Phosphorylation of STATs in the IFNb signaling cascade may not only be counter-regulated by phosphatases but also by other cellular factors, such as proteins of the suppressors of cytokine signaling (SOCS) family. Action of these proteins is mainly controlled on the level of transcriptional activation. SOCS proteins are described to have high affinity for JAK and STAT proteins and to inhibit the transmission of IFNa and IFNb induced signaling [44, 45] . To examine whether expression of SOCS genes is induced in influenza virus infected cells, A549 cells were infected with PR8 for different time points. Subsequently total RNA was analyzed for the amount of SOCS-1 and SOCS-3 mRNA by means of quantitative real time-PCR (qRT-PCR). The mRNA Table 1 for accession numbers of viral genes) using L2000 according to manufacturer's instructions. Note that the Pol II constructs in use also give rise to expression of second reading frames in the NS, M and PB1 genes (NS2, M2, PB1-F2). 48 h post transfection cells were stimulated with human IFNb (500 U/ml) for 15 minutes. Total protein lysates were subjected to Western blot analysis using anti-phospho-STAT1, anti-phospho-STAT2, anti-STAT1 antibodies. Expression of influenza viral proteins was monitored with antibodies against NP, M1, NS1, PA, PB1 or PB2. (E) HEK293 cells were infected with the human influenza virus PR8 (H1N1) (MOI = 5) for the indicated time points and were subsequently stimulated for 15 min with either human IFNb at a concentration of 100 U/ml. Cell lysates were subjected to Western blots as described. (B, D, F) Quantification of relative pSTAT1 and pSTAT2 band intensities in A, C and E using AIDA software and 2D densitometry (Fuji). doi:10.1371/journal.ppat.1000196.g002 levels of SOCS-1 and SOCS-3 differed notably in the time course ( Figure 4A ). While SOCS-3 mRNA is strongly and transiently elevated in the early phases of infection, SOCS-1 gene transcription is only significantly induced 15 h p.i.. Elevated SOCS-3 mRNA levels were also observed in other host cell types, such as HUVEC starting 3 h p.i. ( Figure S1A ). Although elevation of SOCS-3 mRNA levels in infected cells was rather transient, there appears to be a robust induction on protein level ( Figure 4C ). First detected at 4 h p.i., SOCS-3 protein levels increased and stayed on a high level throughout the observation period. Strikingly, expression kinetics of the SOCS-3 protein perfectly matched the kinetics of virus-induced inhibition of STAT1 phosphorylation ( Figure 4C ), indicating that both processes are functionally linked.