High Resolution of Structural Studies of Insect Viruses

我们的团队和合作者对昆虫诺达病毒和小核糖核酸样病毒进行了晶体学、生物物理学和分子生物学研究。许多观察结果显然与该粒子在病毒生命周期中的特定功能有关。该更新申请描述了细胞生物学以及晶体学、生物物理学和分子病毒学领域的实验，因为在原子分辨率和体外研究的化学基础上努力定义病毒表型。该提案的实质是跨越从原子到细胞的范围，用于简单而优雅的生物体，昆虫和植物的无包膜ssRNA病毒。观察到的结构和假设功能的变化自然会导致对这些粒子进化的研究。具体而言，我们力求：（1）使用荧光和电子显微镜来确定鸡舍病毒（FHV）的进入模式，并使用生物化学和生物物理学来确定受体在RNA释放中的作用。(2)利用GFP融合蛋白在哺乳动物和果蝇细胞中的体内表达来建立通过成熟切割从FHV的衣壳蛋白衍生的膜活性肽的作用。(3)确定FHV的RNA指导的RNA聚合酶的结构，并建立其允许其在昆虫、植物、酵母和哺乳动物细胞中复制和CAP RNA分子的新特征。(4)通过晶体学、cryoEM、杆状病毒表达和定点诱变，研究影响昆虫和鱼类野田病毒组装、成熟和基因组组织的因素。(5)通过荧光和电子显微镜确定FHV RNA复制、衣壳蛋白合成和颗粒组装的位点。(6)建立昆虫和鱼类诺达病毒之间以及昆虫和植物小核糖核酸和小核糖核酸样病毒之间的进化关系。

Crystallographic, biophysical, and molecular biological studies were performed by our group and collaborators on insect nodaviruses and picorna-like 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 variations observed in structure and hypothesized function lead naturally to an investigation of the evolution of these particles. Specifically we seek to: (1) Employ fluorescence 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 pf a membrane active peptide derived from the capsid protein of FHV by a maturation cleavage. (3) Determine the structure of the RNA directed RNA polymerase 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 fluorescence and electron microscopy. (6) Establish evolutionary relationships among insect and fish nodaviruses and among insect and plant picorna and picorna-like viruses.