STRUCTURAL BIOLOGY OF VIRUS ASSEMBLY

病毒组装的结构生物学

• 批准号: 6100383
• 负责人: ALASDAIR C. STEVEN
• 金额: --
• 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/6100383
• 关键词: bacteriophage T7; bovine papillomavirus; capsid; coliphages; conformation; crosslink; cryoscience; double stranded RNA; hepatitis B virus group; herpes simplex virus 1; image processing; protein biosynthesis; protein folding; protein structure function; scanning transmission electron microscopy; structural biology; virus DNA; virus RNA; virus assembly; virus protein

We aim to elucidate the molecular mechanisms that control the assembly of viral capsids with the ultimate goals of defining targets for antiviral compounds and of gaining insight into the mechanisms that control the assembly of macromolecular complexes in general. In particular, we focus on the large-scale conformational changes that accompany capsid maturation and interaction of virions with host cells. Our major progress over the past year was as follows: (i) Hepatitis B Virus Capsid ("Core Antigen"). Despite the availability of effective vaccines, HBV remains a public health problem of immense proportions, motivating further efforts to elucidate its replicative cycle. We have been studying its capsid protein (183 amino acids), which we first found to form dimers that are capable of self-assembly into icosahedral particles. These particles are of two different sizes - one with T=3 symmetry (90 dimers; 28nm diameter), the other with T=4 (120 dimers; 34nm diameter). Unlike most other viral capsid proteins, that of HBV was found to be predominantly alfa-helical. In 1997, we calculated a density map from cryo-electron micrographs at the unprecedentedly high resolution of 0.9nm, in which much of the secondary structure was directly visible, including the 4-helix bundle that forms the dimerization motif. Our continuing research has aimed at establishing the precise locations of particular amino acid residues to help delineate the overall path of the polypeptide chain through our density map. Our first success, reported in last year's report and since published, was to localize the C-terminus by gold-cluster labelling. More recently, we have pinpointed a surface loop comprising residues 78-83 by characterizing the binding of Fab fragments of a monoclonal antibody that recognizes this peptide. Localization of the N- terminus was also accomplished by appending an extraneous octapeptide at this site, which we then visualized as additional density in a difference map. We are continuing to extend the resolution of our map of the capsid and to impose more constraints for chain-tracing. (ii) Structure and Tegumentation of the Cytomegalovirus Capsid. Cytomegalovirus (CMV) is a clinically important member of the herpesvirus family, causing infections in immunosuppressed individuals. It is also unresponsive to the antiviral, acyclovir, which is effective against other herpesviruses. In this context, we have been studying cytomegalovirus capsid assembly, using simian CMV as a model for the similar but less tractable human CMV. The main thrust of this project over the past year has been to exploit the opportunity offered by the SCMV system to study capsid-tegument interactions. The tegument is an extensive compartment of proteins situated between the herpesvirus capsid and its envelope. Despite longstanding interest, the molecular architecture and functionale rationale of the tegument have remained obscure. At late times after the infection of cultured fibroblasts with SCMV, tegumented capsids appear in the cytoplasm. By isolating these capsids and comparing their structure and protein composition with those of conventional nuclear capsids, we have characterized the modes of binding of two tegument proteins and identified provisional candidates for them as the "basic phosphoprotein" (119 kDa) and the "upper matrix protein" (69 kDa), respectively. These observations represent the first identification of such linkages for any herpesvirus. (iii) Maturation Dynamics of Bacteriophage HK97. Our primary interest in bacteriophage capsid assembly lies in the monumental conformational changes that accompany maturation of precursor capsids. These changes are irreversible, frequently involve partial refolding of the subunits, and are stringently controlled. As such, they afford unique opportunities for insight into large-scale conformational changes. HK97 represents an advantageous system to study these reactions. The earliest precursor, Prohead I, is converted to Prohead II by proteolysis, then expands to Head I, facilitating covalent cross-linking of Head II, the end-state. Our first study on HK97 compared the 3- dimensional structures of all four particles at a resolution of ~ 2.5 nm. We have since gone on to investigate the dynamic progression of expansion (Prohead II -< Head I) by inducing this event in vitro at low pH, and monitoring subsequent events by time-lapse cryo-electron microscopy. Analysis of the immense volume of data generated in these experiments is proving to be time-consuming but rewarding. Last year, we reported our first results whereby a semi-expanded particle was observed after ~ 30 minutes. We have now characterized intermediates from three later time points and succeeded in obtaining a reconstruction of the spherical "balloon" particle that predominates after 3 hours. Our current account of the acid-induced maturation pathway is that a semi-expanded state is reached rapidly, i.e. within a few minutes; thereafter there is little change in size for ~ 2 hrs, although small changes in capsomer shape continue to take place; then, these intermediates switch into the "balloon" particle; finally, on restoring to neutral pH, the polyhedral Head I state is achieved. A movie has been made of this dynamic process, using the graphical technique of "morphing" to connect experimentally visualized states. (iv) Conformational Changes in Poliovirus upon Interaction with Host Cell. When picornaviruses such as the human pathogen, poliovirus type 1, encounter susceptible cells, the viral capsid binds to a cellular receptor. Ensuing events leading to the intracellular propagation of the infecting virus are not well understood. However, with poliovirus, interaction with its receptor induces a conformational change in the virion, as manifested by a change in its sedimentation coefficient from 160S to 135S. RNA is then released, leaving an 80S capsid. We are investigating the structural basis of these transitions, which may be simulated in vitro by brief heating in an appropriate buffer. The 160S virion has been solved to high resolution by X- ray crystallography, but the 135S and 80S states have not been conducive to such analysis.

