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

Figure 4.

Figure 4

Effects of SARS-CoV-2 ORF8 on expression and downregulation of HLA-A2, ACE2, and CoV-2 S proteins.A–C, Western blot analyses of total ORF8 in cells. Briefly, 293T cells were cotransfected with each target expression plasmid (500 ng), (A) HLA-A0201-HIS, (B)ACE2, or (C) CoV-2 S, and different amounts of the expression plasmid encoding ORF8-mycFLAG (CO3) (0, 1500, 1000, and 500 ng). The total plasmid amount for each transfection was normalized with the empty vector. Next, 293T cells were cotransfected with each target plasmid (125 ng), GFP expression plasmid pAcGFP1-N1 (125 ng), plus pQCXIP empty vector control (C), ORF8-mycFLAG (WT), or ORF8 ΔSP-mycFLAG (ΔSP)-expressing plasmid (750 ng). D–F, FACS analyses of cell surface proteins. D, endogenous HLA-2 surface expression. 293T cells were cotransfected with pAcGFP1-N1 (125 ng) and the ORF8 expression plasmid (875 ng) for analysis. E, ACE2 and (F) CoV-2 S expression on the cell surface. Cells were transfected with each expression plasmid and collected at 40 h after transfection for flow cytometry analysis. The mean fluorescence intensities (MFI) of HLA-, ACE2-, and CoV-2 S-positive cells (gated on GFP-positive cells) were shown (n = 3). The bar graphs on the right represent the mean ± SD (error bars). Student’s t test and one-way ANOVA were used. NS, not significant; ∗p < 0.05, ∗∗p < 0.005. ORF8, open reading frame 8; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; WT, wild-type.