FIG. 8.

ORF57 prevents the miRNA-mediated translational repression of vIL-6 in vitro. (A) A representative of multiple gels in in vitro translation assays. The mRNA and miRNA duplex were denatured at 70°C for 3 min and quenched on ice. hAgo2 (25 nM) with or without ORF57 was then added and incubated at 30°C for 10 min. Each reaction mixture was then mixed with translation mixture and incubated at 30°C for 20 min. The reaction was stopped on ice by the addition of SDS sample buffer. Translated proteins were resolved in a 4% to 12% SDS-PAGE gel and transferred onto a membrane for capturing images on a phosphorimager screen or X-ray film. The relative translation efficiencies in each reaction are shown below the gel. Firefly luciferase RNA (FL) was included as an internal translation control. (B) The bar graph shows relative levels of vIL-6 protein after being normalized to firefly luciferase. Bars represent means ± SD (n = 3). **, P < 0.01; *, P < 0.05 (t test).