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. Author manuscript; available in PMC: 2009 Oct 2.
Published in final edited form as: J Biol Chem. 2007 Aug 30;282(44):32208–32221. doi: 10.1074/jbc.M704870200

Figure 6. SARS-CoV PLpro interacts with IRF-3.

Figure 6

A. HeLa PLpro-4 cells were cultured to repress or induce PLpro-TM expression and subsequently mock-infected or infected with SeV for 16 h. In left panels, expression of p396-IRF-3, total IRF-3, PLpro and actin proteins in cell lysates were determined by immunoblot analysis. In right panels, cell lysates were subjected to immunoprecipitation with a rabbit anti-IRF-3 antibody (lanes 1 through 4) or a control rabbit serum (lanes 5 through 8). The immunoprecipitates were separated on SDS-PAGE, followed by immunoblot analysis of IRF-3 (using an mAb anti-IRF-3) and PLpro (using an mAb anti-V5 tag). B. HEK293 cells were transfected with WT (lanes 1 through 3), C1651A (lanes 4 through 6), or D1826A (lanes 7 through 9) PLpro-TM, respectively, and where indicated, along with an empty vector (lanes 1, 4, and 7), or a vector expressing Flag-IRF-3 (lanes 2, 5, and 8) or untagged IRF-3 (lanes 3, 6, and 9). Cell lysates were immunprecipitated with anti-V5 (PLpro-TM), followed by immunodetection of Flag-IRF-3 (anti-Flag) or PLpro-TM (anti-V5) (right panels). * denotes IgG heavy chain. Immunoblot analysis of input for Flag-IRF-3 and PLpro-TM is shown in left panels. All 3 forms of PLpro-TM interacted with IRF-3. C. HEK293 cells were transfected with WT (lanes 1 and 2), C1651A (lanes 3 and 4), or D1826A (lanes 5 and 6) PLpro-Sol, respectively, and where indicated, along with an empty vector (lanes 1, 3, and 5) or a vector expressing Flag-IRF-3 (lanes 2, 4, and 6). Cell lysates were immunoprecipitated with anti-V5 (PLpro-Sol), followed by immunodetection of Flag-IRF-3 (anti-Flag) or PLpro-Sol (anti-V5) (lower panels). Immunoblot analysis of input for Flag-IRF-3 and PLpro-Sol is shown in upper panels. Note that WT and D1826A PLpro-Sol interacted with IRF-3, while C1651A PLpro-Sol did not. D. HeLa PLpro-4 cells induced for PLpro-TM expression were transfected with GFP, GFP-IRF-3, or GFP-IRF3-5D, respectively. Cell lysates were subjected to Co-IP experiments using either GFP antibody or V5 antibody for immunoprecipitation, followed by immunoblot analysis of the immunoprecipitates using anti-GFP or anti-V5 antibodies. Note that both GFP-IRF3 and GFP-IRF3-5D were associated with PLpro-TM (detected by anti-V5), while free GFP was not.