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.

As previous studies have shown that ORF8b forms vesicle-like puncta in the cytosol, we used the bioinformatics aggregation prediction server PASTA2 (http:// protein.bio.unipd.it/pasta2/) 20 to investigate the intrinsic aggregation propensity of ORF8b. Analysis of the ORF8b sequence predicted a tendency to aggregate based on a carboxy-terminal VLVVL motif (aa 75-79), while reanalyzing the ORF8b sequence with V77 mutated to K (V77K) reduced its predicted propensity to aggregate (Fig. 1a) . To empirically test the aggregation tendency of ORF8b, we expressed GFP-ORF8b (GFP-8b) in Hela cells overnight and imaged the transfected cells by confocal microscopy. While some cells had a homogenous pattern of expression, other GFP-8b expressing cells showed intracellular patch and fiber aggregation patterns (Fig. 1b) . Quantification showed a considerably higher percentage of cells with intracellular aggregates in GFP-8b expressing cells than GFP vector (GFP) expressing cells (Fig. 1c) . Presumably, the homogenous expression of GFP-8b precedes the development of intracellular aggregates. These findings were replicated in A549 cells, a human lung epithelial cell line (Fig. 1b) .