IRF3(5D) activity is dependent on the de-ubiquitination activity of PLpro. A. HEK293T cells were transfected with either empty plasmid, HA-tagged Ubiquitin alone or HA-tagged Ubiquitin in combination with either wildtype PLpro (PLpro/wt) or active site mutant PLpro (PLpro/mt). At 24 hours post-transfection, lysate was extracted and analyzed by western blot with anti-HA and anti-GFP antibodies to confirm that the PLpro/mt was unable to deubiquitinate cellular proteins. B. Plasmid expressing SARS-CoV PLpro/wt and PLpro/mt were tested for the ability to inhibit the induction of an IFNβ promoter/luciferase reporter induced by IRF3(5D). The average induction with standard deviation across 3 transfections is graphed relative to mock transfection. HEK293T cells were transfected with plasmids encoding IFNβ promoter/luciferase, Flag-tagged IRF3(5D), empty GFP vector, HA-tagged SARS-CoV PLpro/wt or PLpro/mt in the combinations noted. At 18 hours post-transfection cells were lysed and firefly luciferase expression quantified.