MOLECULAR ANALYSIS OF VIRUS ASSEMBLY AND UNCOATING

无包膜的二十面体动物病毒外壳蛋白执行 病毒复制过程中的多种功能，包括 亚基组装形成病毒衣壳，特异性囊化 病毒基因组，适当成熟的裂解，与细胞受体结合 和分离。这些活动中的每一个都是 抗病毒治疗，但合理设计药物来控制病毒 疾病需要详细的化学相互作用知识和 每一步都涉及到反应。到目前为止，结构功能 病毒外壳蛋白与完整病毒粒子的关系 仅对包膜的正粘病毒科(主要是 流感病毒)和无包膜微冠状病毒科。许多已知的 病毒组装、稳定性、成熟度和稳定性的机制细节 反汇编是从这些病毒和基本概念衍生而来的 通常适用于其他病毒系统。在此应用程序中，我们 建议扩大这个数据库，扩大我们对 非常简单和容易获得的动物诺达病毒。本组织的成员 族显示了许多结构相似但更复杂的属性 病毒和相关的原型来指导医学研究 重要的病毒病原体。 我们之前使用过X射线结晶学和低温电子 用显微镜以高分辨率鉴定三种诺达病毒。这些 研究已经导致了对分子机制的具体建议 颗粒的组装、成熟和脱膜。我们将测试这些 通过分子遗传学的方法详细提出建议， 生物化学和生物物理学。具体地说，(1) 病毒粒子组装中选定的诺达病毒外壳蛋白区域，稳定性 成熟度将通过表征一种 一系列构建的突变体。高分辨率结构中的区域 已经确定了诺达病毒FHV的突变可能产生的地方 可预测的，生物相关的表型。(2)分子过程 将调查与FHV脱涂层相关的问题。这些措施包括 热处理颗粒释放核糖核酸的分析 特别强调封装物的特殊性 基因组被解放了。此外，切割产物伽马在细胞分裂过程中的作用 将确定脱膜工艺。这将主要通过以下方式完成 研究伽马链突变对病毒结合的影响， 内化和RNA释放到胞浆中。我们的结果是 研究将有助于更好地理解 二十面体结构-功能关系的基本原理 病毒。

Coat proteins of non-enveloped, icosahedral animal viruses perform a multitude of functions during the course of viral replication, including subunit assembly to form the viral capsid, specific encapsidation of the viral genome, proper maturation cleavage, binding to a cellular receptor and disassociation. Each of these activities is a potential target for antiviral therapy, but the rational design of agents to control viral disease requires detailed knowledge of the chemical interactions and reaction involved at each step. To date, structure function relationships of viral coat proteins and intact virions have been studied in detail only for the enveloped orthomyxoviridae (primarily influenza virus) and the non-enveloped picornaviridae. Much of the known mechanistic details of virus assembly, stability, maturation and disassembly have been derived from these viruses and the basic concepts are commonly applied to other virus systems. In this application we propose to expand this data base by extending our studies of the remarkably simple and accessible animal nodaviruses. Members of this family display many properties of structurally similar but more complex viruses and are relevant as prototypes to guide investigation medically important virus pathogens. We have previously used X-ray crystallography and cryo-electron microscopy to characterize three nodaviruses at high resolution. These studies have led to specific proposals for molecular mechanisms of particle assembly, maturation, and uncoating. We will test these proposals in detail through the methods of molecular genetics, biochemistry and biophysics. Specifically, (1) the importance of selected nodaviral coat protein regions in virion assembly, stability and maturation will be tested by characterizing the properties of a series of constructed mutants. Regions in the high resolution structure of nodavirus FHV have been identified where mutations may produce predictable, biologically relevant phenotypes. (2) Molecular processes associated with FHV uncoating will be investigated. These will include an analysis of release of RNA from heat-treated particles with particular emphasis on the specificity with which the encapsidated genome is liberated. Further , the role of cleavage product gamma in the uncoating process will be determined. This will be done primarily by studying the effect of gamma chain mutations on viral binding, internalization and RNA release into the cytosol. The results of our studies will contribute to a better understanding of the general principles underlying structure-function relationships in icosahedral virus.