SARS-Coronavirus Open Reading Frame-8b triggers intracellular stress pathways and activates NLRP3 inflammasomes

Shi, Chong-Shan; Nabar, Neel R.; Huang, Ning-Na; Kehrl, John H.

Intracellular protein aggregates and misfolded proteins cause ER stress and induction of the unfolded protein response (UPR), which can be quantified by detecting the upregulation of the UPR protein CHOP 21, 22 . Confocal imaging showed that aggregated GFP-8b caused ER dispersion as assessed by immunostaining for ERp72 (Fig. 2a) . The expression of GFP did not affect the usual juxta nuclear localization of the ER while expressing GFP-8b disrupted the usual ERp72 localization. Immunoblotting showed greater CHOP induction after transfection of GFP-8b than either GFP or GFP-8b V77K (Fig. 2b) , indicating that GFP-8b forms protein aggregates dependent on its valine 77. As intracellular protein aggregation is toxic once the aggregates overwhelm cellular homeostatic mechanisms, we looked at cell death by Trypan blue uptake following expression of GFP, GFP-8b, and GFP-8b V77K. Expression of GFP-8b induced significantly more cell death than did GFP, while the GFP-8b V77K was partially protective (Fig. 2c) . Taken together, this data suggests that GFP-8b forms insoluble protein aggregates and that can cause cell death, which at least partially depends on its ability to form intracellular aggregates.