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.

在2012财年期间，我们主要关注以下四个子项目： （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重要抗原（CAG，CAPSID）和E-抗原（EAG，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，在该图中，我们可视化形成二聚体基序的4螺旋束。这是Cryo-Em首次获得此类详细信息。我们继续使用冷冻电子显微镜（EM）来研究HBV的抗原多样性，以通过冷冻EM和分子建模来表征7种单克隆抗体的构象表位。在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）个中，我们试图通过研究患者的多克隆抗CAG抗体的结合，将我们先前与鼠单克隆的作品与感染人类的​​免疫反应联系起来。观察到的与Fab相关的密度可以通过仅使用五个Fab进行建模，其位置与先前研究的鼠单克隆群体的位置相匹配。这些结果将鼠标验证为模型系统。我们还研究了患有暴发性肝炎的患者肝脏中大量抗体发现的抗体的反应性，发现该抗体与尖峰侧面的新部位切线结合。这些结果支持这样一种观念，即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的性质），这与CAG不同，在其N末端还有另外十个残基，这是其丙肽的残留物。 EAG和CAG在抗原上是不同的，但具有交叉反应性（1）。一个EAG特异性的Fab形成了具有EAG的稳定复合物，其产生衍射为0.33 nm的晶体。在所得的结构中，EAG单体被认为具有与CAG单体相似的折叠，但二聚化模式完全不同，与140度的旋转相关。该转换说明了两种蛋白质之间的组装特性和抗原性的深刻差异。这些结果已提交出版。 （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，反映了常见的进化起源。在2012财年，我们进行了以下调查。 （2a）基因组包装的调节。双链RNA病毒的衣壳是病毒基因组复制和转录的隔室。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，我们报告了病毒procapsid中P2聚合酶的位置。在FY10中，我们完成了一项研究使用冷冻电子断层扫描的研究，以绘制包装ATPase P4占据的P2占地位点和外部位点的分布。在FY11中，我们研究了RNA包装期间的Procapsid进行的扩展转换。 为了研究procapsid转化，我们通过酸化，加热和盐浓度诱导体外诱导扩张。 结果确定了procapsid和成熟的衣壳之间的两个结构中间体（7）。 PHI6具有额外的蛋白质成分P7，该蛋白质成分充当包装辅助蛋白，并且也参与了组装。但是，它的位置尚不清楚。在2012财年，我们使用冷冻EM来定位P7，通过具有不同组成的procapsids之间的差图（8）。我们发现，P7位于与P2重叠的位点的procapsid的内部表面上，表明这两种蛋白质之间的竞争，并表明两者的化学计量量足以实现其生物学功能。 P7结合位点在三倍轴周围排列，表明该蛋白质通过稳定起始复合物来促进组装。 （2B）Phikz是一种大而复杂的病毒，可感染铜绿假单胞菌的致病细菌。该病毒粒子具有大型二十二个胶囊，其中包含密度堆积的DNA（280kbp）以及一个蛋白质的“内体”，由于与周围DNA的对比匹配，因此在低温电子显微照片中是看不见的。我们偶然发现，内体对电子辐射非常敏感，并以剂量​​的剂量爆炸成气泡的气泡，而周围的衣壳仅略微模糊。我们开发了一种计算方法，用于分析这些气泡图，以在单个核素中定位内体，然后确定其结构（5）。内体宽24 nm，长105 nm，由多个带有6倍对称性的堆叠层组成。质谱和SDS-PAGE表明，内体有五种主要蛋白质，每个蛋白质都有100-200份，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包装过程中起了组织者的作用。一个有效的假设是，内体蛋白与DNA一起注射到宿主细胞中，它们在感染早期所需的功能。 （3）疱疹病毒的二十面体核素被无定形的te和脂蛋白包膜包围（3）。 Tegument至少包含20个注定要输送到宿主细胞的蛋白质。由于Tegument没有规律的结构，因此出现了其蛋白质的募集方式。已知HSV-1 Tegument在其顶点上与Capsid接触Capsid，并且已将两种蛋白质UL36和UL37鉴定为这种相互作用的候选者。我们通过冷冻EM证明，带有和没有UL37的衣壳表现出与顶点相关的密度，代表了UL36的有序部分（336 kDa）。这些观察结果（4）支持UL36为包括UL37（包括UL37）结合的其他Tegument蛋白提供的灵活支架。它们还表明了成熟核素的顺序构象如何控制UL36和UL37的有序结合。 （4）虽然上述许多研究是用高分辨率图像重建可能成像的IcosheDral Capsids进行的，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研究。我们在2003年发表了对多形病毒 - 单纯形病毒1型疱疹病毒的首次分析，并继续进行了许多其他应用。除了完成对风疹病毒的研究（13），纽卡斯尔病毒病毒（12），未成熟腺病毒（15）和登记登革热病毒（14）之外，我们研究了流感病毒对酸性pH的反应（9）。 流感病毒通过内吞作用进入宿主细胞。内体的低pH值触发介导病毒和内体膜融合的血凝素（HA）的构象变化。在pH 4.9时，我们观察到形态学的急剧变化：不再观察到伸长的颗粒；出现了代表融合病毒体的较大颗粒； HA尖峰变得明显混乱。在大多数病毒体上不再解决一层M1基质蛋白。核糖核蛋白复合物（RNP）凝结在病毒座的内部表面。这些观察结果阐明了流感病毒的细胞进入途径。