Structural Biology Of Virus Assembly

病毒组装的结构生物学

• 批准号: 8746487
• 负责人: ALASDAIR C. STEVEN
• 金额: $ 136.36万
• 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/8746487
• 关键词: ATP phosphohydrolase; Accounting; Affect; Allosteric Regulation; Antibodies; Antigenic Diversity; Antigens; Antiviral Agents; Bacteriophage T7; Bacteriophages; Binding; Biochemical; Breathing; Capsid; Capsid Proteins; Cells; Complex; Coupling; Cryoelectron Microscopy; Crystallography; Cystovirus; DNA; DNA Packaging; Deuterium; Dimerization; Distant; Dose; Double Stranded RNA Virus; Electrons; Endosomes; Epitopes; Exhibits; Floor; Genetic Transcription; Genome; Genomics; Goals; Hemagglutinin; Hepatitis B Virus; Hepatitis B e Antigens; Herpesviridae; Herpesvirus 1; Human; Hydrogen; Image; Infection; Injection of therapeutic agent; Investigation; Left; Lipoprotein (a); Macromolecular Complexes; Maps; Mass Spectrum Analysis; Mediating; Membrane; Membrane Fusion; Minor; Modeling; Molecular; Molecular Conformation; Molecular Machines; Molecular Models; Monoclonal Antibodies; Morphology; Motion; Newcastle disease virus; Nucleocapsid; Patients; Play; Pliability; Polymerase; Positioning Attribute; Process; Property; Proteins; Pseudomonas aeruginosa; Publishing; RNA; Recruitment Activity; Research; Resolution; Role; Rotation; Rubella virus; Sampling; Shapes; Site; Structure; Surface; System; Tail; Techniques; Time; Trapezoid bone structure; Viral; Viral Genome; Virion; Virus; Virus Assembly; base; conformer; density; dimer; ds-DNA; electron tomography; flexibility; influenzavirus; insight; interest; irradiation; molecular modeling; monomer; novel; particle; pathogen; pathogenic bacteria; scaffold; structural biology; uptake

During FY12, we focused 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 was achieved by cryo-EM. We went on to investigate the antigenic diversity of HBV by using cryo-EM and molecular modeling to characterize the epitopes of a total of eight monoclonal antibodies. These studies indicated, on statistical sampling grounds, that the total number of distinct epitopes is in the range of 15-20. They lie in two regions on the capsid surface: on the protruding spikes and on the low-lying 'floor' region (15). Similar analyses of polyclonal Fabs from a human patient indicated the presence of epitopes in both regions. We also investigated by x-ray crystallography the structure of the e-antigen which differs from cAg in having at its N-terminus an additional ten residues (1). eAg and cAg are antigenically distinct but are cross-reactive. The eAg structure revealed a monomer with a similar fold to the cAg monomer but an entirely different mode of dimerization. This switch accounts for the profound differences in assembly properties and antigenicity between the two proteins. These results were published in FY13 (11). We also completed and published a study in which native mass spectrometry was combined with hydrogen-deuterium exchange to investigate the effects on HBV capsids of binding two antibodies. By focusing on the capsid protein, we were able to acquire deuterium uptake profiles covering its entire 149-residue sequence and to reveal, in localized detail, the changes in H/D exchange rates that accompanied antibody binding. We observed protection of two epitopes upon Fab binding, but also found that regions distant from the epitopes are also affected, i.e allosterically transmitted effects on conformation. Even at sub-stoichiometric Fab binding, an overall increase in the rigidity of the capsid structure was detected, as well as a general damping of the breathing motions of the capsid (10). (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 FY13, we pursued the following investigations. (2a) Coupling of genome packaging to capsid maturation in the cystovirus system. 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 for phage phi6, which has a tripartite genome. In FY08, we located 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 and identified two structural intermediates. In FY12, we located the P7 packaging facilitator, which turned out to compete with the P2 polymerase for sites on the interior surface of the capsid (5). In FY13, we determined the crystal structure of the capsid protein P1, revealing a flattened trapezoid subunit with a novel alfa-helical fold (14). We also solved the procapsid by cryo-electron microscopy to 0.45 nm resolution. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation which correlates with sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine. (2b) Packaging and injection of internal proteins. Infection by double-stranded DNA phages involves the injection of proteins as well as genomic DNA into the host cell. 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 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. In conventional cryo-EM, internal proteins are hard to detect because they are contrast-matched with the DNA, but they can be visualized by bubblegram imaging (3). This technique revealed that that the inner body is 24 nm wide, 105 nm long, and consists of stacked tiers with 6-fold symmetry. Biochemical analysis indicated that the inner body has five major proteins as well as a number of minor proteins (4). The shape and position of the inner body suggest that it plays a role of organizer in the DNA packaging process. We then went on to apply the same approach to define the corresponding structure in the genetically better defined T7 coliphage (work in progress). (3) Herpesviruses have an icosahedral nucleocapsid surrounded by an amorphous tegument and a lipoprotein envelope. 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 (2) 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 icosahedral capsids which may be imaged by high-resolution cryo-EM, many viruses do not conform to icosahedral symmetry. Nevertheless, their 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 FY12, we completed such studies of Rubella virus (8) and Newcastle disease virus (7). In FY12 we studied how influenza virus responds to acidic pH (9). 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 (6); and larger particles representing fused virions appeared to shed their layer of matrix protein, which we infer makes the particle more pliable and conducive to membrane fusion. In a more detailed analysis we also found that shedding is preceded by a conformational change in M1 protein (12).

