To further assess a functional role of SOCS-3 in virus-induced suppression of STAT1 phosphorylation we analyzed mouse cells with a targeted deletion of the SOCS-3 gene [56] . Wild type and SOCS-3 deficient mouse embryonic fibroblasts (MEF) were infected for different time points with PR8. The time of infection was prolonged in comparison to the infection of A549 cells because the human PR8 replicates less efficiently in mouse than in human cells. Following infection lysates of these cells were assessed for STAT1 phosphorylation ( Figure 7A ). Both cell types showed no phosphorylation of STAT1 in the uninfected state. In contrast, infection of SOCS-3 knock out cells resulted in strongly elevated phosphorylation of STAT1 in a sustained fashion. To rule out that this STAT1 phosphorylation is due to altered secretion of IFNb or Figure 7 . Enhanced STAT1 phosphorylation in infected SOCS-3 deficient MEF correlates with elevated induction of IFNb-stimulated genes. Wild type MEF and SOCS-3 knock out MEF were infected with PR8 (MOI = 5) for the indicated times. Subsequently, cell lysates were analyzed for STAT1 phosphorylation (A). For control of productive virus replication, cell lysates were analyzed for viral protein PB1 expression. In (E, F, G) wild type and knock out cells were lysed at indicated time-points of infection. Subsequently RNA was subjected to reverse transcription. cDNA was analyzed in quantitative real time PCR to assess mRNA amounts of three prototype type I IFN-stimulated genes, SP110 (E), interferon regulatory factor-1 (IRF-1) (F) and OAS1 (G). 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. In (C) wild type MEF and knock out MEF were infected with PR8 (MOI = 5) or left uninfected. Supernatants were taken 6 p.i. and used for stimulation of wild type MEF for 15 minutes. As control wild type MEF were stimulated with 500 U/ml mouse IFNb for 15 minutes. Cells were harvested and analyzed for the amount of STAT1 and phospho-STAT1 in Western blot analysis by specific antibodies. In (B) and (D) the relative band intensities of phospho-STAT1 of the blots in (A) and (C) were quantified as described. doi:10.1371/journal.ppat.1000196.g007 other STAT1-activating cytokines in SOCS-3 deficient cells, we performed conditioned medium experiments ( Figure 7C ). MEF wild type and MEF SOCS-3 deficient cells were infected for 6 h and supernatants were subsequently harvested. Stimulation of MEF wild type cells with these different supernatants for 15 min. revealed no differences in STAT1 phosphorylation, indicating that both infected cell types secrete similar amounts of IFNb and other STAT1 activating cytokines. This is a strong indication that the observed differences in virus-induced STAT phosphorylation are directly due to the presence or absence of SOCS-3 in wild type and knock out MEF, respectively. To answer the question whether enhanced STAT phosphorylation in SOCS-3 deficient cells would also lead to enhanced expression of ISGs, total RNA was isolated at different time points p.i. from infected wild type and knock out cells and monitored for induction of SP110, IRF-1 and OAS1 ( Figure 7E, 7F and 7G ). These genes are described as type I IFN-induced genes [18] . Indeed mRNA levels of all three representative ISGs were elevated in SOCS-3 knock out versus wild type cells at almost every time point during the course of infection. This indicates that enhanced STAT1 phosphorylation and activation in SOCS-3 deficient cells results in elevated expression of ISGs.