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

Figure 3.

Figure 3

ORF8 covalently interacts with Spike and hampers Spike translocation to the Golgi apparatus.A and D–G, HEK293T cells cotransfected with plasmids encoding nontagged Spike (A, D, F and G), Spike-Strep (E), GFP-Flag (A), ORF8-Flag (A, E, F and G), ORF8-Flag I9P (non-ER mutant) (A), or ORF8-Flag Δ1-17 (ER signal deletion) (A) were not infected (A and D–G) or infected (D) with VSVΔG-GFP, for production of a hybrid VSVΔG-GFP that incorporated fully mature SARS-CoV-2 Spike (S-VSV). The cells above expressing Spike and/or ORF8 constructs or S-VSV collected from the culture medium were lysed and directly analyzed by immunoblots using antibodies against S2 (detects uncleaved and S2 fragment of Spike) (A, D and F), N-terminus of S2 (detects S2 fragment only) (A, D and F), Flag (detects GFP or ORF8) (A, E, F and G), Strep (detects Spike) (E), and β-actin, under reducing or nonreducing (protein interactions through disulfide bonds were preserved) conditions or further incubated with anti-Flag magnetic beads (A, F and G), without (A, F and G) or with pretreatment (F and G) (denaturation; 2% SDS, 5 min at 95 °C, reduction: 0.02% β-ME) of the cell lysates. The proteins that were immunoprecipitated were analyzed by immunoblots under reducing (A, F and G) or nonreducing condition (F and G). B and C, A549 (B) or HEK293T (C) cells cotransfected with a plasmid encoding Spike-Flag and a bicistronic plasmid encoding ORF8 and eGFP (ORF8-Strep-IRES-eGFP) were fixed, permeabilized, and immunostained using antibodies against S2 (detects uncleaved and S2 fragment of Spike) and TGN46 (Golgi marker), which were analyzed by fluorescence confocal microscopy imaging. B, the cells that were not expressing or expressing ORF8-Strep (identified by eGFP signals, pseudo-colored to white) were counterstained using DAPI (upper left), with the dashed box that is digitally enlarged to evaluate colocalization (bottom right) of Spike (upper right) and Golgi (TGN46) (bottom left). White scale bars represent 2 μm. C, colocalization of Spike and Golgi signals within the circular area encompassing Golgi (dashed circles in B) was analyzed by measuring Pearson’s coefficient in 60 cells (combined from three independent experiments, in which 20 cells were randomly selected). The data represent or are combined from three independent experiments and are presented as mean ± s.d. Statistical significance was analyzed using two-tailed Student’s t test (C) or one-way ANOVA (Dunnett’s test) (G). ER, endoplasmic reticulum; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; VSV, vesicular stomatitis virus.