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.

ORF8b induces lysosomal stress, the nuclear translocation of TFEB, and the activation of Transcription factor EB target genes Aggregated proteins are typically cleared from the cytosol by the autophagy-lysosome pathway, which engulfs bulk cargo in the cytoplasm and delivers it to the lysosome for degradation 23, 24 . Formation of intracellular protein aggregates can disrupt this pathway, as aggregated proteins are poorly degraded by lysosomes. Lysosomal protein buildup then results in lysosomal stress and compensatory activation of transcription factor EB (TFEB), the master transcriptional regulator of autophagic and lysosomal machinery 25, 26 . To determine if ORF8b disrupts autophagic and lysosomal homeostasis, we expressed GFP-8b and mCherry-galectin 3 in HeLa cells followed by imaging via confocal microscopy. Under homeostatic conditions galectin-3 is localized diffusely throughout the cytosol, but rapidly forms puncta on lysosomes following lysosomal stress/damage 27 . Accordingly, GFP expressing cells showed diffuse cytosolic galectin-3 distribution, while many GFP-8b expressing cells showed clear galectin-3 puncta formation, some of which co-localized with GFP-8b (Fig. 3a) .