High Resolution of Structural Studies of Insect Viruses

结晶学、生物物理学和分子生物学研究由我们的团队和合作者进行 昆虫诺达病毒和类小核糖核酸病毒。一些观察结果显然与特定的 粒子在病毒生命周期中的作用。这份续订申请描述了细胞领域的实验 生物学，以及结晶学、生物物理学和分子病毒学，因为努力定义病毒表型 在原子分辨和体外研究的化学基础上。这项提议的实质是跨越 对于简单而优雅的生物体，从原子到细胞，从昆虫的无包膜单链RNA病毒到 植物。在结构和假设功能上的变化自然导致了对 这些粒子的演化。具体来说，我们试图：(1)使用荧光和电子显微镜来确定 鸡舍病毒(FHV)的进入方式，并利用生物化学和生物物理来确定 RNA释放中的受体。(2)利用GFP融合蛋白在哺乳动物和果蝇细胞中的体内表达 通过成熟的裂解，确定从口蹄疫病毒衣壳蛋白中提取的膜活性多肽的作用。(3) 确定FHV的RNA导向的RNA聚合酶的结构，并建立其新的特征，使其能够 在昆虫、植物、酵母和哺乳动物细胞中复制和CAP RNA分子。(4)用结晶学的方法研究 冷冻EM，杆状病毒表达和定点突变，影响组装、成熟和基因组的因素 昆虫和鱼类诺达病毒的组织。(5)确定FHV RNA复制、衣壳蛋白合成的部位 以及荧光和电子显微镜下的颗粒组装。(6)建立昆虫进化关系 和鱼类诺达病毒，以及昆虫和植物微微核糖核酸和类微微核糖核酸病毒。

Crystallographic, biophysical,and molecular biological studies were performed by our group and collaborators on insect nodaviruses and picorna-likc viruses. A number of observations were made that apparently relate to specific functions of the particle in the virus life cycle. This renewal application describes experiments in the area of cell biology, as well as crystallography,biophysics and molecular virology,as efforts are made to define viral phenotypes on the basis of chemistry discerned from atomic resolution and in vitro studies. The essence of the proposal is to span the range from atoms to cells for simple, yet elegant, organisms, the non enveloped, ssRNA viruses of insects and plants. The variationsobserved in structure and hypothesized function lead naturally to an investigation of the evolution of these particles. Specifically we seek to: (1) Employ fluoresencc and electron microscopy to determine the mode of entry of flock house virus (FHV) and, to employ biochemistry and biophysics to determine the role of the receptor in RNA release. (2) Employ in vivo expression of GFP fusion proteins in mammalian and drosophila cells to establish the role of a membrane active peptide derived from the capsid protein of FHV by a maturationcleavage. (3) Determine the structure of the RNA directed RNA polymerasc of FHV and establish its novel features that allow it to replicate and CAP RNA molecules in insect, plant, yeast and mammalian cells. (4) Investigate, by crystallography, cryoEM, baculovirus expression and site directed mutagenesis, factors that affect assembly, maturation and genome organization of insect and fish nodaviruses. (5) Determine the sites of FHV RNA replication, capsid protein synthesis and particle assembly by fluorcsence and electron microscopy. (6) Establish evolutionaryrelationships among insect and fish nodaviruses and among insect and plant picorna and picorna-like viruses.