Posttranscriptional nature of IL-12 suppression by HHV-6. IL-12 suppression by HHV-6 occurs predominantly at the posttranscriptional level. (A) RPA analysis of IL-12 p40 (□), IL-12 p35 (▦), TNF-α (▪), and IL-1β (▧) mRNA in HHV-6–pretreated Mϕs. Mϕs were exposed to HHV-6 as described in “Materials and methods.” Cells were stimulated for 17 hours with IFN-γ and then with LPS and harvested at either 3 hours (mean ± SD of 6 donors) or 6 hours (mean ± SD of 4 donors) after LPS addition. The data are shown as induction percentages relative to the positive control (dotted line), which was set as 100%. Data obtained 24 hours after stimulation are not shown, since at this time point IL-12 mRNA levels returned to the baseline level seen in unstimulated cells. (B) Lack of intracellular accumulation of IL-12 p40/p70 protein in Mϕs after HHV-6 pretreatment. Mϕs from a representative donor were isolated and exposed to HHV-6A for 17 hours, after which the cells were stimulated for 17 hours with IFN-γ and an additional 8 hours with LPS. The cells were fixed/permeabilized and stained with a specific anti–IL-12 p40/p70 antibody. Intracellular IL-12 levels were analyzed by fluorocytometry; the monocyte gate was selected with the use of the physical parameters (forward scatter [FSC] and side scatter [SSC]) and CD14 positivity. (C) Mϕs from the same donor as in panel B were harvested after 8 hours of LPS stimulation for RPA analysis of IL-12 p35 and IL-12 p40 mRNA expression. Dotted line indicates positive control level (100%).