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. 2008 Aug 13;82(21):10444–10454. doi: 10.1128/JVI.00833-08

FIG. 7.

FIG. 7.

Effect of the carboxyl-terminal SUMO-1 fusion of IE1 on the abilities of IE1 to bind to STAT2 and to repress the IFN-induced gene expression. (A) Interaction of the IE1-SUMO-1 fusion with HDAC3 in cotransfected cells. 293T cells were cotransfected with plasmids encoding myc-tagged HDAC3 and hemagglutinin (HA)-tagged wild-type IE1 or IE1-SUMO1 fusion. The total cell lysates were prepared at 48 h and immunoprecipitated with anti-myc antibody, and SDS-PAGE and immunoblotting with anti-HA antibody were performed (top panel). Immunoblots of the total cell extracts with anti-myc or anti-HA antibody to show the expression levels are shown (bottom panels). (B) Disruption of PML-NBs by IE1-SUMO-1 fusion. HF cells expressing wild-type IE1 or IE1-SUMO1 fusion were fixed in paraformaldehyde, followed by double-label IFA with anti-IE1 (6E1) and anti-PML (PML-C) antibodies. (C) GST pull-down assays. The bacterially purified GST, GST-IE1, and GST-IE1-SUMO-1 proteins immobilized to glutathione-Sepharose beads were incubated with in vitro-translated STAT2 protein. The bound proteins were fractioned by SDS-PAGE and visualized by immunoblotting with anti-STAT2 antibody. One-fifth of the GST or GST fusion proteins used in the binding reaction were shown by Coomassie blue staining, and one-tenth of those in STAT2 were shown by immunoblotting as input controls. (D) CoIP assays using cotransfected cells. 293T cells were cotransfected with myc-STAT2 and the indicated IE1 constructs. At 48 h, CoIP assays were performed as described for Fig. 1B. (E) The reporter assays using the ISG54 ISRE-luciferase construct. 293T cells were cotransfected with 0.5 μg of the ISG54 ISRE-luciferase reporter construct and 0.1 μg of empty vector or plasmid expressing the intact IE1 or IE1-SUMO-1 fusion protein. At 24 h, cells were left untreated or were treated with IFN-β (1,000 U/ml) for 8 h, and luciferase reporter assays were then conducted. The results shown are the mean values and standard errors of three independent experiments. The expression levels of IE1 proteins in the extracts were shown by immunoblotting.