Fig. 1.
Downregulation of MHC-I by SARS-CoV-2 WA1 and its ER-localized gene products. (A) Genome organization of SARS-CoV-2. SARS-CoV-2 accessory proteins are depicted at the 3′ end of the genome. ORF3a, ORF7a, and ORF8 are highlighted in red. (B) Vero E6 or HEK293T-Ace2 cells were infected with SARS-CoV-2WA1 (MOI = 10). 24 h post infection, cells were collected for flow cytometry analysis for surface MHC-I (n = 4). Histograms (Left) are summarized (Right) where mean fluorescence intensity of MHC-I staining with W6/32 was normalized to uninfected group. (C) Transient expression of ORF3a, ORF7a, and ORF8 in HeLaM cells. Western Blot analysis of expression of accessory proteins, MHC-I heavy chain (HC), or GRP94 as a loading control, 24 h post transfection (Left). Flow cytometric analysis of surface MHC-I (Middle) and EGFR (Right) in cells expressing ORF3a, ORF7a, and ORF8 in HeLaM cells (n = 4). (D) Transient expression of ORF3a, ORF7a, and ORF8 in HEK293T cells. Western Blot analysis of expression of accessory proteins, MHC-I heavy chain (HC), or GRP94 as a loading control 24 h post transfection (Left). Flow cytometric analysis of surface MHC-I (Middle) and EGFR (Right) in cells expressing ORF3a, ORF7a, and ORF8 in HEK293T cells (n = 4). (E) Effect of ORF3a on the Golgi morphology. HeLaM cells were transfected with plasmids encoding SARS-CoV-1 ORF3a or SARS-CoV-2 ORF3a for 24 h. Fixed cells were stained with Hoechst (blue), for ORF3a (green), and anti-GM130 to visualize the Golgi (red). (F) HelaM cells were transfected with plasmids encoding SARS-CoV-ORF3a or SARS-CoV-2-ORF3a for 24 h, followed by analysis of expression by western blotting (Left) or flow cytometric analysis of surface MHC-I (Right, n = 4). Quantitative data shown are mean ± SD (error bars), and the dashed line indicates the 70% mark. Statistical significance was evaluated using the unpaired Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.