Fig. 5.
Cleavage of ATF6 by the proteolytic activity of 3Cpro. a Examination of the protease catalytic activity of Wt and individual mutants of 3Cpro. HEK293T cells (1 × 106 cells per well in six-well plates) were transfected with the empty vector AS3w or with constructs carrying Wt 3C, the protease-deficient H40D or C147S single mutant, the H40D/C147S double mutant, or the R84Q RNA-binding mutant of 3C. Cell lysates were prepared 24 h post transfection and were subjected to SDS-PAGE and immunoblotting using antibodies against CstF-64, 3C, and GAPDH. A control viral infection was performed at an MOI of 200, and the cells were harvested at 16 h p.i. The cleavage products (CP) of CstF-64 are indicated by an arrow. b Cleavage of ATF6 by Wt or mutant 3Cpro. HEK293T cells were first transfected with a plasmid encoding HA-ATF6-mycHis or the control vector pcDNA3.1/mycHis B(+)::FLuc. After 24 h, the cells were transfected with the control vector or plasmids encoding Wt or mutant 3Cpro. Cells were harvested and lysates were prepared 24 h post transfection. Equal amounts of cell lysates (30 μg/lane) were analyzed with 10% SDS-PAGE, followed by immunoblotting using antibodies against HA, FLAG, or GAPDH. The data shown here are representative of three independent experiments. c Predicted cleavage sites of 3Cpro at the dipeptide glutamine–glycine (QG) near amino acids 512 and 517. Plasmids encoding point mutations (QG→QA) were constructed to generate the G512A, G517A, and G512A/G517A mutations of HA-ATF6-mycHis. d Mapping of the cleavage sites. HEK293T cells were first transfected with plasmids encoding HA-ATF6-mycHis or the point mutation variants. After 24 h, the cells were transfected with the control vector or a plasmid encoding Wt 3C tagged with FLAG. Cells were harvested and lysates were prepared 24 h post transfection. Equal amounts of cell lysates (30 μg/lane) were analyzed with 10% SDS-PAGE, followed by immunoblotting using antibodies against HA, FLAG, or GAPDH. The asterisk indicates the cleavage product N-70. The results shown here are representative of at least four independent experiments