在2012财年期间，我们主要关注以下四个子标题： （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。我们继续使用低温EM和分子建模来研究HBV的抗原多样性，以表征总共八种单克隆抗体的表位。这些研究表明，从统计抽样的角度来看，不同表位的总数在15-20范围内。它们位于衣壳表面上的两个区域：在突出的尖峰和低洼的“地板”区域上（15）。对人类患者的多克隆Fab的类似分析表明，这两个区域都有表位。我们还通过X射线晶体学研究了E-Antigen的结构，该结构与CAG不同，在其N末端还有十个残基（1）。 EAG和CAG在抗原上是不同的，但具有交叉反应性。 EAG结构揭示了与CAG单体相似的单体，但二聚化模式完全不同。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。这些结果发表在2013财年（11）中。我们还完成并发表了一项研究，其中将天然质谱与氢 - 占率交换结合在一起，以研究对两种结合两种抗体的HBV衣壳的影响。通过专注于衣壳蛋白，我们能够获取覆盖其整个149个占用序列的氘吸收曲线，并以局部细节揭示伴随抗体结合的H/D汇率的变化。我们观察到在FAB结合时保护了两个表位，但也发现远离表位的区域也受到影响，即对构象的变构透射作用。即使在亚化学计量的FAB结合下，也检测到衣壳结构的刚度的总体增加，以及帽骨的呼吸运动的一般阻尼（10）。 （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2013财年，我们进行了以下调查。 （2a）基因组包装与膀胱病毒系统中衣壳成熟的偶联。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的这种非凡现象的结构基础。在08财年，我们在病毒procapsid内找到了P2聚合酶。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Procapsid的扩张转换，并确定了两个结构中间体。在2012财年，我们找到了P7包装促进剂，该促进剂与CAPSID内部表面上的位点的P2聚合酶竞争（5）。在2013财年，我们确定了衣壳蛋白P1的晶体结构，揭示了具有新颖的阿尔法螺旋折叠的扁平梯形亚基（14）。我们还通过冷冻电子显微镜求解了procapsid，以分辨率为0.45 nm。将晶体结构拟合到Procapsid中揭示了两个P1构象体之间的实质构象差异。除了巨大的刚体旋转外，通过两个中间状态通过两个中间状态的成熟涉及重塑。衣壳结构及其逐步成熟与三个RNA段的顺序包装相关的逐步成熟，将半胱氨酸病毒与其他DSRNA病毒分为动态分子机。 （2B）包装和注射内部蛋白质。双链DNA噬菌体感染涉及蛋白质以及基因组DNA的注射到宿主细胞中。 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。我们发现，内体对电子辐射非常敏感，并以剂量​​的剂量爆炸到气态产物的气泡中，这些剂量使周围的衣壳仅略微模糊。在常规的冷冻EM中，由于它们与DNA匹配，因此很难检测到内部蛋白质，但可以通过泡泡糖成像来可视化它们（3）。该技术表明，内体宽24 nm，长105 nm，由堆叠的层组成6倍对称性。生化分析表明，内体具有五种主要蛋白质以及许多次要蛋白质（4）。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。然后，我们继续采用相同的方法来定义遗传上定义的T7 Coliphage（正在进行的工作）中的相应结构。 （3）疱疹病毒的二十面膜核包皮被无定形的te和脂蛋白包膜包围。 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（2）支持UL36为包括UL37（包括Ul37）结合的其他Tegument Proteins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冷冻EM成像的二十面体capsids进行的，但许多病毒不符合二十面体对称性。然而，它们的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。在2012财年，我们完成了风疹病毒（8）和纽卡斯尔病毒（7）的研究。在2012财年，我们研究了流感病毒对酸性pH的反应（9）。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（6）；代表融合病毒体的较大颗粒似乎脱落了其基质蛋白层，我们推断出这使颗粒更加柔韧和有利于膜融合。在更详细的分析中，我们还发现脱落之前是M1蛋白的构象变化（12）。