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

Figure 4.

Figure 4.

Inhibition of METTL3 activity suppresses β-coronavirus replication. (A) MRC-5 cells were infected with HCoV-OC43 at MOI = 0.001 for 48 h in the presence of METTL3 inhibitor (STM2457, yellow), inactive control compound (STM2120, gray), or vehicle (DMSO, white) at the indicated concentrations. Identification of infected cells by indirect immunofluorescence for nucleocapsid protein was as described in Figure 1A. (B) A549+ACE2 cells were infected with icSARS-CoV-2-mNG at MOI = 0.1 for 48 h, and the percentage of cells infected was established by green fluorescence and normalized to infection of nontreated cells. (C, D) The viability of MRC-5 cells (C) and A549+ACE2 cells (D) in the presence of concentrations of STM2120 or STM2457 used in the infection assays shown in A and B was assessed using a commercial ATP quantitation assay. Cells were maintained at either 33°C or 37°C, respectively, in culture medium containing diluted compound for 48 h prior to lysis. Each experiment was conducted three times with internal duplicates, normalized to DMSO-treated cells processed in parallel and plotted as the mean ± SEM. (E) Representative montages showing wells from the infections quantified in A and B that were treated with 30 µM STM2120 or STM2457 and infected with either icSARS-CoV-2-mNG or HCoV-OC43 as indicated. The signal for the OC43-N antibody and Alexa Fluor 647 secondary antibody is represented in green. (F) Infectious viral titers from MRC-5 cells infected with HCoV-OC43 at MOI = 0.001 treated with 30 µM either STM2120 or STM2457 was determined by TCID50 assay. (G) Infectious virus titers from A549+ACE2 cells infected with icSARS-CoV-2-mNG at MOI = 0.1 and treated with 30 µM either STM2120 or STM2457 was determined by plaque assay. (H,I) MRC-5 and A549+ACE2 cells were infected with OC43 or icSARS-CoV-2-mNG, respectively, as in (A) and (B) in the presence of 30 µM STM2120 or STM2457 and 10 µM JAK inhibitor (pyridone-6) or vehicle control (DMSO) and the percent infected cells quantified. Each experiment was conducted three times with internal duplicates, normalized to DMSO-treated cells processed in parallel, and plotted as the mean ± SEM. (J) Immunoblot analysis of lysates from MRC-5 cells infected with HCoV-OC43 at MOI = 0.001 in the presence of 30 µM STM2120 or STM2457 as in A and collected at 48 hpi and probed for viral N protein, or host ISGs (ISG15, RIG-I, PKR, and MDA5) and GAPDH.