Structural analysis of 3’UTRs in insect flaviviruses reveals novel determinant of sfRNA biogenesis and provides new insights into flavivirus evolution

Abstract

Insect-specific flaviviruses (ISFs) circulate in nature due to vertical transmission in mosquitoes and do not infect vertebrates. ISFs include two distinct lineages - classical ISFs (cISFs) that evolved independently and dual host associated ISFs (dISFs) that are proposed to diverge from mosquito-borne flaviviruses (MBFs). Compared to pathogenic flaviviruses, ISFs are relatively poorly studied, and their molecular biology remains largely unexplored. In this study we focused on the characterisation of ISF 3′UTRs and their ability to produce subgenomic flaviviral RNAs — noncoding viral RNAs that are known as important determinants of transmission and replication of pathogenetic flaviviruses. We demonstrated that cISFs and dISFs produce sfRNAs by employing a highly conserved mechanism of resistance to degradation by the cellular 5′-3′ exoribonuclease XRN1. We determined the secondary structures of complete 3′UTRs and experimentally identified structured RNA elements that resist degradation by XRN1 (xrRNAs) in divergent representatives of cISF and dISF clades. We discovered a novel class of xrRNAs in dISFs and identified structurally divergent xrRNA in Anopheles-associated cISFs. Phylogenetic analyses based on sequences and secondary structures of xrRNAs and complete 3′UTRs reveal that xrRNAs of cISFs and MBFs/dISFs evolved from a common xrRNA ancestor similar to the xrRNA of Anopheles-associated cISFs. Additionally, we found that duplications of xrRNAs occurred independently in ISF and MBF clades. Using ISF mutants deficient in the production of sfRNAs, we found that individual sfRNAs of ISFs have redundant functions. We conclude that duplicated xrRNAs were selected in the evolution of flaviviruses to ensure that sfRNA is produced if one of the xrRNAs lose XRN1 resistance due to mutations or misfolding.

A tangled threesome: understanding arbovirus infection in Aedes spp. and the effect of the mosquito microbiota

“…These non-coding regions have complex secondary structures that interfere with helicases from the host cell, recruiting and kidnapping the machinery for the recognition and degradation of vRNA, thus helping the virus spread (Pompon et al, 2017;Yeh and Pompon, 2018;Göertz et al, 2019). This evasion mechanism is conserved in ISV flaviviruses (Slonchak and Khromykh, 2018;Slonchak et al, 2022). It is possible that co-infection with ISV could also facilitate subsequent arbovirus infections by reducing the activity of iRNA with their sfRNA, this could also help to evade response through vDNA.…”

RNase L-induced bodies sequester subgenomic flavivirus RNAs and re-establish host RNA decay

“…sfRNAs are generated when exoribonuclease 1 (XRN1), which is the primary 5'->3' cellular RNA decay exoribonuclease that degrades decapped mRNAs, [10][11][12][13][14][15] stalls on a dumbbell hairpin structural element that is conserved across the genus flavivirus during 5'-3' decay of the flavivirus genome. [16][17][18][19] The sfRNA structures contribute to cytopathogenic effects in culture mammalian cells and pathogenicity in mouse models. 9 However, the functions of sfRNAs are not fully understood.…”