Fig 2: ORF8 N78 glycosylation blocks its interaction with IL17RA.(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.

Fig 3: N-linked glycosylation of ORF8 protects mice from cytokine storm.(A) Unglycosylated ORF8 (ORF8) or synthetic N-linked-glycosylated ORF8 (ORF8-N-Glyc) (1 µg/mL) was added into the culture medium of THP-1 DM cells to stimulate IL-17 pathway. The interaction of ORF8 and IL17RA was detected by co-immunoprecipitation. (B) ORF8 or ORF8-N-Glyc protein was added into the culture medium of THP-1 DM cells at a concentration of 10 ng/mL. The release of cytokines and chemokines was detected by ELISA. (C) Schematic diagram of ORF8 protein treatment model. Mice were treated with aerosols of PBS (n = 13), unglycosylated ORF8 (n = 18) or synthetic N-linked-glycosylated ORF8 proteins (n = 17) (200 µg/mouse) every other day. For IL17RA antibody injections (n = 12), each mouse received 200 µg per injection every three days as indicated. (D-G) Survival curves were calculated using the Kaplan-Meier method (D). Lung lesions were detected by H&E staining at day 7 post infection (dpi) (E). The release of cytokines and chemokines in lungs (F) and livers (G) were detected by ELISA at 7 dpi. (H-J) Hamsters were intranasally infected with 4×105 PFU SARS-CoV-2 (n = 18) or ORF8-deleted SARS-CoV-2 variant (n = 18). Weight loss data were documented daily (H). Lung lesions were detected by H&E staining at 7 dpi (I). The relative expression of cytokines and chemokines in lungs were detected by qRT-PCR at 2, 4 and 7 dpi (J). Representative images from n = 3 biological replicates are shown (A, E, I). Data are shown as the mean ± s.e.m. of n = 6 biological replicates (B, F, G, J). Log-rank (Mantel-Cox) test (D) and one-way ANOVA followed by Bonferroni post hoc test (B, F, G, J) were used for data analysis. Scale bar = 500 µm. *, p < 0.05, **, p < 0.01. Abbreviations: n.s., not significant.