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.

Aggregated proteins often form detergent insoluble deposits. Thus, we separated the triton soluble and insoluble fractions of HeLa cells expressing either GFP, GFP-ORF8b, or mutant GFP-8b V77K to determine if ORF8b forms insoluble aggregates. To separate these fractions, we lysed the cells in a Triton-X based buffer and spun down the resultant lysates at 14,000 rpm for 10 min (min). The supernatant was collected and considered the triton soluble fraction, while the pellet was reconstituted in an LDS based buffer and heated at 100°C for 30 min. While preparing the triton insoluble fraction, we consistently observed that addition of LDS and heating for 30 min was unable to completely solubilize the pellet from GFP-8b expressing cells, but completely solubilized the pellet from GFP and GFP-8b V77K expressing cells. Accordingly, analysis of each fraction by SDS-PAGE followed by immunoblotting for GFP showed considerable monomeric and oligomerized GFP-8b in the insoluble fraction, while significantly less monomeric and almost no oligomerized protein was seen in the GFP-8b V77K insoluble fraction (Fig. 1d) . The control GFP was undetectable in the insoluble fraction and seen only in the soluble fraction (Fig. 1d) . Moreover, we observed consistently less GFP-8b V77K expression in the soluble fraction compared to GFP-8b, indicating that GFP-8b's ability to aggregate may be protective against degradation (Fig. 1d) .