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. 2016 Jul 7;12(7):e1005748. doi: 10.1371/journal.ppat.1005748

Fig 8. IE1 rewires IL6 signaling to STAT1 activation during hCMV infection.

Fig 8

(A) MRC-5 cells were infected with TBwt, TBIE1dl410-420 or TBrvIE1dl410-420 at low input multiplicity (0.1 PFU/cell) in the absence (w/o) or presence of exogenous IFNβ. Culture media were replaced every 24 h, and viral replication was assessed by qPCR-based relative quantification of hCMV DNA from culture supernatants at the indicated times post infection with primers specific for the viral UL86 sequence. Data are presented as means and standard deviations from three independent infections. (B) MRC-5 cells were mock-infected or infected with TBwt, TBIE1dl410-420 or TBrvIE1dl410-420 at a high input multiplicity (5 PFU/cell). At 6 h post infection, cultures were treated with solvent or IL6 plus IL6R (IL6/Rα). At 24 h post infection, whole cell protein extracts were prepared and analyzed by immunoblotting for IE1/IE2, total STAT1, pSTAT1 (Y701) and GAPDH. (C) MRC-5 cells were mock-infected or infected with TBwt, TBIE1dl410-420 or TBrvIE1dl410-420 at a high input multiplicity (5 PFU/cell). At 6 h post infection, cultures were treated with solvent or IL6 plus IL6R (IL6/Rα). At 24 h post infection, relative mRNA levels were determined by RT-qPCR for the STAT1 target genes CXCL9, CXCL10, CXCL11 and IDO. Results were normalized to TUBB, and means and standard deviations of biological triplicates are shown in comparison to solvent-treated mock-infected cells (set to 1). (D) STAT2-deficient human skin fibroblasts were infected with gTBwt, gTBIE1dl410-420 or gTBrvIE1dl410-420 at low input multiplicity (0.1 PFU/cell) in the absence (w/o) or presence of IL6 plus IL6Rα (IL6/Rα). Every 48 h, half of the culture media was replaced and viral replication was assessed at day 6 post infection by fluorescence microscopy. (E) STAT2-deficient human skin fibroblasts were infected with gTBwt, gTBIE1dl410-420 or gTBrvIE1dl410-420 at low input multiplicity (0.1 PFU/cell) in the absence (w/o) or presence of IL6 plus IL6Rα (IL6/Rα). Every 48 h, half of the culture media was replaced and viral replication was assessed by qPCR-based relative quantification of hCMV DNA from culture supernatants with primers specific for the viral UL86 sequence. Data are presented as means and standard deviations from two biological and two technical replicates.