CRYO-EM TOMOGRAPHY OF ISOLATED MITOCHONDRIA FROM FHV INFECTED CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 生物学问题概述：鸡舍病毒(FHV)是一种典型的两部分正链RNA病毒，已成为研究病毒基因组复制的良好生物化学和遗传学模型系统。病毒RNA是由病毒RNA依赖的RNA聚合酶(RdRp)复制的，它在线粒体膜外膜上形成小的(~60 nm)内陷，称为球体。每个球体通过一个小脖子向细胞质开放，据推测，这是新合成的RNA释放的地方。未染膜的样本的电子显微镜断层扫描显示，病毒聚合酶蛋白和RNA在球体内形成环状复合体。虽然这些研究提供了关于细胞内线粒体形态的信息，但由于用于对EM生物成分进行染色的重金属，从这些样本中获得的结构信息有限。这项研究的第一个目标是使用冷冻-EM断层扫描技术来确定从接近自然状态的FHV感染的果蝇细胞分离的线粒体中RdRp复合体的三维结构。第二个目标是从断层图像中提取这些RdRp复合体的密度，并使用它们来构建更高分辨率的平均结构。最终，这些更高分辨率的冷冻-EM断层图像将与FHV感染细胞内线粒体和分离的RdRp蛋白质的原子结构(John Johnson目前正在研究)的细胞断层研究数据相结合，创建一个从细胞分辨率到原子分辨率的全面模型。 意义：所有的RNA病毒都使用病毒编码的RNA聚合酶复制它们的基因组，这种聚合酶主要作为寡聚体发挥作用。破坏这些聚合酶复合体的形成是许多病毒的新治疗靶点。然而，低聚物的结构还不是很清楚，因为它们通常与膜细胞器结合形成。鸡舍病毒的独特之处在于，病毒聚合酶与线粒体形成复制复合体，线粒体是一种细胞器，可以从细胞中分离出来进行EM断层扫描研究。

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.