Figure 3.

hcmv-miR-US33as-5p downregulates IFNAR1 and Jak-STAT, limiting the release of ISGs. (A) MRC-5 and HFF cells were cultured in 24-well plates and transiently transfected with hcmv-US33as-5p mimics or a negative control RNA (NC-RNA) at a concentration of 100 nM for 24 h. The cells were treated with 10 μg/mL CHX, followed by stimulation by 1,500 U/mL IFNα for 1 h until harvest. The expression of IFNAR1 and IFNAR2 was determined by qPCR. (B) The mRNA stability of IFNAR1 and IFNAR2 in MRC-5 cells transfected with US33as-5p mimics or NC-RNA and incubated with actinomycin D to inhibit transcription is presented as the amount of mRNA detected at a given time relative to that at 0 h, which was set as 100%. (C) Immunoblotting of lysates from MRC-5 cells underwent treatment similar to that described in Figure 3A and prolonged incubation with 1,500 U/mL IFN-α for 6 h. was performed. Blots were probed for IFNAR1, IFNAR2, STAT1, STAT2, Jak1, Tyk2 and their phosphorylated forms. β-Actin was run as a loading control. (D) The expression of multiple ISGs (Mx1, RSAD2, DDX58, BST2, IFIT2, and ISG20) in MRC-5 cells was determined by qPCR. The RNA harvested from MRC-5 cells underwent treatment similar to that described in Figure 3C. GAPDH was amplified as a loading control. The assays were performed in triplicate wells, and data were collected from two different experiments and are represented as the means ± SDs; **p < 0.05.