Date of Award

Fall 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Pyle, Anna Marie

Abstract

Single-stranded, positive-sense RNA viruses are a class of pathogens that pose a serious danger to human health. As a group, they have been the focus of research exploring how they infect, evade, and hijack host cellular machinery to propagate. Though these studies have primarily focused on viral proteins, the past two decades have seen a resurgence of interest in the RNA genomes themselves. This is because RNA viral genomes contain functional RNA structures that expand their functional repertoire. While studies of viral RNA structure were originally restricted to 5’ and 3’ viral termini, recent methodological advancements have facilitated the search for functional structure within extensive viral open-reading frames. Here, these methods are applied to the genomes of two RNA viruses, SARS-CoV-2 and West Nile virus. In pursuit of this work, several methodological advancements were made that will facilitate future studies of functional RNA structure. In Chapter 2, this methodology is applied to the genome of SARS-CoV-2, the etiological agent responsible for the ongoing global pandemic. We develop a novel long-amplicon strategy for the collection of SHAPE-MaP data using a highly processive reverse transcriptase, greatly facilitating structural studies of extremely long viral RNAs. The resulting genomic secondary structure model reveals functional motifs at the viral termini that are structurally homologous to other coronaviruses, thereby fast-tracking our understanding of the SARS-CoV-2 life cycle. We uncover elaborate networks of well-folded RNA secondary structures and reveal features of the SARS-CoV-2 genome architecture that distinguish it from other single-stranded, positive-sense RNA viruses. Evolutionary analysis of the full-length SARS-CoV-2 secondary structure model suggests that, not only do these architectural features appear to be conserved across the β-coronavirus family, but individual regions of well-folded RNA may be as well. Using structure-disrupting, antisense locked nucleic acids (LNAs), we demonstrate that RNA motifs within these well-folded regions play functional roles in the SARS-CoV-2 life cycle. In Chapter 3, we extend this methodology to the genome of West Nile virus, an arthropod-borne virus that, due to climate change, poses an increasing global health risk. We report for the first time the complete secondary structure of the WNV genome in both arthropod and mammalian cell lines. The resulting genomic secondary structure model recapitulates a conserved motif in the 5’UTR required for viral replication. Along with our SHAPE-MaP data, our structural models provide novel insights into previously studied but poorly understood aspects of flaviviral biology. We describe a global genome architecture that, along with specific regions of well-folded RNA, folds with minimal host dependence. Owing to weak signals of evolutionary conservation, we instead relied on patterns of structural homology to prioritize specific RNA structures for functional validation. Using a highly optimized workflow, we used structure-disrupting LNAs to demonstrate that a subset of novel well-folded RNA structures plays both conserved and host-specific functional roles. Taken together, the work presented in this dissertation deepens our understanding of viral biology and functional RNA structure, identifies conserved aspects of the viral life cycle that are readily targetable by a novel class of nucleic acids, and therefore represents an important step forward in our fight against expanding global health threats. Methodological improvements and innovations presented in this dissertation have broad applications beyond the study of viral RNAs and will therefore greatly facilitate the discovery and study of functional RNA structure.

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