New hypotheses derived from the structure of a flaviviral Xrn1-resistant RNA: Conservation, folding, and host adaptation

Abstract

Arthropod-borne flaviviruses (FVs) are a growing world-wide health threat whose incidence and range are increasing. The pathogenicity and cytopathicity of these single-stranded RNA viruses are influenced by viral subgenomic non-protein-coding RNAs (sfRNAs) that the viruses produce to high levels during infection. To generate sfRNAs the virus co-opts the action of the abundant cellular exonuclease Xrn1, which is part of the cell's normal RNA turnover machinery. This exploitation of the cellular machinery is enabled by discrete, highly structured, Xrn1-resistant RNA elements (xrRNAs) in the 3′UTR that interact with Xrn1 to halt processive 5′ to 3′ decay of the viral genomic RNA. We recently solved the crystal structure of a functional xrRNA, revealing a novel fold that provides a mechanistic model for Xrn1 resistance. Continued analysis and interpretation of the structure reveals that the tertiary contacts that knit the xrRNA fold together are shared by a wide variety of arthropod-borne FVs, conferring robust Xrn1 resistance in all tested. However, there is some variability in the structures that correlates with unexplained patterns in the viral 3′ UTRs. Finally, examination of these structures and their behavior in the context of viral infection leads to a new hypothesis linking RNA tertiary structure, overall 3′ UTR architecture, sfRNA production, and host adaptation.

RNA Structure Duplications and Flavivirus Host Adaptation

“…After removal of the cap structure present at the 5’ end of flavivirus genomes, the RNA is degraded by the host exoribonuclease XRN1 from the 5’ to the 3’ direction [42]. The biogenesis of sfRNAs is linked to highly structured RNA elements (xrRNAs) present at the viral 3’ UTR, which stall the XRN1 exonuclease activity [51–53]. The process results in the accumulation of small RNA molecules with nucleotide sequences corresponding to viral 3’ UTRs.…”

“…Most MBFV genomes display conserved structural blocks that were defined as SLs or DBs [31]. For clarity the names SLI and SLII are used to refer to the duplicated conserved elements present in region I of MBFV 3’ UTRs, included in the structures also known as xrRNA1 and xrRNA2, respectively [52–53]. …”

RNA Localization in Bacteria

“…In this approach, the 3’UTR is engineered to include two viral pseudoknots (PKs) flanked by PP7 and MS2 binding sites (Figure 3E). Since the PK structures block 5’-to-3’ degradation of the transcript by the 5’−3’ exoribonuclease Xrn1 (142), TREAT allows one to distinguish between intact and partially degraded mRNAs. The feasibility of applying such functional reporters to bacterial systems remains to be tested.…”

Noncoding Subgenomic Flavivirus RNA Is Processed by the Mosquito RNA Interference Machinery and Determines West Nile Virus Transmission by Culex pipiens Mosquitoes

“…sfRNA is produced via a unique mechanism involving degradation of the viral genomic RNA (vgRNA) by the host 5=¡3= exoribonuclease XRN1/Pacman (25). XRN1 stalls at stem-loop (SL) and dumbbell (DB) RNA structures within the flaviviral 3= untranslated region (UTR), resulting in the accumulation of sfRNA (26)(27)(28)(29). The stalling of XRN1 occurs due to steric hindrance caused by interactions of pseudoknots (PK) and other tertiary RNA structures (30).…”