FIG. 8.

Replication of NS5:S653F KUN is associated with greater IFN resistance than WT KUN. (A) Western blot demonstrating increased efficiency of IFN antagonism by KUN NS5:S653F. HEK293 cells were left uninfected or were infected with WT or NS5:S653F KUN at an MOI of 1. At the times indicated, cells were treated with 1,000 U/ml of IFN-β for 15 min, and cell lysates were analyzed for virus protein expression (NS5 and E) and STAT1 phosphorylation. (B) Effect of S653F mutation on ISRE-dependent gene expression during KUN replication. HEK293 cells were transfected with the pISRE-luc reporter plasmid and a plasmid constitutively expressing Renilla luciferase. Cells were infected 24 h later with WT or NS5:S653F KUN at an MOI of 1 and incubated for a further 24 h. Cells were treated with 1,000 U of IFN-β for 7 to 8 h prior to assaying for dual luciferase activities. Fold induction of firefly luciferase activity was determined relative to the normalized luciferase activity value of virus-infected cells not treated with IFN. Error bars indicate SEM from three individual experiments; asterisks indicate significant differences from cells transfected with the empty vector and not treated with IFN (P < 0.005, by Student's t test). (C) KUN replication in the presence of IFN. Vero cells were infected with WT or NS5:S653F KUN at an MOI of 0.001 and treated with high-dose IFN-β at 12 hpi. Supernatants were assayed for infectious virus by focus-forming assay at the times indicated. Error bars indicate SEM from five independent experiments performed in triplicate; an asterisk indicates significant difference (P = 0.0079, Mann-Whitney test). FFU, focus forming units.