RNA BIOLOGY 2021, VOL. 18, NO. 5, 709–717 https://doi.org/10.1080/15476286.2020.1830238 RESEARCH PAPER Xrn1-resistant RNA structures are well-conserved within the genus flavivirus Ivar W. Dilweg a, Assia Bouabdab, Tim Daleboutb, Alexander P. Gultyaevc,d, Peter J. Bredenbeekb, and R. C. L. Olsthoorna aLeiden Institute of Chemistry, Leiden University, Leiden, The Netherlands; bDepartment of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands; cLeiden Institute of Advanced Computer Science, Leiden University, Leiden, The Netherlands; dDepartment of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands ABSTRACT ARTICLE HISTORY Subgenomic RNAs are produced by several RNA viruses through incomplete degradation of their Received 13 August 2020 genomic RNA by the exoribonuclease Xrn1, and have been shown to be essential for viral growth and Revised 24 September 2020 pathogenicity. Within the flavivirus genus of the Flaviviridae family, two distinct classes of Xrn1-resistant Accepted RNA motifs have been proposed; one for mosquito-borne and insect-specific flaviviruses, and one for 25 September 2020 tick-borne flaviviruses and no-known-vector flaviviruses. We investigated tick-borne and no-known- KEYWORDS vector flavivirus Xrn1-resistant RNA motifs through systematic in vitro mutational analysis and showed Xrn1; flavivirus; RNA that both classes actually possess very similar structural configurations, including a double pseudoknot pseudoknot; and a base-triple at identical, conserved locations. For the no-known-vector flavivirus Modoc virus, we exoribonuclease-resistant show that in vivo generation of subgenomic flaviviral RNA was affected by mutations targeted at RNA; sfRNA nucleotides involved in the structural features of flaviviral Xrn1-resistant RNA motifs that were defined in this work. Our results suggest that throughout the genus flavivirus Xrn1-resistant RNA motifs adopt the same topologically conserved structure. ARTICLE HISTORY Received 13 August 2020; Revised 24 September 2020; Accepted 25 September 2020 Introduction stem-loop and pseudoknot structures that are generally well conserved, especially among flaviviruses belonging to the same The family Flaviviridae comprises four genera: flaviviruses, subgroup. This region has been shown to be responsible for the pestiviruses and the more recently added genera hepaci- and accumulation of noncoding subgenomic flaviviral RNA pegiviruses [1–3]. They are all enveloped, positive-stranded (sfRNA) in infected cells, resulting from stalling of the host RNA viruses with genomes ranging between 10 to 12.5 kb. exoribonuclease Xrn1 on Xrn1-resistant RNA (xrRNA) struc­ Based on their mode of transmission, the flaviviruses can be tures after degrading roughly 95% of the flavivirus genome [9– further subdivided into mosquito-borne flaviviruses (MBFV), 12]. These sfRNAs are implicated to interfere with cellular tick-borne flaviviruses (TBFV), no-known-vector flaviviruses RNA decay, RNAi pathway, and the immune response [9,13– (NKVFV) and insect-specific flaviviruses (ISFV). Infection of 15]. Following a flavivirus infection, Xrn1 activity is decreased, a suitable host by either the mosquito- or tick-borne flavivirus which suggests that it may be sequestered by stalling on xrRNA strictly depends on an arthropod vector, whereas infection by structures, thereby altering cellular RNA homeostasis to benefit the mainly bat- or small rodent-infecting NKVFVs appears to viral replication [16]. Currently, the generation of sfRNA due occur via direct contact with infected individuals. ISFVs repli­ to xrRNA in the 3ʹ UTR has only been demonstrated in vivo by cate solely in insects and are unable to replicate in members of the flavivirus genus. Analysis of intracellular RNA a mammalian host. The arthropod-borne flaviviruses are par­ isolated from cells infected with the pestivirus BVDV and ticularly associated with microcephaly and harmful diseases hepacivirus HCV previously did not show the production of like haemorrhagic fever, encephalitis and dengue fever in sfRNA [9]. However, structures in the 5ʹ UTR of BVDV and humans. HCV are capable of stalling and sequestering Xrn1, thereby The flavivirus genome is flanked by a capped 5ʹ untranslated altering mRNA longevity [17]. Production of sfRNA was region (UTR) that mediates translation initiation and revealed in nematodes infected with the unclassified soybean- a relatively long, highly structured, non-polyadenylated 3ʹ cyst nematode virus 5 [18]. Due to its homology, this virus was UTR [4,5]. The RNA structure of the distal part of this 3ʹ initially identified as a pestivirus, however it was then moved UTR is relatively well conserved within each of the four distin­ into the flavivirus genus, in part because of its production of guishable groups of flaviviruses. It has been shown to contain subgenomic RNA. conserved RNA sequences for base pairing with complemen­ The production of sfRNA requires the presence of an intri­ tary sequences at the 5ʹ end of the genome, which is critical for cate xrRNA structure that consists of five stem elements includ­ replication [6–8]. The flavivirus 3ʹ UTR carries several RNA ing two pseudoknot interactions [19]. These are oriented in CONTACT R. C. L. Olsthoorn [email protected] Leiden Institute of Chemistry, Leiden University, Leiden 2300 RA, The Netherlands Supplemental data for this article can be accessed here. