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.

Activation of TFEB increases both lysosomal biogenesis and autophagic flux 31 , thus we checked to see whether GFP-8b affects autophagy downstream of TFEB. Expression of GFP-8b induced clear formation of RFP-LC3 puncta, while GFP did not (Fig. 4a) . In some cells the RFP-LC3 defined autophagosomes strongly co-localized with the GFP-8b aggregates while in other cells many of the aggregates did not (Fig. 4a ). An increase in LC3 puncta can be due to an increase in autophagosome formation or a decrease in autophagosome/lysosome fusion 32 . During autophagosome formation, LC3 is lipidated and converted from LC3-I to LC3-II. We expressed GFP or GPF-8b in the presence or absence of Bafilomycin A1, which blocks autophagosome-lysosome fusion, and immunoblotted the cell lysates for LC3. We found that the cell lysates from the GFP-8b and GFP expressing cells had similar LC3-II Fig. 2 ORF8b induces ER stress and cell death. a Confocal microscopy images of HeLa cells transiently transfected with either GFP or GFP-8b and immunostained for the endoplasmic reticulum marker ERp72 (red). Nuclei were counterstained with DAPI and cells were fixed prior to imaging. Individual and merged images are shown, and the scale bar is 5 μm. b Immunoblot looking at the accumulation of the ER stress marker CHOP after transient transfection of GFP, GFP-8b, or GFP-8b V77K in HeLa cells. c Quantification of cell death by Tryptan Blue uptake after overnight transfection of HeLa cells with the indicated proteins. A minimum of three fields with >50 cells per field were quantified. Statistical significance evaluated with Student's t-test (*p < 0.05, **p < 0.01). Each experiment repeated a minimum of three times levels. However, bafilomycin A1 treatment increased the LC3-II levels in the GFP-8b expressing cells more than it did in the control cells (Fig. 4b) . These results indicate that GFP-8b induces autophagic flux. Treatment with bafilomycin A1 increased the GFP-8b levels, but not GFP levels suggesting that ORF8b aggregates are partially degraded by the autophagy-lysosome pathway. Taken together, the GFP-8b intracellular aggregates trigger cellular stress. This results in a calcineurin dependent activation of TFEB and TFEB target genes, which leads to an increase in autophagic flux.