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

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.