FIG. 2.

Influence of SARS-CoV proteins on the UPR. (A and B) SARS-CoV S protein activates GRP94 and GRP78 promoters. 293FT cells were transiently cotransfected with pGRP78/94-Luc plus a pLenti-based expression vector for the indicated protein. Control cells transfected with pGRP78/94-Luc alone were treated with dimethyl sulfoxide (DMSO), Tg (300 nM), or Tu (5 μg/ml) for 16 h. Cells were harvested 48 hpi for dual luciferase assay. Expression levels of GRP94 and α-tubulin (α-tub) in β-galactosidase (β-gal)- and S-expressing cells were verified by Western blotting (inset). (C) SARS-CoV S protein modestly activates CHOP promoter. 293FT cells were cotransfected with pCHOP-Luc and escalating amounts of pLenti-S. Cells were harvested for dual luciferase assay as described for panel A. A DA form of eIF2α was used as a positive control in this analysis, as were Tg (300 nM) and Tu (5 μg/ml). Transcription from the CHOP promoter is significantly induced by eIF2α DA, Tg, and Tu. (D) SARS-CoV S protein does not significantly affect ATF4 translation. 293FT cells were cotransfected with pATF4-UTR-Luc and escalating amounts of pLenti-S. Cells were harvested for dual luciferase assay as described for panel A. Tg (300 nM) and Tu (5 μg/ml) were used as positive controls in this analysis. pATF4-UTR-Luc contains the 5′ UTR of human ATF4 fused to the coding region of firefly luciferase. This construct is similar to those described for mouse ATF4 (32, 52).