The Membrane Protein of Severe Acute Respiratory Syndrome Coronavirus Functions as a Novel Cytosolic Pathogen-Associated Molecular Pattern To Promote Beta Interferon Induction via a Toll-Like-Receptor-Related TRAF3-Independent Mechanism

Wang, Yi; Liu, Li

HEK293T cells. After 0, 6, 12, and 24 h of transfection, dual-luciferase assays were performed to detect IFN-␤ expression. Each value represents the mean Ϯ standard deviation from three independent tests. *, P Յ 0.05; **, P Յ 0.01. In all data presented above, the relative luciferase activity was determined as firefly luciferase activity divided by Renilla luciferase activity. The effect of TBK1 siRNA (siTBK1) on the expression of endogenous TBK1 by semiquantitative RT-PCR. The increased doses of pBS/U6-siTBK1 plasmid DNAs (0, 0.5, and 2 g) were transiently transfected into HEK293ET cells. Total RNAs or whole-cell lysates were isolated or harvested at 48 h posttransfection. One-step RT-PCR (RT) was conducted to detect the TBK1 mRNA expression with specific primers (upper panel), while Western blotting (WB) was performed to detect TBK1 protein expression using specific anti-TBK1 antibody (lower panel). The expression of ␤-actin served as a loading control. The relative band intensity was quantitated with the Image J program in comparison with the ␤-actin control. The result is representative of at least 2 identical experiments. (C) Effect of siTBK1 on the expression of TBK1 mRNAs by real-time qRT-PCR analysis. Total RNAs isolated in panel B were subjected to qRT-PCR analysis using specific TBK1 primers. Each value represents the mean Ϯ standard deviation from three reactions. The result is representative of at least 2 identical experiments. (D) Effect of siTBK1 on M-mediated IFN-␤ induction. Plasmid pGL3-IFN-␤-luc reporter was cotransfected with 2 g of pCMV-Myc-M or 2 g of pCMV-Myc-M plus increased doses of siTBK1 (0, 0.5, and 2 g) into HEK293ET cells grown on a 12-well plate. At 48 h posttransfection, the dual-luciferase assay was conducted to assay fold induction of M-mediated IFN-␤ expression. Each value represents the mean Ϯ standard deviation from three independent tests. (E) Effect of IRF3 siRNA (siIRF3) on expression of endogenous IFR3 by semiquantitative RT-PCR. The increased doses of pBS/U6-siIRF3 plasmid DNAs (0, 0.5, and 2 g) were transiently transfected into HEK293T cells. Total RNAs or whole-cell lysates were isolated or harvested at 48 h posttransfection. One-step RT-PCR was conducted to detect IRF3 expression with specific primers (upper panel), while Western blotting was performed to detect IRF3 protein expression (lower panel). The expression of ␤-actin served as a loading control. The result is representative of at least 2 identical experiments. (F) Effect of siIRF3 on M-mediated IFN-␤ mRNA expression by real-time qRT-PCR analysis. Real-time qRT-PCR was performed to detect the IRF3 mRNA (isolated in panel E) expression. Each value represents the mean Ϯ standard deviation from three reactions. The result is representative of at least 2 identical experiments. (G) Effect of siIRF3 on M-mediated IFN-␤ induction by luciferase assay. Plasmid pGL3-IFN-␤-luc reporter was cotransfected with 2 g of pCMV-Myc-M or 2 g of pCMV-Myc-M plus increased doses of siIRF3 (0, 0.5, and 2 g) into HEK293ET cells grown on a 12-well plate. At 48 h posttransfection, the dual-luciferase assay was conducted to assay fold induction of M-mediated IFN-␤ expression. Each value represents the mean Ϯ standard deviation from three independent tests. recognizing the majority of extracellular and intracellular PAMPs. Upon the ligation of a PRR with its specific PAMP, both RLR and TLR pathways transmit the signal to a common class of adaptors called tumor nec