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. 710 I. W. DILWEG ET AL. such a way that they can halt Xrn1’s processive degradation in 10 μl volumes, containing 5 μl PCR product (~250 ng), mode. The xrRNA crystal structures of the MBFVs Zika virus 5 mM of each rNTP, 1 μl Enzyme mix, in 1x Transcription (ZIKV) and Murray Valley encephalitis virus (MVEV) have Optimized buffer (40 mM Tris-HCl, 6 mM MgCl2, 2 mM been resolved and appear to form a small ring-like RNA struc­ spermidine, 10 mM NaCl, pH7.9 @ 25°C). After incubation at tural fold in which the 5ʹ end of the viral RNA protrudes from 37°C for 30 mins, 1 unit RQ1 RNase-Free DNAse was added the back to the front [20,21]. The diameter of the ring is too to the reaction and incubation proceeded at 37°C for 20 mins. small for Xrn1 to protrude and continue to hydrolyse the RNA; Reaction samples were checked on agarose gel in order to therefore the enzyme is stalled. This structural analysis, com­ establish subsequent usage of equal amounts of RNA. Xrn1 bined with subsequent mutational studies, has revealed the digestion reactions were performed with ~400 ng RNA in 1x requirement of additional nucleotide interactions that are criti­ NEB3 buffer totalling 10 μl, which was equally divided over cal for a functional stalling site, including a strongly conserved two tubes. To one of the tubes, 0.2 units of Xrn1 and 0.3 units base triple [20,21]. Analysis of the xrRNA present in cell-fusing of RppH (New England Biolabs) were added. Both tubes were agent virus (CFAV), a typical ISFV, has indicated a highly incubated for 30 mins at 37°C and the reactions were termi­ similar structure and the absolute requirement of nated by adding 5 μl formamide containing trace amounts of a homologous base triple, thus implying that MBFV and ISFV bromophenol blue and xylene cyanol FF. After addition of xrRNA stall Xrn1 in a similar fashion [19]. In contrast, the tick- 5 μl denaturing loading buffer (8 M urea, 20 mM Tris-HCl, borne and no-known-vector flaviviruses have been suggested to 20 mM EDTA, trace amounts of bromophenol blue and employ a slightly different structure to resist Xrn1, which xylene cyanol FF) samples were denatured for five minutes endorsed a distinction between these two classes of xrRNA at 75°C. These were run on 8 M urea 10% polyacrylamide gels structures (‘class I’ for MBFV and ISFV xrRNAs and ‘class II’ in TBE buffer, equilibrated at 60–65°C. Gels were stained with for TBFV and NKVFV xrRNAs). However, in the absence of EtBr and each construct was subjected to this assay at least a solved 3D structure for these class II xrRNAs, this distinction twice. Bands were quantified using the Quantity One may be preliminary. 1-D analysis software. Xrn1 stalling positions were estimated In this study, we examined the generation of sfRNA by using the y-positions of untreated RNAs and their corre­ tick-borne encephalitis virus (TBEV), Modoc virus (MODV) sponding sizes, from which an exponential function was for­ and Rio Bravo virus (RBV) – viruses harbouring xrRNA mulated that was used for estimating the sizes of Xrn1-treated motifs of the proposed class II-type – using manual modelling RNAs. based on phylogeny, and in vitro Xrn1-degradation assays. Through site-directed mutagenesis, important features in the In vitro RNA transcription of infectious full length MODV three-dimensional structures responsible for stalling Xrn1 genome RNA were investigated. In doing so, conserved structural elements were identified, which raises the question whether the distinc­ Details on the construction and characterization of the tion between flaviviral xrRNA classes is justified. Using MODV infectious cDNA clone that was fused at the 5ʹ end MODV, the effect of these mutations was further verified by to a T7 ø2.5 promoter [22] in the low copy number vector implementing them into an infectious clone in order to assess pACNR, will be published elsewhere (Jiang et al., manuscript their ability to generate sfRNA in vivo. in preparation). Plasmid DNA for in vitro run-off RNA transcription was purified using the Nucleobond Xtra Maxi DNA isolation kit (Macherey-Nagel, Düren, Germany) and Materials and methods linearized with AflII at a unique site directly adjacent to the MODV 3ʹ UTR.