SUMO1-mediated regulation of IRF3 modulates IFN-β expression during seasonal and pandemic H1N1 Influenza infection.

Figure 3.- The IRF3 protein is differentially modified by addition of SUMO1 during pandemic and seasonal viral infection. A) A549 cells either mock-infected or infected with pandemic or seasonal viral strains were harvest at different times post-infection (12, 16 and 24 hpi), and cytoplasmic and nuclear subcellular fractions were prepared. Subsequently, fractions lysates were analyzed by immunoprecipitation and Western blot. The immunoprecipitation of IRF3 was performed using anti-IRF3, resolved on 7.5% SDS-PAGE, and visualized by immunoblotting using anti-SUMO1 mAb or anti-IRF3, as input control. Specific IRF3-SUMO1 bands are indicated by (*). B) To evaluate the interaction between IRF3 and NS1 viral protein in different cellular compartments, co-immunoprecipitations assays were performed. Cytoplasmic or nucleus lysates from mock-infected cells or influenza infected cells at different times post-infection, were immunoprecipitated using anti-IRF3, resolved on 10% SDS-PAGE and then were evaluated by western blot with anti-NS1. C) Western blotting from cytoplasmic and nuclear fractions was performed using anti-pIRF3, as active IRF3 control. NS1 was included as infection control. Histone H3 and β-tubulin were used to demonstrate the purity of the nucleus and cytoplasmic subcellular fractions, respectively. Results were reproduced in biological triplicates.