我们旨在阐明分子机制 控制病毒式衣壳的组装 定义抗病毒化合物的靶标的 控制大分子组装的机制 一般而言。特别是，我们专注于大规模 伴随着山皮成熟和的构象变化和 病毒体与宿主细胞的相互作用。我们在 过去一年如下：（i）丙型肝炎病毒capsid（“核心 抗原”）。尽管有效疫苗可用，但HBV 仍然是一个巨大比例的公共卫生问题， 激发进一步的努力来阐明其复制周期。我们有 一直在研究其衣壳蛋白（183个氨基酸），我们首先 发现形成能够自组装成的二聚体 二十面体颗粒。这些粒子有两种不同的尺寸 - 一个 t = 3对称（90二聚体；直径28nm），另一个与 t = 4（120二聚体；直径34nm）。与大多数其他病毒式衣壳不同 发现HBV的蛋白质主要是阿尔法螺旋。 1997年，我们计算了一个来自低温电子的密度图 在前所未有的高分辨率为0.9nm的显微照片， 哪些二级结构是直接可见的， 包括形成二聚体基序的4螺旋束。我们的 继续研究旨在建立确切的位置 特定的氨基酸残基有助于描绘整个路径 通过我们的密度图的多肽链的链。我们的第一个成功， 在去年的报告中报道，此后发表，是要本地化 通过金簇标记的C末端。最近，我们有 精确指出了包含残基78-83的表面循环 表征单克隆的Fab片段的结合 识别该肽的抗体。 n-的本地化 终端也通过附加无关 该站点的八肽，然后我们将其视为附加 差异图中的密度。我们正在继续扩展 解决我们的衣壳地图并施加更多约束 用于链条追踪。 （ii）结构和提示 巨细胞病毒衣壳。巨细胞病毒（CMV）是临床上的 疱疹病毒家族的重要成员，引起感染 免疫抑制的人。这也没有反应 抗病毒，阿昔洛韦，可有效防止其他疱疹病毒。 在这种情况下，我们一直在研究巨细胞病毒capsid 汇编，使用Simian CMV作为类似但更少的模型 可处理的人CMV。过去这个项目的主要推力 一年是利用SCMV提供的机会 研究Capsid-tegument相互作用的系统。 tegument是一个 位于疱疹病毒之间的蛋白质的广泛隔室 Capsid及其信封。尽管很感兴趣，但 Tegument的分子体系结构和功能原理 保持晦涩难懂。在感染培养后的后期 带有SCMV的成纤维细胞，temumented Capsids出现在 细胞质。通过隔离这些衣壳并比较它们的结构 和蛋白质成分与常规核衣壳的成分， 我们已经表征了两个tegument的绑定模式 蛋白质并确定了他们的临时候选人为“基本 磷蛋白”（119 kDa）和“上基质蛋白”（69 KDA）。这些观察是第一个 标识任何疱疹病毒的这种联系。 （iii）成熟 噬菌体HK97的动力学。我们对 噬菌体衣壳组件位于纪念性 前体成熟的构象变化 衣壳。这些变化是不可逆的，经常涉及部分 重折叠亚基，并受到严格控制。像这样， 他们提供了独特的机会，可以洞悉大规模 构象变化。 HK97代表一个有利的系统 研究这些反应。最早的前身Prohead I是 通过蛋白水解转换为Prohead II，然后扩展到头I， 促进端州II的共价交联。我们的第一个 HK97的研究比较了所有四个的3维结构 分辨率约为2.5 nm的颗粒。从那以后我们继续 研究扩展的动态进程（Prohead II - < 头i）通过在低pH下在体外诱导此事件并进行监测 随后通过延时冷冻电子显微镜进行的事件。分析 这些实验中产生的大量数据是 事实证明是耗时但很有意义的。去年，我们 报道了我们的第一个结果，半膨胀的粒子是 大约30分钟后观察到。我们现在已经描述了 从以后的三个时间点开始，并取得了成功 获得球形“气球”粒子的重建 三个小时后占主导地位。我们的当前帐户 酸引起的成熟途径是半膨胀的状态是 迅速到达，即几分钟之内；此后几乎没有 尺寸的变化约2小时，尽管胶囊的变化很小 形状继续进行；然后，这些中间体切换到 “气球”粒子；最后，在恢复中性pH时， 多面部头I状态已达到。电影已经制作了 动态过程，使用“变形”的图形技术 连接实验可视化的状态。 （iv）构象 与宿主细胞相互作用时脊髓灰质炎病毒的变化。什么时候 PICORNAVIRES，例如人类病原体，脊髓灰质炎病毒1型， 遇到易感细胞，病毒衣壳与细胞结合 受体。随之而来的事件导致细胞内传播 感染病毒尚不清楚。但是，与 脊髓灰质炎病毒与其受体的相互作用诱导构象 病毒体的变化，这是由于其沉积物的变化所表现出的 系数从160到135s。然后释放RNA，留下 80年代的衣壳。我们正在研究这些结构性基础 过渡，可以在体外通过短暂加热在体外模拟 适当的缓冲区。 160年代的病毒体已被求解到高 X-Ray晶体学的分辨率，但135s和80年代的状态 不利于这种分析。