Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ
Figure 8
ISG induction by IE1 depends on STAT1 tyrosine phosphorylation.
A) IE1 expression leads to increased steady-state levels of Y701- and S727-phosphorylated STAT1. TetR and TetR-IE1 cells were treated for 72 h with doxycycline and for 1 h with solvent (–) or IFN-γ. Whole cell protein extracts were prepared and subjected to immunoblotting with anti-STAT1, anti-pSTAT1 (Y701), anti-pSTAT1 (S727), anti-GAPDH, and anti-IE1 antibodies. B) Verification of knock-down resistance and phosphorylation deficiency of STAT1 variants. TetR-IE1 cells without (–) and with stable expression of ectopic wild-type STAT1 (STAT1), siRNA-resistant wild-type STAT1 (STAT1*), and siRNA-resistant phosphorylation-deficient STAT1 (STAT1*Y701F and STAT1*S727A) were transfected with negative control (#149) or STAT1-specific (#146) siRNA duplexes. Two days post transfection cells were treated for 1 h with IFN-γ. Whole cell protein extracts were prepared and subjected to immunoblotting with anti-STAT1, anti-pSTAT1 (Y701), anti-pSTAT1 (S727), and anti-GAPDH antibodies. C) Ectopic wild-type STAT1 but not phosphorylation-deficient STAT1 mutants efficiently rescue IE1-dependent ISG induction in cells depleted of endogenous STAT1. TetR-IE1 cells without (–) and with stable expression of the indicated ectopic STAT1s were transfected with control (#149) or STAT1-specific (#146) siRNA duplexes. Two days post transfection cells were treated for 72 h with doxycycline. Relative mRNA expression levels were determined by qRT-PCR with primers specific for the CXCL10, GBP4, IE1, and STAT1 genes. Results were normalized to TUBB and mean values with standard deviations from two biological and two technical replicates are shown. Expression is shown in comparison to control siRNA-transfected TetR-IE1 cells without ectopic STAT1 expression (set to 1).