Abstract
Subgenomic flaviviral RNAs (sfRNAs) are virus-derived noncoding RNAs produced by pathogenic mosquito-borne flaviviruses (MBF) to counteract the host antiviral response. To date, the ability of non-pathogenic flaviviruses to produce and utilise sfRNAs remains largely unexplored, and it is unclear what role XRN1 resistance plays in flavivirus evolution and host adaptation. Herein the production of sfRNAs by several insect-specific flaviviruses (ISFs) that replicate exclusively in mosquitoes is shown, and the secondary structures of their complete 3’UTRs are determined. The xrRNAs responsible for the biogenesis of ISF sfRNAs are also identified, and the role of these sfRNAs in virus replication is demonstrated. We demonstrate that 3’UTRs of all classical ISFs, except Anopheles spp-asscoaited viruses, and of the dual-host associated ISF Binjari virus contain duplicated xrRNAs. We also reveal novel structural elements in the 3’UTRs of dual host-associated and Anopheles-associated classical ISFs. Structure-based phylogenetic analysis demonstrates that xrRNAs identified in Anopheles spp-associated ISF are likely ancestral to xrRNAs of ISFs and MBFs. In addition, our data provide evidence that duplicated xrRNAs are selected in the evolution of flaviviruses to provide functional redundancy, which preserves the production of sfRNAs if one of the structures is disabled by mutations or misfolding.
Highlights
The Flavivirus genus can be divided into the following ecological groups: mosquito-borne flaviviruses (MBFs), which circulate between mosquito and vertebrate hosts; tick-borne flaviviruses (TBFs) that are maintained in nature in tick-vertebrate cycle[1]; viruses that only infect vertebrates and are thought to be transmitted horizontally[2] and insect-specific flaviviruses (ISFs) that infect mosquitoes or sand flies and are believed to circulate predominantly via vertical transmission[3]
To determine whether phylogenetically divergent classical and dual host-associated ISFs are capable of subgenomic flaviviral RNA (sfRNA) production, we performed a Northern blot of total RNA isolated from C6/36 cells infected with Aedes-associated classical ISFs (cISFs) Parramatta River Virus (PaRV)[29], Culex-associated cISF Palm Creek virus (PCV)[30] and dISFs Binjari virus (BinJV)[31,32] and Hidden valley virus (HVV)[32]
Through the time course of the experiment (Supplementary Fig. 2B) and reduced production of sfRNAs by PaRV and PCV by approximately 50% (Fig. 1B, Supplementary Fig. 2C). It resulted in decreased accumulation of BinJV sfRNA-1 and -2 by 50% and 80%, respectively (Fig. 1B, Supplementary Fig. 2C). These results show that sfRNAs in cISF and major sfRNA species in dISFs are produced via incomplete digestion of 3’UTRs by XRN1
Summary
Hall[1,2], Subgenomic flaviviral RNAs (sfRNAs) are virus-derived noncoding RNAs produced by pathogenic mosquito-borne flaviviruses (MBF) to counteract the host antiviral response. The ability of non-pathogenic flaviviruses to produce and utilise sfRNAs remains largely unexplored, and it is unclear what role XRN1 resistance plays in flavivirus evolution and host adaptation. The production of sfRNAs by several insect-specific flaviviruses (ISFs) that replicate exclusively in mosquitoes is shown, and the secondary structures of their complete 3’UTRs are determined. Viral genomic RNA is subjected to degradation by the host 5’-3’ exoribonuclease XRN1. Stalling of XRN1 prevents complete degradation of viral genomic RNA and results in accumulation of 3’UTR-derived sfRNAs in the infected cells[7] Multiple studies have demonstrated that sfRNAs facilitate viral replication and pathogenesis by inhibiting IFN response in vertebrates[8–12] and RNAi13,14 or apoptosis[15] in mosquitoes. All flavivirus xrRNAs characterized to date are formed by the stem-loops (SL) that contain a three-way junction between three RNA helices (P1, P2 and P3) and a pseudoknot (PK) formed by the terminal loop (L2)
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