Fig 1: Functional impact of tRNA-modifying enzymes in coronavirus infection.A–C Representative western blots showing siRNA-mediated knockdown of ELP3 (mcm⁵U/mcm⁵s ≤ U pathway), QTRT1 (Q pathway), and NSUN2 (m⁵C pathway) in HCoV-OC43–infected A549 cells (MOI 0.1). HCoV-OC43 N protein (NP) levels were measured by western blot at 48 hpi. A non-targeting siRNA (siRNA-scr) was used as a control. D–F Quantification of ELP3, QTRT1, NSUN2, and viral NP protein levels in siRNA-treated cells relative to non-targeting siRNA (scr) controls. Vinculin and GAPDH were used as loading controls. Measurements were obtained from independent biological replicates (n = 4 for panel D; n = 2 for panels E and F). Statistical significance was assessed using a two-sided, nonparametric Mann–Whitney U test (unpaired). D ELP3 levels: p = 0.003 for mock siRNA-scr vs mock siRNA-ELP3; p = 0.0137 for HCoV-OC43 siRNA-scr vs HCoV-OC43 siRNA-ELP3; NP levels: p = 0.0057 for HCoV-OC43 siRNA-scr vs HCoV-OC43 siRNA-ELP3. E QTRT1 levels: p = 0.17 for mock siRNA-scr vs mock siRNA-QTRT1; p = 0.0724 for HCoV-OC43 siRNA-scr vs HCoV-OC43 siRNA-QTRT1; NP levels: p = 0.042 for HCoV-OC43 siRNA-scr vs HCoV-OC43 siRNA-QTRT1. F NSUN2 levels: p = 0.043 for mock siRNA-scr vs mock siRNA-NSUN2; p = 0.071 for HCoV-OC43 siRNA-scr vs HCoV-OC43 siRNA-NSUN2; NP levels: p = 0.91 for HCoV-OC43 siRNA-scr vs HCoV-OC43 siRNA-NSUN2. Data are presented as mean ± s.d. Individual data points are shown. G Plasmid-mediated overexpression (OVE) of ADAT2 (I pathway) and ALKBH1 (hm⁵C/f⁵C pathway) in HCoV-OC43 (MOI 0.1) infected A549 cells. HCoV-OC43 NP levels was measured by western blot at 48 hpi. Empty vector transfection (pcDNA3) was used as a control. Vinculin was used as loading control. H Quantification of viral NP protein levels from independent biological replicates in plasmid-transfected cells relative to empty vector–transfected controls. Statistical significance was assessed using a two-sided, nonparametric Mann–Whitney U test (unpaired; n = 4). Exact p-values are p < 0.0001 for HCoV-OC43–empty vs HCoV-OC43–ADAT2 overexpression (OVE) and p < 0.0001 for HCoV-OC43–empty vs HCoV-OC43–ALKBH1 overexpression (OVE). Data are presented as mean ± s.d. Individual data points are shown.