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.

我们的目标是阐明其分子机制 控制病毒衣壳的组装，最终目标是 确定抗病毒化合物的目标并深入了解 控制大分子组装的机制 综合体一般。我们特别关注大规模 伴随衣壳成熟的构象变化和 病毒粒子与宿主细胞的相互作用。我们的主要进展 去年的情况如下： (i) 乙型肝炎病毒衣壳（“核心 抗原”）。尽管有有效的疫苗，乙型肝炎病毒 仍然是一个巨大的公共卫生问题， 激励进一步努力阐明其复制周期。我们有 一直在研究它的衣壳蛋白（183 个氨基酸），我们首先 发现形成能够自组装成的二聚体 二十面体粒子。这些颗粒有两种不同的尺寸 - 一种 具有 T=3 对称性（90 个二聚体；28nm 直径），另一个具有 T=4（120 个二聚体；34nm 直径）。与大多数其他病毒衣壳不同 乙肝病毒的蛋白质主要是α螺旋。 1997年，我们通过低温电子计算了密度图 0.9nm 前所未有的高分辨率显微照片 其中大部分二级结构是直接可见的， 包括形成二聚化基序的 4 螺旋束。我们的 持续的研究旨在确定精确的位置 特定氨基酸残基有助于描绘整体路径 通过我们的密度图来了解多肽链。我们的第一次成功， 去年的报告中提到并自发布以来，目标是本地化 C 末端采用金簇标记。最近，我们有 通过以下方法精确定位了包含残基 78-83 的表面环 表征单克隆抗体 Fab 片段的结合 识别该肽的抗体。 N-的本地化 终点站也是通过附加一个无关的来完成的 该位点上的八肽，然后我们将其可视化为附加的 差异图中的密度。我们正在继续延长 分辨率我们的衣壳图并施加更多约束 用于链追踪。 (ii) 的结构和覆盖 巨细胞病毒衣壳。巨细胞病毒（CMV）是一种临床上 疱疹病毒家族的重要成员，引起感染 免疫抑制个体。它也没有反应 抗病毒药物阿昔洛韦，对其他疱疹病毒有效。 在此背景下，我们一直在研究巨细胞病毒衣壳 装配，使用 simian CMV 作为类似但较少的模型 易于处理的人类 CMV。该项目过去的主要目标 今年是利用 SCMV 提供的机会 研究衣壳-外皮相互作用的系统。外皮是一个 位于疱疹病毒之间的广泛蛋白质区室 衣壳及其包膜。尽管长期以来的兴趣， 外皮的分子结构和功能原理 一直默默无闻。在培养物感染后的晚期 带有 SCMV 的成纤维细胞，被膜衣壳出现在 细胞质。通过分离这些衣壳并比较它们的结构 以及与常规核衣壳的蛋白质组成， 我们已经表征了两个外皮的结合模式 蛋白质并确定了它们的临时候选者作为“基本 磷蛋白”（119 kDa）和“上基质蛋白”（69 kDa），分别。这些观察结果代表了第一个 鉴定任何疱疹病毒的此类联系。 (三) 成熟度 噬菌体 HK97 的动力学。我们的主要兴趣是 噬菌体衣壳组装位于巨大的 伴随前体成熟的构象变化 衣壳。这些变化是不可逆转的，通常涉及部分 亚基的重折叠，并受到严格控制。像这样， 他们提供了洞察大规模的独特机会 构象变化。 HK97代表了一个有利的系统 研究这些反应。最早的前身 Prohead I 是 通过蛋白水解转化为 Prohead II，然后扩展为 Head I， 促进Head II（最终状态）的共价交联。我们的第一个 HK97 的研究比较了所有四种的 3 维结构 颗粒分辨率约为 2.5 nm。我们从那时起就继续 研究扩张的动态进展（Prohead II -< Head I) 通过在低 pH 条件下体外诱导该事件，并监测 通过延时冷冻电子显微镜观察后续事件。分析 这些实验中产生的大量数据是 事实证明，这很耗时，但很有回报。去年，我们 报告了我们的第一个结果，其中半膨胀颗粒 约30分钟后观察。我们现在已经表征了 中间三个时间点并成功 获得球形“气球”粒子的重建 3小时后占主导地位。我们的经常账户 酸诱导的成熟途径是半膨胀状态 快速到达，即几分钟内；此后就很少有 尽管壳粒变化很小，但尺寸变化约 2 小时 形状继续发生；然后，这些中间体转变成 “气球”粒子；最后，当pH值恢复到中性时， 实现多面体头I状态。还拍过一部电影 动态过程，使用“变形”的图形技术 连接实验可视化状态。 (iv) 构象 脊髓灰质炎病毒与宿主细胞相互作用后的变化。什么时候 小核糖核酸病毒，例如人类病原体、1 型脊髓灰质炎病毒、 遇到易感细胞，病毒衣壳与细胞结合 受体。随后发生的事件导致细胞内传播 感染病毒尚不清楚。然而，随着 脊髓灰质炎病毒与其受体相互作用会诱导构象 病毒粒子的变化，表现为其沉降的变化 系数从160S到135S。然后释放 RNA，留下 80S衣壳。我们正在研究这些的结构基础 转变，可以通过在体外短暂加热来模拟 适当的缓冲液。 160S病毒粒子已被解析为高 X 射线晶体学分辨率，但 135S 和 80S 状态 不利于此类分析。