Negative Regulation of Interferon-β Gene Expression during Acute and Persistent Virus Infections (original) (raw)
Figure 5
Interferon signaling is defective in PI-MEFs.
A. ISGs are not activated by recombinant IFNβ in PI-MEFs, but ribavirin treatment activated the expression of ISGs and IFNβ. Control MEFs and PI-MEFs were treated with recombinant IFNβ (1000 U/ml) or ribavirin (100 µg/ml) either alone or in combination for 11 and 24 hrs. Cells were harvested for total RNA extraction, and the expression of IFNβ, Stat1, IRF7 and β-Actin mRNAs were analyzed by RT-PCR. B. ISGF3 complex formation is blocked in PI-MEFs. Control MEFs and PI-MEFs were stimulated with recombinant IFNβ protein for 1 hr, and total cell extracts prepared and assayed for the ISGF3 formation on ISRE DNA probes derived from the ADAR1 and MX1 genes. C. Reduced phosphorylation of STAT1 tyrosine 701 in PI-MEFs after IFN treatment. Top: Control MEFs and PI-MEFs were transfected with a plasmid encoding Stat1α protein for 24 hrs, transfected and non-transfected cells were stimulated with recombinant IFNβ for 1 hr. Total protein was prepared for western blot analysis with antibodies against Stat1, phospho-tyrosine 701 Stat1, Stat1α and MAP kinase p42/p44. Bottom: the same experiments were conducted in IFNAR1 deficient MEFs and the expression of Stat1 and phospho-Y701 Stat1 were analyzed by western blotting. GFP was also transfected as a control. D. Plasma membrane localization of Stat1 protein in PI-MEFs. Control MEFs and PI-MEFs were transfected with a plasmid encoding Flag-tagged Stat1 protein. 24 hrs later cells were fixed and subjected to immunofluorescent staining with an anti-Flag antibody (green). Blue: DAPI staining for nuclei. Scale bar, 10 µm. E. SeV C protein specifically interacts with Stat1 protein. Constructs for Flag-tagged SeV C, V, P and NP proteins were transfected into MEFs, 24 hrs later, whole cell extracts were prepared and subjected to anti-Flag M2 bead immunoprecipitation (IP). The associated proteins were eluted and separated on SDS-PAGE, blotted and probed with an anti-Stat1 antibody. An hnrnp U expression construct was also included as a control. IB: immunoblot.