Figure 5. MARV VP40 inhibits ISRE- and GAS-induced gene expression.
(A) MARV VP40 inhibits type I IFN-induced gene expression. 293T cells were co-transfected with a construct expressing the CAT gene driven by an ISG54 promoter along with a constitutively expressed Renilla luciferase gene and 1 µg of plasmids that express the indicated viral proteins and 24h.p.t. treated with 1000 IU/ml IFNα/β for 18 h and assayed for CAT and luciferase activities. The IFN-induced CAT activity was normalized to Renilla luciferase activity. Presented is the mean fold induction of 3 experiments compared to the untreated negative control, error bars represent the standard deviations and the asterisks the p-values (**p-val = 0.016; ***p = 0.0006). Lysates were analyzed for viral protein expression (data not shown). (B) MARV VP40 inhibits IFNγ dependent gene expression. Huh-7 cells were co-transfected with the IFNγ inducible firefly luciferase reporter plasmid pGAS-Luc along with a constitutively expressed Renilla luciferase gene and 0.6 µg of the indicated expression plasmids. Cells were treated with a 1000 IU/ml of IFNγ for 18 h and assayed for dual luciferase activities. The IFN-induced firefly luciferase activity was normalized to Renilla luciferase activity. The bars represent the mean fold induction of 3 experiments compared to the untreated negative control, and the error bars represent the standard deviations. (C) MARV VP40 inhibits the IFNγ dependent IP-10 production. 2×105 HUVECs were transfected with 2 µg of the indicated expression plasmids. Cells were treated with 100 IU/ml IFNγ for 24 h; and supernatants were collected, cleared by centrifugation and analyzed by ELISA for IP-10. (D) MARV VP40 does not inhibit TNFα-induced IL-8 production. HUVECs were transfected as in (C) and treated with 50 ng/ml of TNFα for 24 h. Supernatants were collected, cleared by centrifugation and analyzed by ELISA for IL-8 concentrations.