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. 2021 Jul 1;35(13-14):1005–1019. doi: 10.1101/gad.348320.121

Figure 5.

Figure 5.

Initial HCoV-OC43 RNA synthesis and protein expression is reduced by inhibition of METTL3 catalysis. (A) Immunoblot analysis of lysates from MRC-5 cells infected with HCoV-OC43 at MOI = 3 in the presence of 30 µM STM2120 or STM2457 collected at 24, 48, or 72 hpi and probed for viral N protein, or host METTL3 and GAPDH. (B) Representative images showing detection of viral N protein or viral dsRNA by indirect immunofluorescence assay (green) in HCoV-OC43-infected MRC-5 cells at 24 hpi in the presence of 30 µM STM2120 or STM2457. Cell nuclei were stained with DAPI. Scale bar, 20 µm. (C). The relative abundance of HCoV-OC43 gRNA (ORF1ab) or sgRNA (N) or host GAPDH at 24 hpi MOI = 3 in the presence of 30 µM STM2120 or STM2457 was determined by RT-qPCR using transcript-specific primers and normalizing to 18S rRNA and plotted as the mean ± SEM (n = 3). (D) MRC-5 cells infected with HCoV-OC43 at MOI = 3 in the presence of 30 µM STM2120 or STM2457 were metabolically pulse-labeled with 35S amino acids for 1 h. Lysates were separated by SDS-PAGE and the fixed, dried gel exposed to film. Migration of molecular weight standards is shown at the left. The arrow at the right indicates migration of virus-encoded N protein. Additionally, the relative levels of total eIF2α and phospho-eIF2α in the same lysates was assessed by immunoblotting. Note that an ultrasensitive enhanced chemiluminescent substrate (SuperSignal West Femto) was required to visualize the very low levels of phospho-eIF2α. (E, left) Cytoplasmic lysates from HCoV-OC43-infected MRC-5 cells treated with STM2120 (gray) or STM2457 (yellow) for 24 h prior to harvest were fractionated over a 10%–50% sucrose gradient, and the absorbance at 254 nm is shown with ribosomal peaks indicated. (Right) The relative abundance of viral N and ORF1a/b RNAs and host GAPDH mRNA in each gradient fraction was determined by RT-qPCR, and the sum of polysomal mRNA (fractions 13–20) under each drug treatment is presented. A representative experiment of two biological replicates in which similar results were obtained is shown.