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.

To assess the impact of GFP-8b on the localization of NLRP3, we expressed NLRP3-DsRed in HeLa cells in the presence or absence of GFP-8b. In the absence of GFP-8b NLRP3 expressed homogenously throughout the cytosol of transfected HeLa cells, however the presence of GFP-8b dramatically altered NLRP3 localization (Fig. 5d) . We found that both proteins co-localized in the cytosol in small cytoplasmic dots as well as in a large perinuclear aggregate. We performed a similar experiment using HEK 293T cells (Fig. 5e ). In the left panel the images show a HEK 293T cell expressing NLRP3 alone and one expressing both proteins, while the right panel shows another cell co-expressing NLRP3 and GFP-8b. We found that GFP-8b forms large aggregates in HEK 293T cells and that its expression disturbed the homogenous cytosolic pattern of NLRP3, and as we had observed with HeLa cells both proteins showed an overlapping expression pattern. We also checked the distribution of the trans-Golgi network in HeLa cells co-expressing GFP-8b and NLRP3-DsRed by immunostaining for TGN38 (Fig. 5f ). We found partial co-localization between the NLRP3-DsRed and GFP-8b cytoplasmic dots and TGN38.