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.

Correspondingly, expression of GFP-8b with mCherry-TFEB showed prominent TFEB nuclear localization compared to the control (Fig. 3b) . Immunoblotting endogenous TFEB after separation of the nuclear and cytosolic fractions in GFP-8b expressing HeLa cells showed significantly more TFEB in the nucleus than control cells, and levels of the TFEB target gene LAMP1 were consistently increased (Fig. 3c) . Confocal microscopy of GFP-8b expressing cells showed large, prominent lysosomes as assessed by LAMP1 immunofluorescence (Fig. 3d) . However, we noted only moderate overlap between the LAMP1 immunostaining and the GFP-8b aggregates. TFEB nuclear translocation following both ER and lysosomal stress has been reported to depend upon dephosphorylation of its S211 residue by the phosphatase calcineurin (PPP3CB) [28] [29] [30] . To determine the mechanism of TFEB activation in GFP-8b overexpressing cells, we immunoblotted endogenous TFEB following nuclear fractionation of GFP, GFP-8b, and GFP-8b V77K expressing cells. The GFP-8b expressing cells were treated or not with the calcineurin inhibitor cyclosporin A. Again, GFP-8b induced TFEB nuclear translocation and increased LAMP1 expression, both of which were reduced by treatment with cyclosporin A. Expression of GFP-8b V77K did not increase nuclear TFEB levels or increase the level of LAMP1 in the cytosolic fraction (Fig. 3e) . These data indicate that ORF8b aggregates causes ER and lysosomal stress, which leads to the nuclear translocation of TFEB, and the upregulation of TFEB target genes.