Structural Dynamics of Rotavirus Transcription

描述（由申请人提供）：RNA病毒在人类中引起广泛的疾病，并具有重大的医学，经济和社会影响。本申请的总体目标是详细了解轮状病毒（导致儿童严重胃肠炎的RNA病毒）如何在完整的亚病毒双层颗粒背景下转录其基因组。虽然非转录轮状病毒双层颗粒存在高分辨率结构，但对进行mRNA合成的颗粒的结构知之甚少。核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。这一假设将通过两个综合但独立的具体目标进行检验：（i）使用冷冻电子显微术确定主动转录轮状病毒双层颗粒的三维结构以优于10-A的分辨率，和（ii）确定主动转录轮状病毒双层颗粒的三维结构以优于20-A的分辨率，一种使用新型液体成像平台的分辨率，称为原位分子显微镜。具体地，将从轮状病毒感染的细胞中分离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成像之前在玻璃冰中快速冷冻或浸没在微流体室中。国家的最先进的图像处理软件，依赖于贝叶斯推理将获得三维重建的双层粒子在冰或液体。这一建议是创新的，因为它采用了新技术来研究酶活性轮状病毒转录酶复合物的结构，目前几乎没有信息。这项工作意义重大，因为它有望成为旨在开发消除RNA病毒转录的药理学策略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件的技术能力，从而使我们更接近“实时”EM成像。

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.