Structural Biology Of Virus Assembly

病毒组装的结构生物学

• 批准号: 8157127
• 负责人: ALASDAIR C. STEVEN
• 金额: $ 241.15万
• 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/8157127
• 关键词: Virus Assembly; structural biology

During FY10, we focussed on three subprojects relating respectively to hepatitis B virus, bacteriophage HK97, and bacteriophage phi6. (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 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. 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 FY10, we followed three main lines of investigation. (i) We completed and published a study 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-antigen and e-antigen, including several whose epitopes we previously identified 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) We followed 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%. Specifically, we exploited this high mass discrimination to measure the rates at which both capsids exchange dimers with the unassembled pool, and found this rate to be slow but significant (10% exchange in 3 months) for the smaller T=3 capsid and undetectably slow for the T=4 capsid. (iii) We also 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) and their mechanical properties can be successfully modelled using both finite element and molecular dynamics formalisms. (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 FY10, we focussed mainly on two projects. (2a) All genomes cycle between a condensed state assumed during replication of the organism and a decondensed states assumed during gene expression, etc. The encapsidated state of phage DNA represents an extreme case of genome condensation. We used a combination of scanning calorimetry and cryo-EM to investigate this phenomenon in the phage HK97 system which is particularly well suited for study 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. (Previously, we detected a similar effect in herpesvirus capsids). 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 was completed and published in FY10. (2b) 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 FY10 we completed and published an extension of these studies by using cryo-electron tomography to map the internal sites occupied by P2 and the external sites occupied by P4, the packaging ATPase. The observed distributions disclosed 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. Our ongoing effort on this system targets the maturational expansion of the procapsid which has been hypothesized to proceed sequentially according to the successive packagings of the three segments of genomic RNA. We are attempting to induce the corresponding transitions in vitro by exposing procapsids to differing solution conditions (pH, ionic strength, temperature, etc). To date, we have succeeded in identifying and characterizing one intermediate state.

在2010财年，我们专注于三个子项目，分别与乙肝病毒、噬菌体HK97和噬菌体phi6有关。 (1)乙肝病毒衣壳组装体。我们研究了乙肝病毒衣壳蛋白，它代表了这一人类主要病原体的三个临床重要抗原中的两个-核心抗原(衣壳)和e抗原(未组装蛋白)。在第一次证明衣壳蛋白从二聚体自我组装成两个不同大小的壳之后，我们在1997年获得了一张冷冻EM密度图，在其中我们可视化了形成二聚化基序的4-螺旋束。这是冷冻-EM首次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。接着，我们用低温电子显微镜研究了七种不同的抗衣壳蛋白单抗的构象表位，从而研究了乙肝病毒的抗原多样性。在2010财年，我们遵循了三条主要调查路线。(I)我们完成并发表了一项研究，其中使用表面等离子激元共振来测量一组通常用于区分核心抗原和e抗原的鼠单抗的结合亲和力，其中包括几个我们先前通过Cryo-EM确定其表位的抗体。出乎意料的是，大多数抗体与这两种抗原都有很高的亲和力。例外的是抗体e6，它检测只能在二聚体上访问并封闭在衣壳上的表位，以及抗体3120，它检测只存在于衣壳上的表位，因为它的表位跨越二聚体间的界面。(Ii)我们跟进了2008财年报道的“本地”高分辨率质谱学实验，在这些实验中，衣壳的两种大小变体的质量被确定为在0.1%以内。具体地说，我们利用这种高质量分辨率来测量两个衣壳与未组装的池交换二聚体的速率，发现对于较小的T=3衣壳，这个速率很慢但很显著(3个月内交换10%)，对于T=4衣壳，这个速率慢得难以检测。(Iii)我们还开展了一个合作项目，通过原子力显微镜进行的纳米压痕方法来测量两个衣壳的机械性能。这两种衣壳具有相似的整体稳定性和弹性(杨氏模数为0.4 Gpa)，其力学性能可以成功地用有限元和分子动力学公式建模。 (2)噬菌体衣壳的组装和成熟。我们对衣壳组装的兴趣在于伴随着它们成熟的大量构象变化。这些转变为变构调控提供了独特的见解。我们研究了几个噬菌体的成熟，以开发每个系统的有利方面。尾巴噬菌体为疱疹病毒衣壳提供了一个很好的模型，反映了共同的进化起源。在2010财年，我们主要关注两个项目。 (2a)所有基因组都在生物体复制过程中呈现的浓缩状态和基因表达过程中呈现的解浓缩状态之间循环。噬菌体DNA的包裹状态代表了基因组浓缩的极端情况。我们使用扫描量热法和冷冻EM相结合的方法来研究噬菌体HK97系统中的这种现象，鉴于对其衣壳结构的详细了解，该系统特别适合研究。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到50度)下释放。加热会增加内部压力，导致衣壳破裂，释放DNA。DNA包装还导致衣壳结构的变化，这既反映在比空衣壳更早的热变性开始，也反映在细微的形态差异上。(此前，我们在疱疹病毒衣壳中检测到了类似的效果)。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。该项目已于2010财年完成并发布。 (2B)双链RNA病毒的衣壳是病毒基因组复制和转录的专门隔间。我们研究了噬菌体phi6系统中这种显著现象的结构基础，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P2聚合酶在病毒前衣壳内部的位置，这是通过野生型和突变颗粒的冷冻-EM确定的。P2是亚化学计量比的，只占据20个潜在位点中的3-10个(取决于突变体)。在2010财年，我们完成并发表了这些研究的扩展，使用冷冻电子断层扫描绘制了P2占据的内部部位和P4占据的外部部位，包装ATPase。观察到的分布表明，这两种蛋白质是随机分布的，因此这两种病毒酶的活性之间不存在直接耦合，这两种酶分别进行RNA包装、复制和转录。我们在这个系统上正在进行的努力以原衣壳的成熟扩张为目标，该扩张已被假设为根据基因组RNA的三个片段的连续包装而顺序进行。我们正试图在体外通过将Proapsids暴露在不同的溶液条件(pH、离子强度、温度等)中来诱导相应的转变。到目前为止，我们已经成功地识别和描述了一个中间状态。