SARS-CoV-2 and MERS-CoV Protein Expression

Vero E6, Huh-7, 293T, and HeLa cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and Calu-3 2B4 cells (Kent Tseng, University of Texas Medical Branch) were grown in MEM supplemented with 20% FBS as previously described (41 (link)). Codon-optimized sMacro (nucleotides 3262 to 3783 of SARS-CoV MA15) and MERS-CoV ORF4a were synthesized and cloned into pUC57 (GenScript). The sMacro and GFP sequences were PCR amplified and ligated into a linearized pcDNA3 plasmid using In-Fusion (Invitrogen) cloning. The ORF4a sequence was PCR amplified and then restriction digested and ligated into the pLKO plasmid. The resulting constructs were confirmed by restriction digestion, PCR, and direct sequencing. Human IFN-α (B/D) and IFN-β were purchased from PBL (Piscataway, NJ). High-molecular-weight poly(I-C) and poly(dA-dT) were purchased from InvivoGen (San Diego, CA). Cells were transfected with either Polyjet (Amgen, Thousand Oaks, CA) or Lipofectamine 2000 (Fisher Scientific, Waltham, MA) per the instructions of the manufacturers.