Figure 2.
HCoV-OC43 infection results in the nuclear accumulation of METTL3, YTHDF1, and YTHDF2. (A) Lysates from uninfected (UI) or HCoV-OC43-infected MRC-5 cells were prepared at 8, 24, 48, and 72 h postinfection at MOI = 3 and probed by immunoblotting using antibodies to methyltransferase subunits (METTL3, METTL14, WTAP, RBM15, and RBM15B), demethylases (ALKBH5, FTO) and m6A binding proteins (YTHDC1, YTHDF1, YTHDF2, and YTHDF3). Arrowheads denote target bands. (B) Indirect immunofluorescence images of representative uninfected or HCoV-OC43-infected MRC5 cells (24 hpi, MOI = 3) with primary antibodies to METTL3, YTHDF1, YTHDF2, and YTHDF3. Scale bar, 10 µm. (C) Quantitation of nuclear signal for each primary antibody used in panel B after normalization to background fluorescence. Analysis was performed on ≥100 cells/condition and statistical significance determined using an F-test. (****) P ≤ 0.0001, (**) P ≤ 0.0047, (ns) not significant. (D) Uninfected and HCoV-OC43-infected MRC-5 cells (as in B) were rapidly lysed to produce insoluble particulate (nuclear [N]) and soluble (cytosolic [C]) fractions and probed by immunoblotting using antibodies to METTL3, YTHDF1, YTHDF2, and YTHDF3. Lysis and subcellular fractionation efficiency were assessed using antibodies to cytoplasmic β-tubulin and nuclear histone H3.