Figure 5.

ORF8 limits the levels of cell-surface Spike.A, a monolayer of HEK293T cells stably expressing ACE2/TMPRSS2 were cotransfected with a plasmid encoding Spike-Flag or ORF8-Strep. The cells were fixed, permeabilized, and immunostained with antibodies against Flag (Spike) and Strep (ORF8). After counterstaining with DAPI, the cells were analyzed using fluorescence confocal microscopy imaging. The dotted boxes were digitally enlarged (upper right inset). The scale bars represent 50 μm. B–H, HEK293T cells cotransfected with a plasmid encoding Spike-Flag (C-terminal–tagged) (B, C and E), Flag-Spike-Flag (N-terminal– and C-terminal–tagged) (D and E), nontagged Spike (F and G), or SARS-CoV–derived Spike (F–H), and a bicistronic plasmid encoding eGFP and different ORF8-Strep genotypes (ORF8-Strep (B–E and H), ORF8-Strep S84L (B and C) or SARS-CoV-derived ORF8ab-Strep, ORF8a-Strep, or ORF8b-Strep (H)). The cells were harvested and directly immunostained for cell surface Spike by incubating with antibodies against S2 (B–D, G and H; detects uncleaved Spike, S2 fragment, or SARS-CoV-derived Spike) or Flag (E) or fixed, permeabilized, and immunostained by incubating with antibodies against S2 (G). Cell-surface Spike levels (B–H) in viable (LIVE/DEAD-negative) cells expressing ORF8-Strep (eGFP-positive) or total cellular Spike levels (G) in cells expressing ORF8-Strep (eGFP-positive) were measured by flow cytometry. Cellular expression levels of SARS-CoV-2 Spike or SARS-CoV Spike were confirmed by immunoblot analysis (F). The data represent or are combined from three independent experiments and presented as mean ± s.d. Statistical significance was analyzed using one-way ANOVA (C, E and H; Dunnett’s test) or two-tailed Student’s t test (D and G). SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.