Fig. 5. Inhibition of host cGAS-STING pathway by Nsp15.
(A and B) A549-ACE2 cells were pretreated with H-151 or SN-011 for 16 h prior to viral infection. Cells were then infected with Nsp15WT (blue line) and Nsp15H234A (red line) rSARS-CoV-2 at MOI of 1 under H-151 or SN-011 treatment for 48 h. Supernatants harvested for viral titration by plaque assay. Cell viability determined by WST-1 assay after incubating with H-151 or SN-011 for 72 h (black dot line). Data are mean ± SD (n = 3). (C and D) HEK293T cells were co-transfected with IFN-β promoter reporter plasmid (C) or NF-κB responsive element reporter plasmid (D), Renilla control plasmid plus the indicated plasmids containing empty vector, cGAS/STING, mCherry and WT and H234A Nsp15 for 48 h. Relative luciferase activity was performed by use of Dual-Glo Luciferase System. Data are mean ± SD (n = 3) and analyzed by two-way ANOVA with Dunnett’s multiple comparison test. (E, F, G) HEK293T transfected with different combinations of plasmids were infected with EV-D68 at MOI of 0.1 for 48 h. Culture supernatants applied for viral titer quantification by plaque assay (E, upper bar graph). Cell lysates collected for protein level determination (E, lower blot graph) and total RNA collected for RT-qPCR (F, G). (H, I, J) HEK293T transfected with different combinations of plasmids were infected with NDV-EGFP at MOI of 1 for 48 h. Culture supernatants applied for viral titer quantification by focus forming assay targeting EGFP (H, upper bar graph). Cell lysates collected for protein level determination (H, lower blot graph) and total RNA collected for RT-qPCR (I, J). Relative target RNA level normalized with that of 18S rRNA or ACTB was shown. Data are mean ± SD (n = 3 or 4). and analyzed by two-way ANOVA with Šídák multiple comparison test. ** P≤0.01; *** P≤0.001; **** P≤0.0001; ns, not significant.