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. 2022 Feb 11;298(3):101724. doi: 10.1016/j.jbc.2022.101724

Figure 1.

Figure 1

Expression of the SARS-CoV-2 ORF8 protein in 293T cells.A, schematic representation of the SARS-CoV-2 ORF8 protein. ORF8 is presumably a secretory protein with an N-terminal signal peptide and a glycosylation site at N78 (green line). Cysteine residues involved in the formation of intermolecular (inter) and intramolecular (intra) disulfide bonds are indicated by the red line. B, Western blot analysis. Briefly, 293T cells were transfected with C-terminally myc-FLAG-tagged ORF8 expression plasmids containing no ORF8 gene (–), ORF8 cDNA (cDNA), codon optimized ORF8 gene 1 (CO1), or the additionally optimized ORF8 gene 2 (CO2) or 3 (CO3). The pcDNA 3.1 or pQCXIP expression vector was used for transient expression. The proteins were detected by Western blotting using anti-FLAG tag mAb and anti-β-tub antibody. The bands corresponding to “FL” ORF8-mycFLAG (FL ORF8) and “spliced form 1” ORF8-mycFLAG (SF1 ORF8) are indicated. C, analysis of mRNA in the transfected 293T cells. DNA fragments amplified by RT-PCR were separated by electrophoresis and stained with ethidium bromide. The fragments amplified from pcDNA 3.1 ORF8-mycFLAG (PC1: 678 bps) and pQCXIP 3.1 ORF8-mycFLAG (PC2: 604 bps) were used as positive controls. D, schematic representation of the cryptic splice sites in the ORF8 cDNA. Based on the fragment sequences, two major sliced forms were found: spliced form 1 (SF1) and spliced form 2 (SF2). The “SFmut1” is the ORF8 cDNA of which the second cryptic donor site (230 nt) was mutated. The major cryptic splice sites in the open reading frame are indicated. E, analysis by pull-down assay. ORF8-mycFLAG proteins were expressed in 293T cells using ORF8-mycFLAG expression plasmids, cDNA-derived ORF8 (cDNA) and optimized ORF8-mycFLAG (CO3). ORF8 proteins in cells (cell) and supernatants (sup) were immunoprecipitated with anti-FLAG mAb beads. The immunoprecipitated (IP) and input fractions were analyzed by Western blot using anti-FLAG mAb and β-tub antibody. F, analysis of ORF8 with or without 2-ME. The gene-optimized ORF8 (CO3) with or without mycFLAG was transiently expressed in 293T cells. The samples treated with and without 2-ME were separated by SDS-PAGE and analyzed by Western blot with an anti-SARS-CoV-2 ORF8 rabbit polyclonal antibody. 2-ME, 2-mercaptoethanol; ORF8, open reading frame 8; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.