Proteolysis of A12L. In order to characterize the proteolytic processing of A12L, we examined the utilization of each AG/X site and determined the responsible proteinase for the processing. A. The A12L ORF with double AG/X site mutations were placed into pRB21 and appended with a C-terminal FLAG epitope (FC). The N-terminal AG/A site and internal AG/K site mutations, the N-terminal AG/A and C-terminal AG/K site mutations, and the internal and C-terminal AG/K site mutations were indicated as SD 1&2, SD 1&3, and SD 2&3, respectively. Each transient expression would result in 4, 8, and 15 kDa cleavage product by cleavages at the C-terminal and internal AG/K residues, and N-terminal AG/A site. B. All of the plasmids contained the same mutations as described above except a FLAG epitope in the N-terminus (FN) of A12L ORF. Ara-C refers to the cells transfected with pA12L-FN in the presence of cytosine arabinoside (Ara-C, 40 μg/mL) as an inhibitor of VV late gene expression. The FLAG tag at the N-terminus of each mutant plasmid would represent the products of 16, 12, and 6 kDa peptides resulted from utilization of the C-terminal, internal AG/K, and N-terminal AG/A site. pA12L-FN: A12L intact ORF under an early/late synthetic promoter. An Arrow indicates a cleavage product near N-terminus. C. BSC-40 cells were transfected with a plasmid containing a FLAG epitope at C-terminus of A12L ORF (pA12L-FC) and infected with WR or Dts-8 (I7L temperature-sensitive mutant virus). Having WR-infected cells as a positive control, Dts-8 infection at the permissive (31°C) and non-permissive (39°C) temperatures showed I7L participation in A12L cleavage event. pRB21: vector plasmid containing an early/late synthetic promoter. pI7L: plasmid born I7L in pRB21. D. To determine another cleavage reaction at N-terminus as indicated with arrow at Fig. 5C, the pA12L-FC and pA12L-FN were transfected into BSC-40 cells and infected with VV WR and Dts-8 at an MOI of 5 PFU/cell. Both infections were incubated at permissive temperature.