MOLECULAR ANALYSIS OF VIRUS ASSEMBLY AND UNCOATING

无包膜的二十面体动物病毒的外壳蛋白执行 在病毒复制过程中的多种功能，包括 亚基组装以形成病毒衣壳， 病毒基因组，适当的成熟裂解，与细胞受体结合 和分离这些活动中的每一项都是潜在的目标， 抗病毒治疗，但药物的合理设计，以控制病毒 疾病需要详细了解化学相互作用， 反应在每一步。到目前为止， 病毒外壳蛋白和完整病毒粒子的关系已经被 仅对包膜正粘病毒科进行了详细研究（主要 流感病毒）和无包膜小核糖核酸病毒科。许多已知的 病毒组装、稳定性、成熟和 反汇编是从这些病毒中衍生出来的， 通常应用于其他病毒系统。在本申请中，我们 我建议通过扩大我们对这些问题的研究来扩大这一数据库。 非常简单和容易获得的动物诺达病毒。议员 家庭显示出结构相似但更复杂的许多属性 病毒和相关的原型，以指导医学调查 重要的病毒病原体。 我们以前使用X射线晶体学和低温电子 显微镜表征三个野田病毒在高分辨率。这些 研究已经导致了具体的建议的分子机制， 颗粒组装、熟化和去包衣。我们将测试这些 通过分子遗传学的方法， 生物化学和生物物理学。具体而言，（1） 选择的野田村病毒外壳蛋白区域在病毒体装配中，稳定性 和成熟度将通过表征一种 一系列的突变体。高分辨率结构中的区域 已经确定了野田病毒FHV的突变可能产生 可预测的生物学相关表型。(2)分子过程 将研究与FHV未涂层相关的问题。这些将包括 从热处理颗粒释放RNA的分析， 特别强调的是， 基因组被解放了此外，裂解产物γ在裂解过程中的作用也被证实。 将确定脱膜工艺。这将主要通过以下方式实现： 研究γ链突变对病毒结合的影响， 内化和RNA释放到胞质溶胶中。的成果 研究将有助于更好地了解 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.