CRYO-EM TOMOGRAPHY OF ISOLATED MITOCHONDRIA FROM FHV INFECTED CELLS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 生物学问题的一般描述：Flock House病毒（FHV）是一种典型的双链正链RNA病毒，它一直是生物化学和遗传学研究病毒基因组复制的极好模型系统。病毒RNA通过病毒RNA依赖性RNA聚合酶（RdRp）复制，该聚合酶在线粒体外膜上形成小的（约60 nm）内陷，称为小球。每个小球通过一个小颈向细胞质开放，这可能是新合成的RNA释放的地方。其中膜未染色的样品的EM断层扫描显示病毒聚合酶蛋白和RNA在小球内形成环形复合物。尽管这些研究提供了有关细胞内线粒体形态的信息，但由于用于EM生物组分染色的重金属，从这些样品中获得的结构信息有限。本研究的第一个目标是使用cryo-EM断层扫描，以确定的三维结构的RdRp复杂的线粒体分离FHV感染的果蝇细胞在接近天然状态。第二个目标是从断层图像中提取这些RdRp复合物的密度，并使用它们来构建更高分辨率的平均结构。最终，这些更高分辨率的cryo-EM断层扫描图将与来自FHV感染细胞内线粒体的细胞断层扫描研究和分离的RdRp蛋白的原子结构的数据（目前正在由John约翰逊研究）相结合，以创建从细胞分辨率到原子分辨率的综合模型。 意义：所有RNA病毒都使用病毒编码的RNA聚合酶复制其基因组，这些RNA聚合酶主要作为寡聚体发挥作用。破坏这些聚合酶复合物的形成代表了许多病毒的新治疗靶点。然而，低聚物的结构还没有很好地理解，因为它们通常与膜细胞器结合形成。Flock House病毒的独特之处在于病毒聚合酶与线粒体形成复制复合物，线粒体是一种可以从细胞中分离出来用于EM断层扫描研究的细胞器。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. General description of the biological problem: Flock House virus (FHV) is a prototypical bipartite positive-stranded RNA virus, that has been an excellent model system for biochemically and genetically investigating virus genome replication. The viral RNA is replicated by the viral RNA dependant RNA Polymerase (RdRp), which forms small (~60 nm) invaginations on the outer mitochondrial membrane called spherules. Each spherule is open to the cytoplasm through a small neck, which is presumably where the newly synthesized RNA is released. EM tomography of samples where the membrane is not stained reveals that the viral polymerase protein and RNA form a ring shaped complex inside the spherules. Although, these studies are informative about the morphology of the mitochondria inside the cell, the structural information obtainable from these samples are limited due to the heavy metals used for staining the biological components for EM. The first goal of this study is to use cryo-EM tomography to determine the three-dimensional structure of the RdRp complex in mitochondria isolated from FHV infected Drosophila cells in a near to native state. The second goal will be to extract the density for these RdRp complexes from the tomogram and use them to construct a higher resolution averaged structure. Ultimately these higher resolution cryo-EM tomograms will be combined with data from cellular tomography studies of the mitochondria inside FHV infected cells and atomic structures of the isolated RdRp protein (currently being investigated by John Johnson) to create a comprehensive model that spans from the cellular resolution to the atomic resolution. Significance: All RNA viruses replicate their genome using viral encoded RNA polymerases, which primarily function as oligomers. Disrupting the formation of these polymerase complexes represents a new therapeutic target for many viruses. However, the structure of the oligomers are not well understood since they typically form in association with membrane organelles. Flock House virus is unique in that the viral polymerase forms replication complexes in association with the mitochondria, which is an organelle that can be isolated from the cell for EM tomography studies.