Complete Structural Determination of Rice dwarf virus

本项目对分子质量约为7500 kDa的双壳病毒水稻矮缩病毒进行了结构研究，利用x射线晶体学、低温电子显微镜和分子生物学技术揭示了该病毒的整体结构。大多数的结构研究都是在P2缺失的RDV颗粒上进行的，它包含了除P2蛋白外的所有病毒成分，其中P2蛋白在纯化步骤中用CCl_4处理时从样品中去除。通过x射线晶体学测定RDV晶体的3.5Å分辨率电子密度图，揭示了RDV晶体内外壳（P3和P8蛋白）几乎完整的结构以及P7蛋白的一部分。亚基蛋白之间的相互作用表明了900种蛋白质成分是如何构建成一个更高阶的病毒核心结构的，我们提出了结构组装层次的模型。这一模型得到了分子生物学实验的有力支持。采用低温电子显微镜和x射线晶体学相结合的方法对病毒颗粒的内部结构进行了研究。低分辨率低温电镜图显示转录复合体（P1，P5和P7蛋白复合体）对应的清晰分子包膜。该图谱还清楚地显示了dsRNA的至少三层结构。特别是第一层和第二层的dsrna呈棒状结构，排列成液晶状结构。此外，我们已经成功地从病毒颗粒和/或过表达样品中纯化了组分蛋白，这些样品经过结晶处理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.