Viral growth factor- and STAT3 signaling-dependent elevation of the TCA cycle intermediate levels during vaccinia virus infection
Fig 7
VACV infection induces non-canonical STAT3 phosphorylation at S727 in a VGF-dependent manner.
(A) VACV VGF is indispensable to activate STAT3 (S727) phosphorylation. HFFs infected with indicated viruses at an MOI of 5 for the indicated time. Western blotting analysis was performed to measure the levels of various forms of STAT3. (B) Upregulation of STAT3 S727 phosphorylation starts early during VACV infection. HFFs infected with indicated viruses at an MOI of 5. The samples were collected at 10 min post-infection and 8 hpi, respectively, followed by Western blotting analysis. (C) VACV activates STAT3 (S727) phosphorylation in the absence of glutamine in the medium. HFFs were infected with indicated viruses at an MOI of 5 in medium with glucose only (Glc) or with glucose+asparagine (Glc+N). Protein levels were detected by performing a Western blotting analysis at 4 hpi. (D) Inhibition of the EGFR pathway decreases STAT3 S727 phosphorylation in VACV infected cells. HFFs were infected with MOI of 5 of WT-VACV with or without 3 μM afatinib treatment. Western blotting analysis was performed at 4 hpi to test the levels of different forms of STAT3 protein. (E) Inhibition of the MAPK pathway decreases both Y705 and S727 phosphorylation. HFFs were infected with MOI of 5 of VACV in medium with or without 20 μM PD0325901 treatment. Western blotting analysis was performed at 2 hpi to detect the levels of different forms of STAT3 protein. (F) Stattic treatment inhibits S727 phosphorylation. HFFs were infected with MOI of 5 of WT VACV with or without 3 μM stattic. At 4 hpi, Western blotting analysis was performed to detect the levels of different forms of STAT3 protein. (G) STAT3 S727 phosphorylation is independent of the JAK1/2 pathway. HFFs were infected with an MOI of 5 of VACV in medium with or without 5 μM ruxolitinib treatment. Western blotting analysis was performed at 4 hpi to measure different protein levels. (H) Ruxolitinib treatment decreases the induction of citrate level upon VACV infection. HFFs were infected with WT VACV at an MOI of 5 in the presence or absence of ruxolitinib treatment. The citrate level was measured at 4 hpi. (I) Effects of inhibition of STAT3 and its upstream signaling pathways on VACV early protein expression. HFFs infected with WT VACV at an MOI of 2 in the presence or absence of 3 μM afatinib, 20 μM PD0325901, 3 μM stattic, 5 μM ruxolitinib, 40 μg/mL AraC, or 100 μg/mL cycloheximide. The levels of VACV E3 protein was measured at 2 hpi by a Western blotting analysis. (J) Effects of inhibition of STAT3 and its upstream signaling pathways on VACV early protein levels. HFFs infected at an MOI of 2 with a recombinant VACV expressing Gaussia luciferase under virus early VGF promoter in the presence or absence of 3 μM afatinib, 20 μM PD0325901, 3 μM stattic, 5 μM ruxolitinib, 40 μg/mL AraC, or 100 μg/mL cycloheximide. Early gene expression was measured by a Gaussia luciferase activity assay kit at 2 hpi. All experiments were performed in media with glucose plus glutamine unless otherwise stated. Error bars represent the standard deviation of at least three biological replicates. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001. The blots were from different lanes on the same gel and the dashed lines indicate that some non-relevant lanes were removed.