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.

THP-1, HEK293, A549, and HeLa cells were obtained from the American Type Culture Collection (ATCC) and maintained following ATCC's recommendations. THP-1 monocytes were differentiated into macrophages by treating with PMA (50 nM) for 3 h. To generate stable THP-1 cell lines, constructs expressing 8b-GFP or GFP were transfected followed by G418 (200 μg/ml) selection for 6 weeks. GFP-positive cells were FACS sorted twice and expanded. SARS-CoV ORF8b cDNA was generated by PCR from K14 and J17 cDNA clones by Dr. H.Y. Qi and Dr. James Shelhamer (Critical Care Medicine Department, National Institutes of Health), which were produced from the SARS-CoV genome Tor2 isolate (Michael Smith Genome Sciences Centre, Vancouver, Canada) 48 . PCR primers for cDNA generation were derived from the SARS-CoV accessory gene sequences in accession number NC_004718 (NCBI). Constructs expressing N and C terminal GFP-tagged SARS-CoV ORF8b were made using the pEGFP-N1 and C1 vectors (Clontech). PCR mutagenesis of GFP-8b was used to generate the V77K GFP-8b point mutant, and ORF8b-Flag was made by replacing the 8b-GFP c-terminal GFP with a 3x Flag tag. All constructs were verified by DNA sequencing.