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. 2023 Jan 23;19(1):e1011128. doi: 10.1371/journal.ppat.1011128

Fig 4. ORF8 N78 glycosylation blocks its interaction with IL17RA.

Fig 4

(A) Schematic diagram of glycosylation identification based on HLPC-MS/MS. DMSO (control), Brefeldin A (3 μg/mL) or Monensin (2 μM) was used to pretreat Calu-3 cells for 2 hours. The supernatant was collected for HLPC-MS/MS analysis. (B) SARS-CoV-2 ORF8 secreted through conventional pattern has N78 glycosylation. An increase of 2.9890 Da of Asn residue was used to determine N-linked glycosylation. (C-G) Calu-3 cells were infected with SARS-CoV-2 ORF8-N78Q variant (C, D, F), or transfected with ORF8 N78Q plasmid (C, E, G). After 12 hours, the supernatant was collected and divided into three parts. One part was used to purify ORF8 protein, followed by PNGase F digestion and western blotting (C); the second part was used to stimulate THP-1 DM cells (D, E); the third part was used to purified ORF8 protein and then stimulate THP-1 DM cells at a final concentration of 10 ng/mL (F, G). After 12 hours, the release of cytokines and chemokines was detected by ELISA (D-G). (H) Calu-3 cells were infected with SARS-CoV-2 ORF8-N78Q variant, or transfected with ORF8-N78Q plasmid. Twelve hours later, the supernatant was used to stimulate THP-1 DM cells for another 12 hours. The interaction of ORF8 and IL17RA was detected by co-immunoprecipitation. (I, J) Calu-3 cells were infected with SARS-CoV-2 ORF8-N78Q variant (I), or transfected with ORF8-N78Q plasmid (J). After 12 hours, the supernatant was collected to purify ORF8 protein. After PNGase F digestion, the ORF8 protein was used to stimulate THP-1 DM cells for 12 hours, and the interaction of ORF8 and IL17RA was detected by co-immunoprecipitation. (K, L) Brefeldin A or Monensin was used to pretreat Calu-3 cells for 2 hours, followed by infection with SARS-CoV-2 ORF8-N78Q variant (K), or transfection with ORF8-N78Q plasmid (L). Twelve hours later, the supernatant was used to stimulate THP-1 DM cells for another 12 hours. The interaction of ORF8 and IL17RA was detected by co-immunoprecipitation. Representative images from n = 3 biological replicates are shown (C, H-L). Data are shown as the mean ± s.e.m. of n = 6 biological replicates (D-G). One-way ANOVA followed by Bonferroni post hoc test (D-G) was used for data analysis. *, p < 0.05, **, p < 0.01.