Fig. 3

ATF6 is necessary and sufficient for viral replication by stabilizing P1 precursor viral protein. After transfection with scATF6 or siATF6 for 48 h, RD cells were transfected with HA-ATF6-FLAG (a) or HA-ATF6-FLAG and AS3w-P1-cFLAG (right panel, d). At 72 h after siRNA transfection, the cells were infected with EV-A71 at an MOI of 10. Cells were collected at the indicated time points for titer determination (a) and western blotting (d). a HA-ATF6-FLAG alleviated the siATF6-mediated reduction in viral production. The cells were collected and subjected to three freeze-thaw cycles for viral progeny collection. The ratio of viral titers in siATF6-transfected cells in the presence of HA-ATF6-FLAG or vector control was normalized to that of the scATF6-transfected cells, which was set arbitrarily at 1. The data are presented as the means ± SD. n = 3, *P < 0.05, **P < 0.01, and ***P < 0.001. b ATF6 was not associated with viral protein processing. Anti-ATF6 and anti-GAPDH antibodies were used to detect ATF6 and GAPDH. Anti-3D, which recognized both precursors (P3 and 3CD) and the mature viral protein 3D, was used to monitor the processing of the viral P3 region. The processing efficiency was expressed as a ratio of the band intensities of 3CD and (P3 + 3CD) (lanes 3-6) or of 3D and (P3 + 3CD + 3D) (lanes 7-8). The band intensity of 3D at 4 and 5 h p.i. was below the detection limit. The bands at 7 h p.i. were saturated and were not included in the analysis. Representative data from three independent experiments are presented. c ATF6 did not affect IRES activity. A structural diagram of the pcDNA-RHF EV-A71 IRES reporter is shown at the top. siRNA transfection was performed, and the 2231 IRES reporter plasmid and its corresponding control vector (pcDNA-RHF) were transfected 48 h later, followed by lysate preparation for a reporter assay. The relative luciferase activity of siATF6-transfected cells was normalized to that of scATF6 control-treated cells (arbitrarily set at 1). The data are presented as the means ± SD from three independent experiments. d ATF6 was associated with viral protein stability. CHX (10 μg/mL) was added 72 h after siATF6 transfection, and the cells were harvested for western analysis at 150 and 180 min (lanes 1-8) or at 150 min (lanes 9-16). Anti-VP1 and anti-ATF6 antibodies were used to detect P1 and endogenous and exogenous ATF6. The ratio of P1 to GAPDH was normalized to that at the 150 min time point in the absence of CHX in scATF6- or siATF6-transfected cells, which was set arbitrarily at 1. The data are representative of three independent experiments