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.

Initially, two studies had difficulty expressing the ORF8b protein, suggesting it may be rapidly degraded in cells 14, 15 . However, antibodies raised specifically against the ORF8b protein detect low levels of expression as early as 8 h (h) post infection, while expression is prominent by 24 h 16 . Immunostaining ORF8b after expression in Vero E6 cells localized it to cytosolic punctate vesicle-like structures reminiscent of intracellular aggregates 17 . Here, we investigate the cellular mechanisms by which ORF8b contributes to SARS-CoV pathology. We report that ORF8b forms intracellular aggregates dependent on a valine at residue 77, which contributes to the induction of lysosomal stress, autophagy, and eventual cell death. Given the importance of IMMs in SARS-CoV pathology, the link between intracellular aggregates and inflammasome activation 18 , and the observation that SARS-CoV infected patients have elevated serum levels of IL-1 and IL-18 19 , we tested whether ORF8b might affect inflammasome activation in macrophages and lung epithelial cells. We found that ORF8b triggers robust NLRP3 inflammasome activation and IL-1β release. NLRP3 activation was accompanied by direct binding of ORF8b to the LRR domain of NLRP3. In sum, this study suggests that ORF8b activates cell stress pathways by forming intracellular aggregates and may be important in the aberrant activation of macrophages during SARS-CoV infection.