Figure 4

SARS-CoV PLpro does not inhibit IRF3 or IRF3(5D) binding to the IFNβ promoter. A-C. Chromatin immunoprecipitations (ChIP) were performed on HEK293T cells transfected with either HA tagged PLpro, Flag-tagged IRF3, Flag-tagged IRF3(5D) or the noted combinations. At 24 hours post transfection, cells were fixed with 4% Paraformaldehyde and DNA was extracted and sonicated to ~300-1.2Kb in length. Samples were then immunoprecipitated with the designated antibodies and used as template for PCR reactions for GAPDH to control for input template (A, arrow denotes correct PCR product) of the IFNβ promoter (B and C, arrows denote correct PCR products). PCR reactions were run on 2% agarose gels to visualize products. D. HEK293T cells were transfected with either Flag-tagged IRF3(5D) alone or in combination with HA-tagged PLpro. Cells were lysed and lysates incubated with radioactively labeled probe from either the ISG15 promoter or the OAS1b promoter. Samples were run on a polyacrylamide gel and protein complexes visualized on film. Arrow denotes supershifted complex.