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. 2023 Jan 23;17(1):e0011098.
doi: 10.1371/journal.pntd.0011098. eCollection 2023 Jan.

Functional characterization of 5' untranslated region (UTR) secondary RNA structures in the replication of tick-borne encephalitis virus in mammalian cells

Laura Upstone  1 , Robin Colley  2 , Mark Harris  1 , Niluka Goonawardane  1   2
Affiliations

Functional characterization of 5' untranslated region (UTR) secondary RNA structures in the replication of tick-borne encephalitis virus in mammalian cells

Laura Upstone et al. PLoS Negl Trop Dis. .

Abstract

Tick-borne Encephalitis Virus (TBEV) is an emerging flavivirus that causes neurological disorders including viral encephalitis of varying severity. Whilst secondary RNA structures within the 5' untranslated regions (UTRs) of many flaviviruses determine both virus replication and pathogenic outcomes in humans, these elements have not been systematically investigated for TBEV. In this study, we investigated the role of predicted RNA secondary elements of the first 107 nucleotides (nts) of the viral genome forming the stem-loop A (SLA). Experiments were performed in replicons and infectious TBEV system. This region comprises three distinct structures: 5' stem 0 (S0), stem-loop 1 (SL1) and stem-loop 2 (SL2). S0 was found to be essential for virus infection as mutations in the lower stem of this region significantly reduced virus replication. Point mutations in SL1 that preserved the Y-shape confirmation delayed viral RNA replication but did not abolish virus infectivity. Deletion of SL2 did not abolish infectivity but had a negligible effect on virus propagation. No correlation was observed between in vitro translation efficiency and virus infectivity, suggesting that the 5'UTR functions independently to virus translation. Together, these findings reveal distinct RNA elements within the 5'UTR that are essential for the stability and replication of viral RNA. We further identify changes in RNA folding that lead to altered TBEV infectivity and pathogenesis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. mFold structure of the 5′UTR of TBEV.
(A) Computer-simulated predictions of WT TBEV with schematic representation of specific mutations within the SLA (arrows). Single nucleotide changes for M3-M6 and M12 (non-infectious) are circled in red. M11 (infectious) is in green. Double loop deletions (M16-M18) are not shown. Proposed sequence elements within Stem-Loop A (SLA) are labelled. Each deleted region is depicted by a solid line. Non-lethal mutants are highlighted in green. The first 107 nucleotides of the WT (GenBank accession: AF069066) region was assessed using Mfold (available on http://www.unafold.org/mfold/applications/rna-folding-form.php).
Fig 2
Fig 2. Alignment between the 5′UTRs of tick-borne flaviviruses and constructed mutants (M).
Accession numbers are indicated. Reference sequences for Sof [44] are shown. The top line depicts Vs virus that was used to produce the infectious clone. Conserved regions are highlighted in orange. Elements of the RNA secondary structures are specified as S0, SL1, and SL2 within SLA. Deletions and substitutions are highlighted in white letters within black boxes. Numbers in front of the 5’UTR mutants correspond to the numbers in Fig 1.
Fig 3
Fig 3. Replication kinetics of the 5′UTR mutants.
(A) Schematic representation of the TBEV genome (B) and TBEV replicon in which the structural genes were replaced with firefly (cu) luciferase, and Foot-and-mouth disease virus 2A site (FMDV-2A). The NS5 polymerase inactive site GAA is indicated. (C) Luciferase assays in PS cells co-transfected with WT or 5′UTR mutants over the indicated time course (12–72 h post-transfection [hpt]). (D) Representative quantification of pTK-Ren (Renilla) performed 24 h post-transfection (hpt), showing nonsignificant (ns) differences between mutants. Bar heights are the mean ± SEM of three biological replicates. Assays were performed in triplicate. *P = <0.01, **P = <0.001, ****P = <0.0001 versus WT determined using a one-way Anova with Bonferroni’s correction.
Fig 4
Fig 4. Infectivity of mammalian cells by 5′UTR mutants.
(A) Quantification of plaque assays from PS cell supernatants infected with 5’UTR WT and mutant viruses 24 h post-infection (hpi). **P<0.001, ***P<0.001, ****P<0.0001 versus WT from 24 or 48 hpi determined in two biological replicates (n = 3 technical repeats). Data were analysed using a one-way Anova with Bonferroni’s correction. (B) Expression of NS5 in PS cells transfected with WT or mutant viruses assayed over the indicated timeframe. Schematic representation of the mutants within the Y-structure are shown (arrows).
Fig 5
Fig 5. Translation of WT and SLA-mutant replicons.
(A) Translation rates of 5′UTR mutants in cell-free translation assays 30 min post initiation. NS5-GAA RNA was evaluated as a negative control. (B) Translation of input mutant RNA in PS cells at 3 hpt. Normalized luciferase levels are shown relative to WT. Replication-competent NS5-GAA RNA and ΔAUG, evaluated as a negative control. Data were derived from three biological replicates; error bars show standard deviation. ns indicates no significant difference from WT. Data were analysed using a one-way Anova with Bonferroni’s correction.
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