Fig 1. Residues at the 3B-3C boundary are important for replicon replication.

(A) Schematic of the FMDV sub-genomic replicons bearing 3B deletions and tyrosine mutations used in this study. The 3B region is expanded for clarity. (B) BHK-21 cells seeded into 24 well plates were transfected with mCherry replicons bearing 3B deletions or mutations as well as wild-type (wt) and replication-defective polymerase mutant (3D^pol-GNN) controls. Transfections were performed with replicons from which 3B1, 3B2 or 3B3 had been deleted (Δ3B1, Δ3B2, and Δ3B3, respectively), both 3B1 and 3B2 deleted in tandem (Δ3B1+2) or point-mutation to the uridylatable tyrosine of 3B1, 3B2 or 3B3 (3B1^Y3F, 3B2^Y3F or 3B3^Y3F, respectively). Expression of mCherry was monitored hourly over a 24 hour period. Data shown represent mean mCherry positive cells per well at 8 hours post-transfection. Significance compared to wild-type control (n = 3 ± SD, * = p<0.05, ** = p<0.01). (C) Alignment of the replicon 3B amino acid sequences and the chimeric 3B boundary mutations (3B2/3 and 3B3/2) showing the specific amino acid sequences of each mutation underlined. (D) BHK-21 cells seeded into 24 well plates were transfected with a ptGFP replicon with the positions of 3B2 and 3B3 exchanged (3B132) and expression of ptGFP monitored hourly over a 24 hour period. The wild-type, 3D^pol-GNN, Δ3B3 and 3B123^Y3F constructs were included as controls. Data shown represent mean ptGFP positive cells per well at 8 hours post-transfection. (E) BHK-21 cells seeded into 24 well plates were transfected with mCherry replicons bearing chimeric 3B mutations and expression of mCherry monitored hourly over a 24 hour period. The wild-type, 3D^pol-GNN, Δ3B1+2 and Δ3B3 constructs were included as controls. Data shown represent mean mCherry positive cells per well at 8 hours post-transfection. Significance compared to wild-type control (n = 3 ± SD, * = p<0.05, ** = p<0.01).