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

为了更好地控制和治疗病毒爆发，如当前的COVID-19大流行，有必要在分子水平上更好地了解RNA病毒的生命周期——它们如何进入和离开宿主细胞，以及它们如何在这些细胞中复制。该项目主要研究三种最简单、最具特征的RNA病毒，比如SARS-2，它们的基因组是单链RNA，目标细胞将其转化为病毒蛋白质。该项目旨在发现单个RNA基因组如何在数小时内扩增10,000倍的基本方面，新的RNA分子如何被包装成保护性蛋白质外壳（衣壳），以及这些新形成的颗粒（核衣壳）如何离开宿主细胞。所涉及的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、荧光和电子显微镜。这项工作的结果将为推动疫苗设计、抗病毒药物发现和生物技术创新提供基础信息。本研究的三种病毒是：溴状病毒，其四个基因包含在三个不同颗粒包装的三个RNA基因组分子中；一种诺达病毒，其四个基因包含在两个分子中，包装在一个颗粒中；一种阿尔法病毒，它的九个基因都包含在一个RNA分子和一个粒子中。这些病毒说明了将RNA基因复制和包装成病毒颗粒的策略的广度。细胞识别这些病毒基因组，就像它们是信使RNA一样，所以基因组都是自我复制的，因为它们编码RNA复制酶蛋白质，复制基因组。通过比较和对比这些病毒的生命周期，最终目的是控制自我复制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：时间分辨低温电子断层扫描；衣壳形成蛋白的基因工程；单分子/单细胞荧光显微镜。具体实验包括：RNA病毒和VLPs自组装途径的时间分辨层析成像；蛋白质配体功能化的体外重组VLPs的合成以及病毒和非病毒RNA分子对RNA复制酶的竞争。这项研究由生物科学理事会分子和细胞生物科学部的遗传机制项目资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.