FIG 8.

NS4 expression does not significantly affect host cellular protein shutoff, mRNA splicing, or translation. (A) Primary ovEC were mock infected or infected with BTV8wt and BTV8ΔNS4, and nascent proteins were metabolically labeled with [³⁵S]methionine/cysteine for 2 h at the time points indicated. Cell extracts were fractionated by SDS-PAGE, and the gels were stained with Coomassie blue. The dried gels were analyzed by phosphorimaging. The black arrow indicates cellular actin. The signal intensity was quantified using ImageQuant software. The red arrows indicate BTV proteins. (B) CPT-Tert cells were cotransfected with variable amounts of plasmids expressing BTV8 NS4 protein and either a plasmid expressing FLuc under the control of a CMV promoter or a plasmid expressing RLuc downstream of an intron and the CMV promoter. FLuc or RLuc expression was assessed 22 h posttransfection. Cell lysates were analyzed by Western blotting using antibodies to NS4. One-way ANOVA, P < 0.0001. **, P < 0.01; ***, P < 0.001 (Dunnett's multiple-comparison test). (C) CPT-Tert cells were cotransfected with variable amounts of a DNA plasmid expressing BTV8 NS4 and either a plasmid expressing FLuc under the control of a CMV promoter or capped and polyadenylated RNA made in vitro. FLuc activity was assessed 22 h posttransfection. Cell lysates were also analyzed by Western blotting using antibodies to NS4. One-way ANOVA, P < 0.0001. *, P < 0.05; ***, P < 0.001 (Dunnett's multiple-comparison test). The error bars represent standard deviations.