Packaging, Targeting, and Replication of Virus-derived RNA Replicons

为了更好地控制和应对像目前的新冠肺炎疫情这样的病毒爆发，有必要在分子水平上更好地了解核糖核酸病毒的生命周期-它们如何进出宿主细胞，以及它们如何在这些细胞中复制。该项目专注于三种最简单、特征最好的RNA病毒--像SARS-2这样的病毒，它们的基因组是单链RNA，目标细胞将其翻译成病毒蛋白质。该项目旨在发现单个RNA基因组如何在几个小时内放大10,000倍，新的RNA分子如何被包装成保护性的蛋白质壳(衣壳)，以及这些新形成的粒子(核衣壳)如何离开宿主细胞。这项研究将由不同的本科生、研究生和博士后进行，他们正在接受最先进的分子生物学、化学和物理方法的培训，包括基因工程、生化/酶反应以及荧光和电子显微镜。这项工作的结果将提供能够推动疫苗设计、抗病毒药物发现和生物技术创新的基础信息。这项研究中的三种病毒是：一种溴病毒，它的四个基因包含在三个不同颗粒中的三个RNA基因组分子中；一种诺达病毒，它的四个基因包含在一个颗粒中的两个分子中；以及一种甲病毒，它的九个基因都包含在一个RNA分子和一个颗粒中。这些病毒说明了将RNA基因复制和包装到病毒颗粒中的策略的广度。细胞识别这些病毒基因组，就像它们是mRNAs一样，因此基因组都是自我复制的，从某种意义上说，它们编码复制基因组的RNA复制酶蛋白。在比较和对比这些病毒的生命周期时，最终目标是控制自我复制的RNA分子的自我组装。所涉及的最先进的物理和分子生物学技术包括：时间分辨冷冻电子断层扫描；形成衣壳的蛋白质的基因工程；以及单分子/单细胞荧光显微镜。具体的实验包括：RNA病毒和VLP自组装路径的时间分辨断层成像；由蛋白质配体功能化的体外重组VLP的合成；以及病毒和非病毒RNA分子对RNA复制的竞争。这项研究由生物科学局分子和细胞生物科学部门的遗传机制计划资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

In order to better control and treat viral outbreaks such as the current COVID-19 pandemic, it is necessary to better understand on a molecular level the life cycles of RNA viruses – how they get into and out of their host cells, and how they replicate in those cells. This project focuses on three of the simplest and best characterized RNA viruses – ones, like SARS-2, whose genomes are single-stranded RNA that the target cell translates into viral proteins. This project aims to discover fundamental aspects of how a single RNA genome is amplified 10,000-fold within hours, how the new RNA molecules are packaged into protective protein shells (capsids), and how these newly-formed particles (nucleocapsids) exit their host cell. The research involved will be carried out by diverse undergraduate, graduate, and postdoctoral students who are being trained in state-of-the-art molecular biology, chemistry and physics methods, including genetic engineering, biochemical/enzymatic reactions, and fluorescence and electron microscopies. The results of this work will provide foundational information that can propel vaccine design, antiviral pharmaceutical discovery, and innovations in biotechnology. The three viruses featured in this research are: a bromovirus whose four genes are contained in three RNA genome molecules packaged in three different particles; a nodavirus whose four genes are contained in two molecules packaged together in one particle; and an alphavirus whose nine genes are all contained in one RNA molecule and one particle. These viruses illustrate the breadth of strategies for replication of and packaging of RNA genes into virus particles. Cells recognize these viral genomes as if they were mRNAs so that the genomes are all self-replicating in the sense that they encode RNA replicase proteins that replicate the genome. In comparing and contrasting the life cycles of these viruses the ultimate goal is to control the self-assembly of self-replicating RNA molecules. The state-of-the-art physical and molecular biological techniques involved include: time-resolved cryo-electron tomography; genetic engineering of capsid-forming proteins; and single-molecule/single-cell fluorescence microscopy. The particular experiments include: time-resolved tomographic imaging of the self-assembly pathway of RNA viruses and VLPs; syntheses of in vitro reconstituted VLPs functionalized by protein ligands; and competitions of viral and non-viral RNA molecules for RNA replicases.This research is funded by the Genetic Mechanisms program in the Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.