A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally dynamic RNA structure

Abstract

Folded RNA elements that block processive 5' → 3' cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5' end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the dianthovirus xrRNAs undergo conformational changes and can use "codegradational remodeling," exploiting the exoribonucleases' degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.

A widespread Xrn1-resistant RNA motif composed of two short hairpins

“…Previous studies on the 3' UTR of both flavi-and dianthoviruses have indicated that elaborate structures are formed by the xrRNAs they utilize 19,21,35,36 . For instance, the crystal structure of Murray Valley Encephalitis Virus (MVE) flaviviral xrRNA revealed a ring-like conformation through tertiary interactions between its 5' end and a downstream hairpin, which itself forms a pseudoknot with nucleotides even more downstream 36 .…”

“…Base triples play a crucial role in Xrn1-resistant RNAs of flaviviruses 21 and dianthoviruses 19 . In addition to degradation assays, different approaches are necessary to elucidate the three-dimensional structure of coremin xrRNA.…”

“…These xrRNAs are not exclusive to flaviviruses however. The plant-infecting diantho-, beny-and cucumoviruses produce a subgenomic RNA through the action of an Xrn1-like enzyme 14,19 .…”

“…While elaborate tertiary structures are required to block Xrn1 progression in flavivirus and dianthovirus RNAs 19,21 , the role of RNA structure in the production of beny-and cucumovirus subgenomic RNAs has remained enigmatic. During infection of Beta macrocarpa by Beet necrotic yellow vein virus (BNYVV), a member of the Benyviridae family and Benyvirus genus 22 , a non-coding RNA is produced from BNYVV RNA3 23 .…”

Evolution of subgenomic RNA shapes dengue virus adaptation and epidemiological fitness

“…More importantly, these RNA structures halt diverse exoribonucleases to produce sfRNA, not only Xrn1 exoribonuclease (MacFadden A et al, 2018). Therefore, we refer herein to these RNA structures as flaviviral nuclease-resistant RNA (fNR) structures, as initially termed by Piljman et al (2008), and to differentiate them from the unrelated nuclease-resistant RNA structures in dianthoviruses (Steckelberg AL et al, 2018).…”