Structural Dynamics of Rotavirus Transcription

描述(申请人提供)：RNA病毒在人类中引起广泛的疾病，并具有重大的医疗、经济和社会影响。该应用程序的总体目标是详细了解轮状病毒，即导致儿童严重胃肠炎的RNA病毒，如何在完整、亚病毒、双层颗粒的背景下转录其基因组。虽然存在非转录轮状病毒双层颗粒的高分辨率结构，但对合成mRNA的颗粒结构知之甚少。中心假设是轮状病毒双层颗粒在转录过程中经历动态结构重排。这一假说将通过两个完整但独立的特定目标得到验证：(1)使用冷冻电子显微镜确定具有活性转录的轮状病毒双层颗粒的三维结构，使其分辨率高于10-A；(2)使用一种称为原位分子显微镜的新型液体成像平台，确定具有活性转录的轮状病毒双层颗粒的三维结构，使其分辨率高于20-A。具体地说，将从轮状病毒感染的细胞中分离出具有转录能力的双层颗粒，诱导其在体外进行mRNA合成，然后在使用透射电子显微镜进行成像之前，将其快速冷冻在玻璃冰中或浸入微流体室中。依靠贝叶斯推理的最先进的图像处理软件将被用来推导出冰或液体中双层粒子的三维重建。这一建议是创新的，因为它应用了新技术来研究具有酶活性的轮状病毒转录酶复合体的结构，目前关于该复合体的信息很少。这项工作意义重大，因为预计这将是旨在开发药物策略以消除RNA病毒转录的连续研究的第一步。同样重要的是，这项工作将提高我们在液体中可视化生物组装的技术能力，从而使我们离‘实时’电磁成像又近了一步。

DESCRIPTION (provided by applicant): RNA viruses cause widespread disease in humans and have significant medical, economic, and social repercussions. The overall objective of this application is to provide detailed insight into how rotaviruses, RNA viruses that cause severe gastroenteritis in children, transcribe their genomes in the context of intact, subviral, double-layered particles. While high-resolution structures exist for non-transcribing rotavirus double-layered particles, little is known about the structure of particles undergoing mRNA synthesis. The central hypothesis is that the rotavirus double-layered particle undergoes dynamic structural rearrangements during the process of transcription. This hypothesis will be tested through two integrated, yet independent, specific aims: (i) determine the three-dimensional structures of actively-transcribing rotavirus double-layered particles to better than 10-A resolution using cryo-electron miscroscopy and (ii) determine the three-dimensional structures of actively-transcribing rotavirus double-layered particles to better than 20-A resolution using a novel liquid imaging platform called in situ molecular microscopy. Specifically, transcriptionally-competent double-layered particles will be isolated from rotavirus-infected cells, induced to perform mRNA synthesis in vitro, and then either flash-frozen in vitreous ice or submerged in a microfluidic chamber prior to being imaged using a transmission electron microscope. State-of-the-art image processing software that relies on Bayesian inference will be employed to derive three-dimensional reconstructions of double-layered particles in ice or liquid. This proposal is innovative because it applies new technologies to investigate the structure of the enzymatically-active rotavirus transcriptase complex for which little information currently exists. The work is significant because it is expected to be the first step in a continuum of research aimed at developing pharmacological strategies to obliterate RNA virus transcription. Equally important, this work will advance our technical capabilities to visualize biological assemblies in liquid, thereby bringing us one step closer to 'live' EM imaging.