So far, our data suggest that influenza virus-induced transcriptional upregulation of the SOCS-3 gene is not mediated by the . Equivalent mRNA amounts were normalized to GAPDH mRNA levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as 1. To detect SOCS-3 protein expression (C) cells were infected for time points indicated or left uninfected. Total cell lysate was subjected to Western blot analysis using anti-SOCS-3 antibody. To allow better comparison of SOCS-3 protein expression and STAT1 phosphorylation phospho-STAT1 and STAT1 Western blots from figure 1A are shown again here. (E) To functionally test effective knock down of the IFNAR, A459 wild type, A549 vector control cells or A549 cells stably expressing IFNAR II-1specific shRNA were stimulated with human IFNb (100 U/ml) for 15 min. Subsequently cells were lysed and levels of phospho-STAT1 were determined by Western blotting using specific antibodies. In addition, the relative pSTAT1 band intensities were quantified. doi:10.1371/journal.ppat.1000196.g004 autoregulatory action of type I IFNs ( Figure 4D and 4F) but is directly induced through accumulation of viral RNA during infection. This raises the question, which RNA-induced signaling pathways are responsible for SOCS-3 expression. The MKK/p38 mitogen activated protein kinase (MAPK) pathway [47] [48] [49] as well as the IkB kinase (IKK)/nuclear factor of kB (NF-kB) cascade [50] [51] [52] are both known to be activated by RNA or influenza virus infection and to be involved in the control of SOCS-3 expression. To assess whether the MKK6/p38-or the IKK/NF-kB-module is required for SOCS-3 gene induction, we generated A549 cell lines expressing dominant negative forms of either MKK6 (MKK6Ala) or IKK2 (IKK2KD) (Figure 6A to 6D). These mutants have been previously shown to efficiently block p38 or NF-kB signaling, respectively [52] [53] [54] . To monitor SOCS-3 gene induction, wild type, vector or mutant expressing cell lines were infected with PR8 ( Figure 6A ) or stimulated with RNA from virally infected or uninfected A549 cells ( Figure 6C ). Induction of IFNb mRNA was monitored as a control ( Figure 6B and 6D) . While MKK6Ala expression did not result in significant reduction of SOCS-3 in either infected ( Figure 6A ) or RNA-stimulated cells ( Figure 6C ), transcription is markedly reduced in IKK2KD expressing cell lines. To obtain independent evidence for NF-kB dependence of SOCS-3 gene transcription, A549 wild type cells were incubated with the NF-kB specific inhibitor BAY 11-7085 prior to stimulation with RNA from virally infected or uninfected A549 cells ( Figure 6E ). Again, IFNb mRNA levels were assessed for control purposes ( Figure 6F ). Both, SOCS-3 and IFNb mRNA levels were strongly reduced in BAY 11-7085 treated cells. This indicates that virus-induced SOCS-3 expression strongly depends on IKK2 and NF-kB activation, while the MKK6/p38 appears not to play a prominent role.