Figure 5From: Characterization of vaccinia virus A12L protein proteolysis and its participation in virus assemblyProteolysis 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.Back to article page