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.

Enterovirus 71 (EV71) 3D protein stimulates NLRP3 inflammasome activation by interacting with NLRP3 to facilitate inflammasome complex assembly. EV71 3D interacted with NLRP3 through the NACHT and LRR domains. To test whether ORF8b might also interact with NLRP3, we transiently expressed 8b-Flag in PMA differentiated and LPS stimulated THP-1 cells and tested whether we could detect an association with endogenous NLRP3. We found that immunoprecipitated 8b-Flag pulled down endogenous NLRP3, while control immunoprecipitates did not (Fig. 6a) . To characterize the region in NLRP3 domain responsible for the interaction, we examined the binding of Myc-tagged truncated NLRP3 proteins to 8b-Flag. In addition to full-length NLRP3, we used constructs that express NLRP3 with a deleted leucine-rich repeat (LRR) domain (NLRP3ΔLRR), a deleted Pyrin domain (NLRP3ΔPYD), or deleted NACHT and LRR domains (NLRP3ΔLRRΔNACHT). Following transient expression, 8b-Flag interacted with both fulllength NLRP3 and the PYD domain deleted protein, but not the LRR and LRR/NACHT domains deleted proteins (Fig. 6b) To directly show that ORF8b interacts with the LRR domain of NLRP3, we expressed the NLRP3 LRR domain as a Myc tagged protein (amino acids 537-991) and collected Myc immunoprecipitates following GFP-8b or GFP expression. We found GFP-8b, but not GFP in the Myc-immunoprecipitates (Fig. 6c) . Next, we expressed the non-functional NLRP3 LRR domain and determined whether its presence affected the induction of mature IL-1β secretion by ORF8b. We found that expression of the NLRP3 LRR domain impaired the production of mature IL-1β after GFP-8b expression in inflammasome reconstituted HEK 293T cells (Fig. 5e) , confirming the functional importance of this interaction. In summary, our data indicates that ORF8b activates the NLRP3 inflammasome and directly targets the NRLP3 LRR domain.