Figure 3.

RNA replication is necessary for but not affected by miRNA biogenesis. (A) Top: schematic diagram of parental luciferase reporter replicon C17, which contains the first 17 aa of the capsid protein and has the rest of the structural proteins replaced by fluc. The 3′-NCR of each replicon is identical to that of the corresponding TNd/c virus mutant (Figure 1 C). Replication-negative C17 BART(+)GAA differs from C17 BART(+) by a GDD-to-GAA mutation in the polymerase active site of the viral NS5 protein. Below: IF staining with rabbit anti-TBEV serum 48 h post transfection. (B) Replication efficiencies of the C17 BART mutant set monitored by fluc activity in BHK-21 cells. Error bars represent the SD of three independent experiments, each measured in triplicate. (C) Relative Rluc activity in BHK-21 cells co-transfected with miR-BART2 reporter plasmid psiCHECK1-BART2 and the C17 BART mutants C17 BART(+) and C17 BART(+)GAA. Rluc levels are shown as a percentage of the level obtained with the parental replicon C17. Error bars represent the SD calculated from a minimum of three independent experiments, each measured in triplicate. (D) Schematic diagram of the reporter plasmid psiCHECK1-BART2, which contains the miR-BART2 target sequence fused to the Rluc-coding sequence under the control of an SV40 promoter. (E) Relative Rluc activity of C17 BART mutants at 48 h post transfection (top) and northern blot analysis (below) of the same intracellular RNA preparation used in the Rluc assay. The positions of the precursor and the mature miRNA are indicated by arrows. Total RNA of cells EP with synthetic miR-BART2 (syn. miRNA) served as a control. Diagrams (A) and (D) are not to scale.