Fig 3. Mutations at the C-terminus of 3B3 prevent complementation of 3D^pol mutations but not 3B mutations in trans.

(A) BHK-21 cells seeded into 24-well plates were co-transfected with mCherry replicons bearing replication-defective 3B or 3D^pol mutations or controls and wild-type ptGFP, ptGFP-3B123^Y3F or ptGFP-3D^pol-GNN replicon. In (A) all the mCherry replicons contained a full IRES (+). In (B) the mCherry replicons contained a deletion of the entire IRES (Δ), in addition to the indicated non-structural protein mutation (3D^pol-GNN, 3B123^Y3F, 3B3/2). Co-transfections were performed with yeast tRNA (bars labelled ‘na’) as a negative control (i.e. no complementation) in both experiments. Expression of ptGFP is shown representing mean positive cells per well at 8 hours post transfection (n = 3, ± SD). For clarity the statistics were excluded from the figure. The mCherry data is shown in S3 Fig. In (C), after the initial the co-transfection, total RNA was harvested and replicons RNA purified by oligo(dT) precipitation. 100 ng of total oligo(dT) purified RNA was re-transfected into BHK-21 cells seeded into 24-well plates and expression of ptGFP and mCherry were monitored hourly. Data shows mean ptGFP positive cells per well at 8 hours post transfection. Significance compared to plus yeast tRNA negative control. (n = 5 ± SD, * = p<0.05, ** = p<0.01).