Complete Structural Determination of Rice dwarf virus

本研究计划利用X射线晶体学、冷冻电子显微镜和分子生物学技术，对分子量约为7,500 kDa的水稻矮缩病毒进行结构研究，以揭示其整体结构，其中大部分结构研究是对缺失P2的RDV颗粒进行的，该颗粒含有除P2蛋白外的所有病毒组分，其中P2蛋白在纯化步骤中用CCl_4处理期间已从样品中除去。通过X射线晶体学测定的RDV晶体的3.5 μ m分辨率的电子密度图显示了几乎完整的内壳和外壳（P3和P8蛋白）和部分P7蛋白的结构。亚基蛋白之间的相互作用表明900个蛋白质组分是如何构建成一个更高层次的病毒核心结构的，我们提出了结构组装层次模型。利用分子生物学技术对该病毒颗粒的内部结构进行了研究，并结合冷冻电镜和X射线晶体学对病毒颗粒的内部结构进行了分析。低分辨率cryo-EM图谱显示对应于转录复合物（P1、P5和P7蛋白的复合物）的清晰分子包膜。图谱还清晰地显示了dsRNA的至少三层结构。此外，我们还成功地从病毒颗粒和/或过表达样品中纯化了组分蛋白，并对这些样品进行了结晶，以揭示组分蛋白的高分辨率结构。

This project has been worked on the structural studies of Rice dwarf virus, which is a double-shelled virus with its molecular mass of about 7,500 kDa, to reveal its whole structure using x-ray crystallography, cryo-electron micrograph and molecular biological techniques.Most of the structural studies have been done with the P2-deletion RDV particle, which contained all viral components except the P2 protein, where P2 protein had been removed from the sample during treatment with CCl_4 in the purification step. The 3.5Å resolution electron density map of RDV crystal, determined by x-ray crystallography, revealed almost complete structures of the inner- and outer-shells (P3 and P8 proteins) and a part of P7 protein. The interactions between the subunit proteins suggest how the 900 protein components are built into a higher-ordered virus core structure, and we proposed the model for the hierarchy of structural assembly. This model was strongly supported by some experiments using molecular biological techniques.Internal structure of the virus particle was studied by combination of the single-particle analysis using cryo-electron micrograph and x-ray crystallography. Low-resolution cryo-EM map showed clear molecular envelop corresponding to the transcription complex (complex of P1,P5 and P7 proteins). The map also clearly showed at lease three layered-structure of dsRNA. Especially, dsRNAs in the first and second layer showed rod-shaped structure, which are packed into liquid crystal-like structure.In addition, we have succeeded to purify the component proteins from the virus particle and/or from overexpressed sample, these samples were subjected to crystallization to reveal the high-resolution structure of the component proteins.