Several single-stranded RNA viruses make use of Xrn1-resistant RNAs in their 3’ untranslated regions of their genome RNAs in order to increase their pathogenicity. This thesis focuses on two types... Show moreSeveral single-stranded RNA viruses make use of Xrn1-resistant RNAs in their 3’ untranslated regions of their genome RNAs in order to increase their pathogenicity. This thesis focuses on two types of Xrn1-resistant RNAs: those involving the “coremin” motif (xrRNAC) and those found in members of the Flaviviridae family (xrRNAF). While the structure for xrRNAFs has been solved, the xrRNAC structure is yet elusive. Therefore, we employed systematic mutational analysis in order to identify the features that are involved in halting the 5’-3’ exoribonuclease Xrn1 by xrRNAC. This led to the identification of novel variations of xrRNAC in viral families that were not yet known to employ an xrRNA. Regarding xrRNAF, we investigated their distribution and variability throughout the Flaviviridae family, and concluded that a universal xrRNAF structure is responsible for stalling Xrn1. Furthermore, the work in this thesis expands on the known, potential functions of xrRNAs by showing how xrRNAC is able to both inhibit scanning ribosomes and promote frameshifting. Show less
After infection by flaviviruses like Zika and West Nile virus, eukaryotic hosts employ the well-conserved endoribonuclease Xrn1 to degrade the viral genomic RNA. Within the 3MODIFIER LETTER PRIME... Show moreAfter infection by flaviviruses like Zika and West Nile virus, eukaryotic hosts employ the well-conserved endoribonuclease Xrn1 to degrade the viral genomic RNA. Within the 3MODIFIER LETTER PRIME untranslated regions, this enzyme encounters intricate Xrn1-resistant structures. This results in the accumulation of subgenomic flaviviral RNAs, an event that improves viral growth and aggravates viral pathogenicity. Xrn1-resistant RNAs have been established throughout the flaviviral genus, but not yet throughout the entire Flaviviridae family. In this work, we use previously determined characteristics of these structures to identify homologous sequences in many members of the genera pegivirus, hepacivirus and pestivirus. We used structural alignment and mutational analyses to establish that these sequences indeed represent Xrn1-resistant RNA and that they employ the general features of the flaviviral xrRNAs, consisting of a double pseudoknot formed by five base-paired regions stitched together by a crucial triple base interaction. Furthermore, we demonstrate that the pestivirus Bungowannah virus produces subgenomic RNA in vivo. Altogether, these results indicate that viruses make use of a universal Xrn1-resistant RNA throughout the Flaviviridae family. Show less
Subgenomic RNAs are produced by several RNA viruses through incomplete degradation of their genomic RNA by the exoribonuclease Xrn1, and have been shown to be essential for viral growth and... Show moreSubgenomic RNAs are produced by several RNA viruses through incomplete degradation of their genomic RNA by the exoribonuclease Xrn1, and have been shown to be essential for viral growth and pathogenicity. Within the flavivirus genus of theFlaviviridaefamily, two distinct classes of Xrn1-resistant RNA motifs have been proposed; one for mosquito-borne and insect-specific flaviviruses, and one for tick-borne flaviviruses and no-known-vector flaviviruses. We investigated tick-borne and no-known-vector flavivirus Xrn1-resistant RNA motifs through systematicin vitromutational analysis and showed that both classes actually possess very similar structural configurations, including a double pseudoknot and a base-triple at identical, conserved locations. For the no-known-vector flavivirus Modoc virus, we show thatin vivogeneration of subgenomic flaviviral RNA was affected by mutations targeted at nucleotides involved in the structural features of flaviviral Xrn1-resistant RNA motifs that were defined in this work. Our results suggest that throughout the genus flavivirus Xrn1-resistant RNA motifs adopt the same topologically conserved structure. Show less
