Figure 2.

SARS-CoV-2 N protein inhibits TRIM25-mediated RIG-I activation to counteract IFN-β secretion. Lenti-X 293T cells were co-transfected with the IFN-β-Luc Firefly luciferase reporter plasmid and the Renilla luciferase control reporter plasmid along with RIG-I- and TRIM25-expressing plasmids. Where indicated, fixed (A) or increasing (B) amounts of SARS-CoV-2 N were co-expressed. (C) Wild-type RIG-I was replaced by its T₅₅I mutated counterpart. Cells were collected at 48 h post-transfection, prior to 12 h of stimulation with poly(I:C). The luciferase activity of the cell lysates was analyzed with the dual luciferase reporter assay system (Promega). Fold changes in IFN-β promoter activation were calculated with respect to the relative reference sample based on Firefly luciferase values normalized to Renilla luciferase activities. (D) The specificity of SARS-CoV-2 N on TRIM25 was achieved by enhancing RIG-I activation through the over-expression of the stimulatory protein Riplet or (E) solely on TRIM25 expression. Luciferase assays were assessed and the fold change in activation was calculated as previously described. Results were plotted as mean values (n = 4) ± standard deviation (SD), and significance was determined by p-values 0.005 < ** p < 0.01, 0.01 < * p < 0.05). Cell lysates from reporter gene assays were analyzed by immunoblotting. Fifty micrograms of WCLs were loaded on SDS-PAGE and probed with specific monoclonal antibodies for protein detection.