Increased induction of

STATs and IRF9 was also observed after IFN-α treatment in Pol-expressing Huh7 cells but was much weaker than that observed in control cells (Fig. 2B). The levels of STAT1 Ser727 phosphorylation were clearly repressed by transfection of Huh7 cells with Pol; however, tyrosine phosphorylation of STAT1/2 was not affected. To further investigate the effect of Pol on the tyrosine phosphorylation-induced STAT1-STAT2 heterodimerization, we performed co-immunoprecipitation (co-IP) experiments in Huh7 cells transfected with increasing amounts of Pol (Fig. 2C) and in Dox-regulated HepAD38 cells (Fig. 2D). The results showed that STAT1-STAT2 interaction find more in response to IFN-α was consistently observed in cells with or without Pol. Meanwhile, Flag-Pol was not detected in the immune complexes precipitated with anti-STAT1

or Target Selective Inhibitor Library anti-STAT2 Abs, indicating no direct interaction between Pol and activated STAT1/2. Moreover, there was not much difference in IFN-α–induced heterodimer formation between cells expressing Pol and control cells (Fig. 2E,F), indicating that Pol does not affect the IFN-α–stimulated STAT1-STAT2 heterodimerization. IFN-α-induced phosphorylation of the serine residue at position 727 (Ser727) of STATs contributes critically to their transcriptional activity.12, 13 Although still controversial, PKC-δ, p38 and ERK have been reported to function as kinases that regulate Ser727 phosphorylation.14, 15 To elucidate the mechanism by which Pol interferes with STAT1 Ser727 phosphorylation, we examined the effect of Pol on IFN-α–induced phosphorylation of PKC-δ, p38 and ERK (Fig 3A). The results showed that Pol only inhibited PKC-δ but not p38 or ERK phosphorylation in IFN-α–stimulated Huh7 cells. Rottlerin, a selective inhibitor of PKC-δ, was used to verify the role of PKC-δ in Ser727 phosphorylation of STATs (Fig. 3B), and the unless data demonstrate that PKC-δ is specifically required

for the Ser727 phosphorylation, but not for STAT tyrosine phosphorylation. IFN-α–stimulated PKC-δ phosphorylation was also found to be impaired in HepG2.215 cells compared with that in HepG2 cells (Fig 3C), but was restored by Pol siRNA transfection (Supporting Fig. 5A). In addition, we investigated whether Pol inhibits IFN-α signaling by regulating the level of STAT3, as it was reported to be a negative regulator of the type I IFN response.16 Little difference in the basal expression level and IFN-α–induced tyrosine phosphorylation of STAT3 was observed between the cells with or without Pol; however, PKC-δ–dependent Ser727 phosphorylation of STAT3 was inhibited by Pol in a dose-dependent manner (Fig. 3D). Furthermore, less STAT1 was coprecipitated with PKC-δ from lysates of Pol-expressing IFN-α–treated cells (Fig. 3E), and Pol was found to interact with the catalytic domain of PKC-δ (Fig. 3F and Supporting Fig. 5B).