Fig. 2: SARS-CoV-2 ORF10 blocks the RLR pathway by downregulating the expression of MAVS.

a HEK293T cells were cotransfected with the IFN-β promoter plasmid (60 ng), Renilla luciferase reporter plasmid (pRL-TK; 60 ng), HA-MDA5 plasmid (80 ng), Flag-RIG-I(N) plasmid (80 ng), Flag-MAVS plasmid (80 ng), TBK1-HA plasmid (80 ng) or IRF3(5D)-HA plasmid (80 ng) and empty vector or the ORF10-HA plasmid. Luciferase activity in cell lysates was measured 24 h after transfection. b HeLa cells were transfected with ORF10-HA or pCMV-HA for 24 h and were then treated with poly(I:C) (20 μg/mL). The cell lysates were subjected to immunoblot analysis with the indicated antibody. c Immunoblot analysis of the expression of MAVS in HeLa cells transfected with the ORF10-GFP plasmid (0, 400, 800 and 1600 ng) and then treated with poly(I:C) (20 μg/mL). d HeLa cells transfected with the ORF10-GFP plasmid and pEGFP-N1 were stimulated by poly(I:C) transfection and were then treated with MG132 (5 μM), bafilomycin A1 (Baf A1; 5 nM), or chloroquine (CQ; 40 μM) for 24 h. The cell lysates were then analyzed by western blotting. e HeLa cells were transfected with the ORF10-HA or pCMV plasmid for 24 h. The cell lysates were then analyzed by western blotting. f HeLa cells were seeded in 12-well plates and cotransfected with the mCherry-GFP-LC3 plasmid and the ORF10-HA plasmid or transfected with only mCherry-GFP-LC3 (vector). The cells were incubated for 36 h for immunofluorescence analysis. Scale bars: 10 µm. Data from three independent experiments were analyzed using a t test (a) and are presented as the mean ± SD values (*p < 0.05, **p < 0.01, and ***p < 0.001; ns indicates a nonsignificant difference)