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. 2023 Jun 23;299(8):104955. doi: 10.1016/j.jbc.2023.104955

Figure 2.

Figure 2

ORF8 colocalizes with Spike and modulates Spike protein levels and furin-dependent processing.A, A549 cells cotransfected with plasmids encoding Spike-Flag or ORF8-Strep were fixed, permeabilized, and immunostained for Flag (Spike) and Strep (ORF8), which were analyzed by fluorescence confocal microscopy imaging. The cells were counterstained using DAPI and phalloidin (upper left), with ORF8 (upper right), Spike (bottom left), or both ORF8 and Spike signals merged (bottom right). White scale bars represent 10 μm. B, C and E–H, HEK293T cells cotransfected with plasmids encoding Spike-Flag (B, C and E–G) or SARS-CoV–derived Spike (H), or eGFP-Strep (B and C), ORF8-Strep (B, C and E–H), ORF8-Strep S84L (the B lineage genotype) (E and F), or SARS-CoV–derived ORF8-Strep genotypes (ORF8ab, ORF8a, ORF8b, or ORF8a and ORF8b together) (G and H) were lysed for immunoblot analysis using antibodies against S2 (detects uncleaved and S2 fragment of Spike or SARS-CoV Spike) (B, C and E–H), S1 fragment (detects uncleaved and S1 fragment of Spike) (B and C), Strep (detects eGFP or ORF8s) (B, C and E–H), and β-actin (B, C and E–H). D, depiction of whole Spike with the site that can be cleaved by furin, yielding S1 and S2 fragments. Apparent immunoblot mass (kDa) is indicated. The data represent or are combined from three independent experiments and are presented as mean ± s.d. Statistical significance was analyzed by one-way ANOVA (Dunnett’s test).