Fig. 7: SELECT and mutagenesis analyses of DENV RNA reveal no m6A modification.
From: N6-methyladenosine modification is not a general trait of viral RNA genomes
a SELECT was performed using total RNA extracted from either Huh7 cells infected with DENV-2 (wild type) for 48 h at an MOI of 2 or from HEK293T cells infected for 96 h at an MOI of 0.5. Additionally, SELECT reactions were carried out with a ssDNA containing the modified DRACH motif in the SLC39A14 RNA, as well as with DENV-2 in vitro transcribed (IVT) RNA. The threshold cycle difference of amplification (ΔCT) of total RNA versus IVT ΔCT is shown for each motif (1–13). If total RNA ΔCT = IVT ΔCT, values would align on the solid diagonal line. If there is a difference of −1 CT cycle between the total RNA ΔCT and the IVT ΔCT, which would indicate the motif is m6A-modified, values would align on the diagonal discontinuous line. All experiments were performed using three technical replicates (separate SELECT reactions). All CT values from SELECT results can be found in Supplementary Figs. 7 and 8 and Source Data. b Previously elucidated conserved RNA elements in DENV-2 RNA55 are shown in blue. The m6A peak (9114–9414 nt) conserved across all m6A-Seq studies is shown in purple. c Schematic diagram of the conserved RNA loop (yellow circle) in DENV-2 wild type (WT) identified by SHAPE-informed structure analysis55, which contains DRACH motif 8 (AAACA, underlined nucleotides). The blue rectangle highlights the similar structure found in DENV-1, DENV-2, and DENV-4 serotypes55. The putative modified adenosine is at position 9293. This schematic diagram is adapted from Boerneke et al.55. d Schematic diagram of DENV-2 mutant (Mut) virus, carrying a mutation where the cytosine (at position 9294) within the DRACH motif 8 is changed to uridine (shown in red). e SELECT was performed using total RNA extracted from either Huh7 cells or from HEK293T cells (both infected with DENV-2 Mut for 96 h at an MOI of 0.5) to test the DENV-2 mutant motif 8. Additionally, SELECT reactions were carried out with DENV-2 mutant in vitro transcribed (IVT) RNA as control. All experiments were performed using three technical replicates (separate SELECT reactions). The bar chart shows mean values from three SELECT reactions with the error bars showing SD. ΔCT values for SELECT reactions using total RNA extracted from DENV-2 wild type (WT)-infected cells and corresponding DENV-2 WT IVT are shown for comparison. Source data are provided with this paper.
