Influenza A Virus Inhibits Type I IFN Signaling via NF-κB-Dependent Induction of SOCS-3 Expression
Figure 8
Efficiency of influenza A virus replication is dependent on SOCS3 expression levels.
Wild type MEF and SOCS-3 knock-out MEF were infected with PR8 (MOI = 0.01) (A) or A/Victoria/3/75 (MOI = 0.001) (B) for the indicated times. In (C) A549 wt cells were transfected for 48 h with 150 nM human SOCS3 siRNA using Hiperfect according to manufacturers protocol and infected with PR8 (MOI = 0.001) for 9 h. In (D) the highly susceptible cell line HEK293 was transfected with either pSUPER empty vector or pSUPER-mSOCS-3 for 48 h. Subsequently cells were infected with PR8 (MOI = 0.001) for 9 h. For (A), (B), (C) and (D) progeny virus titers were determined from the supernatants of infected cells by means of plaque assay. To determine the effect of over expressed SOCS-3 on STAT1 phosphorylation (E) A549 cells were treated as in (D) and infected with PR8 (MOI = 5) and/or stimulated with human IFNβ (100 U/ml). Cells were lysed and cell extracts were analyzed for levels of phosphorylated STAT1 and over expressed mSOCS-3 using anti phospho-STAT1 and anti flag-antibody in Western blots. Effective of SOCS-3 knock down was determined by Western blot (C, right). Cells were treated as in (C, left) and total cells lysates were analyzed for endogenous SOCS-3 protein levels using anti-SOCS-3 antibody in Western blot. (F) Quantification of relative pSTAT1 band intensities in (E).