Mechanisms of transcriptional and post-transcriptional RNA regulation of positive- and negative-sense RNA viruses
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Abstract
This dissertation examines the mechanisms that govern RNA virus transcription and post-transcriptional regulation and presents the tools developed to address these questions.
I - Analysis of the Properties Governing Coronavirus Subgenomic RNA Expression.
Coronaviruses are nonsegmented, positive-sense RNA viruses which undergo discontinuous transcription. This includes the synthesis of negative-sense (-) subgenomic RNAs (sgRNAs) that are used as transcription intermediates to produce viral subgenomic mRNAs (sgmRNAs). However, little is known about the mechanism that determines the relative stoichiometries of coronavirus (-) sgRNAs. Here we developed a novel RNA-Seq approach to quantify the (-) sgRNAs produced during SARS-CoV-2 and murine hepatitis virus (MHV) infections. Based on our own and published sequencing data, we show that the relative expression levels of the viral (+) sgmRNAs strongly correlate with the expression levels of the (-) sgRNAs, suggesting that mRNA expression rates are mainly determined by (-) sgRNA expression rates. We further determined that the relative expression levels of (-) sgRNAs are independent of time of infection, cell type, and multiplicity of infection (MOI). Moreover, we uncovered a direct correlation between long-range RNA: RNA intragenomic interactions and the relative abundance of (-) sgRNAs. Using recombinant viruses, we demonstrated that this correlation is governed by the thermodynamic stability of these long-range genomic interactions and ultimately dictates the steady-state levels of (+) sgmRNAs used for translation of viral proteins.
II – Investigating Ebola virus mRNA Post-transcriptional Regulation.
Ebola virus (EBOV) is a nonsegmented, negative-sense (NNS) RNA virus which encodes seven monocistronic mRNAs that contain long 5′ and 3′ untranslated regions (UTRs). The 3' UTRs of EBOV mRNAs are enriched in AU content. AU-rich elements (AREs) represent common cis-acting sequences which regulate mRNA stabilization and decay to fine-tune transcript levels and protein production within the cell. We have identified putative AREs within EBOV 3'UTRs that represent binding sites for ARE-binding proteins (ARE-BPs). We demonstrate the potential for these post-transcriptional regulatory elements within in the viral 3' UTRs to regulate EBOV mRNA half-life. Using both mammalian and zebrafish embryo reporter assays, we show that the tested ARE-containing EBOV 3' UTRs increased reporter gene activity, suggesting mRNA stabilizing activity. We also show that the EBOV nucleoprotein (NP) mRNA, whose 3' UTR is enriched in AREs, is targeted by the ARE-BP tristetraprolin (TTP, ZFP36), leading to reduced NP mRNA and protein production and attenuated reporter gene expression in an EBOV minigenome system. In summary, the presence of AREs in EBOV 3' UTRs suggests that they are targets for modulation of RNA stability and translational activity through ARE-BP activity.
III – Developing Tools to Streamline the Identification of Filovirus Genomic 5´ Termini.
Next-generation sequencing (NGS) has been a central methodology in determining the genomic sequences of emerging RNA viruses. Despite this, de novo genome sequencing efforts often do not provide sufficient coverage of the 5′ and 3′ ends of RNA viral genomes. Since the genome ends of RNA viruses contain sequences critical for viral replication and transcription, a lack of terminal sequence information obstructs efforts to study emerging and re-emerging viruses. We developed a novel method named Viral Bona fide End Sequencing (ViBE-Seq) that allows for rapid sequencing of the 5′ termini of viral genomic RNA. We validated ViBE-Seq by successfully capturing the 5′ termini of EBOV and Marburg virus (MARV) RNA isolated from virions, infected cells, and infected animal tissue. We have used ViBE-Seq to sequence the 5′ ends of the Lassa virus (LASV) genome and nucleoprotein (NP) mRNA, validating its efficacy across filovirus and arenavirus families. Finally, we utilized ViBE-Seq to uncover the terminal 5′ genomic nucleotides of Lloviu virus (LLOV), a recently discovered filovirus, to accelerate research regarding its potential pathogenicity. ViBE-Seq has the potential to generate quantitative, nucleotide-level sequencing data of viral 5′ termini and expedite research on novel viruses to combat potential future outbreaks.
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2025