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Structural Analysis of 3′Utrs in Insect Flaviviruses

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Structural Analysis of 3′Utrs in Insect Flaviviruses bioRxiv preprint doi: https://doi.org/10.1101/2021.06.23.449515; this version posted June 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Structural analysis of 3’UTRs in insect flaviviruses reveals novel determinant of sfRNA biogenesis and provides new insights into flavivirus evolution Andrii Slonchak1,#, Rhys Parry1, Brody Pullinger1, Julian D.J. Sng1, Xiaohui Wang1, Teresa F Buck1,2, Francisco J Torres1, Jessica J Harrison1, Agathe M G Colmant1, Jody Hobson- Peters1,3, Roy A Hall1,3, Andrew Tuplin4, Alexander A Khromykh1,3,# 1 School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, Australia 2Current affiliation: Institute for Medical and Marine Biotechnology, University of Lübeck, Lübeck, Germany 3Australian Infectious Diseases Research Centre, Global Virus Network Centre of Excellence, Brisbane, QLD, Australia 4 School of Molecular and Cellular Biology, University of Leeds, Leeds, U.K. # corresponding authors: [email protected]; [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.23.449515; this version posted June 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 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. 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.23.449515; this version posted June 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Introduction Flaviviruses are generally known as important human pathogens capable of causing severe diseases and unpredictable outbreaks. However, the flavivirus genus is very diverse and includes viruses that cannot replicate in vertebrates. This group of viruses, known as insect-specific flaviviruses (ISF), recently attracted significant attention due to their ability to inhibit replication of pathogenic flaviviruses in co-infected mosquitoes1 and their potential use as a safe recombinant vaccine platform2. The Flavivirus genus can be divided into the following ecological groups: mosquito-borne flaviviruses (MBFs), which circulate between mosquito and vertebrate (avian, equine or human) hosts; tick-borne flaviviruses (TBFs) that are maintained in nature in tick-vertebrate cycle3; viruses that only infect vertebrates and are thought to be transmitted horizontally4 (no known vector flaviviruses, NKVFs) and insect- specific flaviviruses (ISFs) that infect mosquitoes or sand flies and are believed to circulate predominantly via vertical transmission5. ISFs include two phylogenetically distinct lineages – (i) lineage I or classical ISFs (cISFs) that independently diverged from an ancient flavivirus ancestor and (ii) lineage II or dual host associated ISFs (dISFs) that are believed to be evolved from MBFs that lost the ability to infect vertebrates5,6. To replicate in diverse hosts, flaviviruses have evolved multiple mechanisms to counteract, subvert and evade host antiviral responses. One of them is by producing noncoding viral RNA named subgenomic flaviviral RNA (sfRNA) (reviewed in7). In infected cells, viral genomic RNA is subjected to degradation by the host 5’-3’ exoribonuclease XRN1. XRN1 is a highly processive enzyme with a helicase activity, which can unwind and fully digest virtually any RNA8. However, flaviviruses contain uniquely folded RNA elements in their 3’UTRs that can halt the progression of XRN19. Stalling of XRN1 prevents complete degradation of viral genomic RNA and results in accumulation of 3’UTR-derived sfRNAs in the infected cells10 (Supplementary figure 1A). Multiple studies have demonstrated that 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.23.449515; this version posted June 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. sfRNAs facilitate viral replication and pathogenesis by inhibiting IFN response in vertebrates11–15 and RNAi16,17 or apoptosis18 in mosquitoes. However, all these results were obtained using vertebrate-infecting dual host flaviviruses, while biogenesis and functions of sfRNAs in ISFs remain poorly characterized. To date, production of sfRNA was reported for only one ISF – cell fusing agent virus (CFAV)19. XRN1-resistant elements are believed to have conserved secondary and tertiary structures (Supplementary figure 1B, C) within each clade of flaviviruses7. Commonly, 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) of P2 helix (Supplementary figure 1C). In addition, xrRNAs contain a small pseudoknot (sPK) within a junction between P1 and P3 helices (Supplementary figure 1C). The pseudoknots and non-canonical interactions within xrRNAs determine their unique tertiary conformation in which the 5’-end of the RNA passes through a ring-like structure20,21 (Supplementary figure 1B). This unique fold determines the resistance of xrRNAs to XRN1 as the RNA ring creates a roadblock for the progression of the enzyme22 (Supplementary figure 1A). To date, secondary structures have been experimentally determined for xrRNAs of MBFs9,23,24, TBFs19, NKVFs19,25 and cISF CFAV19. In addition, crystal structures have been solved for three MBF xrRNAs20,21 and xrRNA of NKVF Tamana Bat virus25. Based on the conserved structural elements, xrRNAs are classified into two classes – class 1 xrRNAs occur in MBF, while class 2 xrRNAs occur in TBFs and NKVFs19. Within class 1, two additional subclasses can be distinguished – subclass 1a includes the typical MBF xrRNAs that contain 5-nt P1 helix, 5-nt pseudoknot and conserved unpaired nucleotide between P2 and P320 (Supplementary Figure 1C). Subclass 1b xrRNAs were identified in NKVF Tamana Bat virus (TABV) and contain 3-4-nt P1 helix, often with non-canonical A-C base pairing at the base of the helix, 3-nt pseudoknot and no 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.23.449515; this version posted June 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. unpaired bases between P2 and P325,26 (Supplementary Figure 1C). Based on covariance models and/or sequence alignments, dISFs were predicted to contain class 1a xrRNAs27, although these predictions have not been experimentally validated. 3’UTRs of MBFs typically contain duplicated SL-based xrRNAs followed by two dumbbell (DB) structures and a 3’-terminal stem-loop (3’SL)27. The duplicated SLs give rise to two sfRNAs of different length28. In addition, two smaller sfRNAs are produced by MBFs at the dumbbell structures. However, the biogenesis mechanism of these RNAs is unclear as dumbbells have been recently shown to lack XRN1 resistance29. Currently, it is not fully understood why MBFs acquired additional xrRNAs and whether different length isoforms of sfRNAs have redundant or specialised functions. One study demonstrated that Dengue virus 2 (DENV2) accumulates mutations in the individual xrRNAs and switch between the production of longer and shorter sfRNAs when switching hosts13. This suggests that duplication of the structural elements within 3’UTR represents an adaptation to the dual host life cycle, and individual sfRNA species are specifically adapted to function in different hosts. Although the production of sfRNAs by dISFs is yet to be shown, their 3’UTRs were predicted
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