDS-PAGE 表明体内有 5 种主要蛋白质，每种蛋白质有 100-200 个拷贝，以及许多次要蛋白质 (6)。内部身体的形状和位置表明它在DNA包装过程中起着组织者的作用。一个可行的假设是，体内蛋白质与 DNA 一起被注入宿主细胞，在那里它们实现感染早期所需的功能。 (3) 疱疹病毒具有被无定形外皮和脂蛋白包膜包围的二十面体核衣壳 (3)。被膜包含至少 20 种用于递送至宿主细胞中的蛋白质。由于外皮没有规则的结构，因此出现了其蛋白质如何招募的问题。已知 HSV-1 外皮在其顶点接触衣壳，并且两种蛋白质 UL36 和 UL37 已被确定为这种相互作用的候选蛋白。我们通过冷冻电镜证明，带有和不带有 UL37 的衣壳表现出与顶点相关的密度，代表 UL36 的有序部分 (336 kDa)。这些观察结果 (4) 支持以下假设：UL36 提供了一个灵活的支架，其他皮层蛋白可以为该支架提供柔性支架。包括 UL37、绑定。它们还表明成熟核衣壳中的连续构象变化如何控制 UL36 和 UL37 的有序结合。 (4) 虽然上述大部分研究是用二十面体衣壳进行的，可以通过高分辨率图像重建来成像，但许多病毒不符合这种对称性。然而，它们的三维结构可以通过冷冻电子断层扫描来研究。我们于 2003 年首次发表了对多形性病毒（1 型单纯疱疹病毒）的此类分析，并继续进行了许多其他应用。除了完成风疹病毒 (13)、新城疫病毒 (12)、未成熟腺病毒 (15) 和成熟登革热病毒 (14) 的研究外，2012 财年我们还研究了流感病毒如何响应酸性 pH 值 (9)。 流感病毒通过内吞作用进入宿主细胞。内体的低 pH 会引发血凝素 (HA) 的构象变化，从而介导病毒和内体膜的融合。在 pH 4.9 时，我们观察到形态的巨大变化：不再观察到拉长的颗粒；代表融合病毒颗粒的较大颗粒出现； HA 尖峰变得明显混乱；大多数病毒体上不再解析 M1 基质蛋白层；核糖核蛋白复合物（RNP）凝结在病毒体的内表面。这些观察结果阐明了流感病毒进入细胞的途径。