FIG. 7.

The transmembrane form of HCoV-NL63 PLP2 and catalytic mutants inhibit RIG-I-mediated IFN-β induction in a dose-dependent manner. The native downstream hydrophobic domain was cloned into the PLP2 plasmid, and the catalytic cysteine or histidine residue was mutated to alanine. (A) HEK293 cells were transfected with the indicated amounts of PLP2-TM, PLP2-TM C1678A, or PLP2-TM H1836A along with the IFN-β-Luc and pRL-TK reporters. N-RIG was used to stimulate IFN-β induction. At 24 h posttransfection, cell lysates were harvested and assayed for luciferase activity via the Dual-Luciferase reporter assay. Values are expressed as percentages of N-RIG-stimulated luciferase controls set to 100. Error bars indicate standard deviations from the means for triplicates. (B) The cell lysates described above were mixed with 2× sample buffer and subjected to 12.5% SDS-PAGE. Following transfer to nitrocellulose, the membrane was blotted with mouse anti-V5 to detect the proteases and antiactin as a loading control.