Expression of full-length epitope-tagged vIRF-2 protein. 293 cells were separately transiently transfected with either 500 ng of pcDNA4/HisMax DNA (lane 1) or 500 ng of pcDNA4/vIRF-2 plasmid DNA (lane 2). Lysates were prepared after 48 h and analyzed by Western blotting with anti-Xpress primary antibody and horseradish peroxidase-conjugated secondary antibody. The position of the epitope-tagged vIRF-2 protein is indicated. M, molecular weight protein marker.

Effect of vIRF-2 protein expression on IRF activation of the full-length IFN-β promoter (p125-luc). 293 cells were harvested 16 h after transfection with the full-length IFN-β promoter (p125-luc) reporter vector. The pRLSV40 plasmid (10 ng) constitutively expressing Renilla luciferase was added as an internal control to which firefly luciferase levels were normalized. The IRF expression plasmids were pIRF-1, pIRF-3(5D), and pIRF-7(477/479), each transfected at 200 ng, or 200 ng of both pIRF-3(5D) and pIRF-7(477/479) plasmid. Each transfection included 50 ng of either the empty parental plasmid backbone, pcDNA4, or the vIRF-2-expressing plasmid, pcDNA4/vIRF-2 and 100 ng of p125-luc.

Inhibition of rIFN-driven expression of pISRE-luc by vIRF-2 protein expression. (A) Inhibition of rIFN-α. 293 cells were transfected with pISRE-luc (250 ng) and increasing amounts of pcDNA4/vIRF-2 plasmid. At 5 h posttransfection the cells were treated with rIFN-α2b (200 U/ml) and harvested 16 h later. The empty parental plasmid backbone, pcDNA4/HisMax, was added as a “stuffer” plasmid to equalize the amount of DNA in each transfection to 800 ng. The pRLSV40 plasmid (10 ng) constitutively expressing Renilla luciferase was added as an internal control to which firefly luciferase levels were normalized. Transfections where pcDNA4/vIRF-2 plasmids were not included were calculated to have 100% luciferase activity. The data represent the mean ± the standard deviation of two independent experiments, each performed in duplicate, but the differences between the experiments are too low for the error bars to be clearly distinguished in this figure. (B) Inhibition of rIL-28A (IFN-λ1). The experiment was performed as described in Fig. 2A, except that the cells were treated with rIL-28A (1,000 ng/ml). The data are presented as mean normalized firefly luciferase activity (±the standard deviation) for three independent experiments. (C) Inhibition of rIL-29 (IFN-λ3). The experiment was performed as described in Fig. 2A, with the exception that cells were treated with rIL-29 (1,000 ng/ml). The data are presented as mean normalized firefly luciferase activity (±the standard deviation) for three independent experiments.

Inhibition of IRF-1-driven expression of pISRE-luc by vIRF-2 protein expression. (A) IRF-1 induces pISRE-luc in a mechanism distinct from rIFN-α induction. 293 cell transfections contained pISRE-luc (250 ng) reporter plasmid. The pIRF-1 plasmid (50 ng) was cotransfected where indicated. After transfection for 5 h, rIFN-α2b (200 U/ml) and/or 400 neutralizing units of each rabbit polyclonal antibody against human IFN-α and IFN-β/ml was added to the cells as indicated. The pRLSV40 plasmid (10 ng) constitutively expressing Renilla luciferase was added as an internal control to which firefly luciferase levels were normalized. The empty parental plasmid backbone, pcDNA4/HisMax, was added as a “stuffer” plasmid to equalize the amount of DNA in each transfection to 800 ng. The data represent the mean ± the standard deviation of two independent experiments, each performed in duplicate. (B) Inhibition of pIRF-1-induced stimulation of pISRE-luc by vIRF-2. 293 cells were transfected with the pISRE-luc reporter plasmid (250 ng), the expression of which was driven by IRF-1, itself constitutively expressed from the cotransfected pIRF-1 plasmid (50 ng). Increasing amounts (0 to 50 ng) of the vIRF-2 expression plasmid pcDNA4/vIRF-2 were also cotransfected. A parallel transfection containing pIRF-2 (50 ng) and pISRE-luc (250 ng) was performed in the absence of pcDNA4/vIRF-2 plasmid. The pRLSV40 plasmid (10 ng) constitutively expressing Renilla luciferase was added as an internal control to which firefly luciferase levels were normalized. The empty parental plasmid backbone, pcDNA4/HisMax, was added as a “stuffer” plasmid to equalize the amount of DNA in each transfection to 800 ng. The cells were harvested 16 h after transfection. Transfections where pcDNA4/vIRF-2 plasmid was not included were calculated to have 100% luciferase activity. The data represent the mean ± the standard deviation of two independent experiments, each performed in duplicate, but the differences between the experiments are too low for the error bars to be clearly distinguished in this figure.