Fig 2: SARS-CoV-2 ORF8 can be secreted by epithelial cells.(A) Schematic diagram of SARS-CoV-2 infectious cDNA clone generated by a reverse genetic system. The cDNA fragments F1-F7 were synthesized and assembled into full-length SARS-CoV-2 cDNA, and RNA transcription, electroporation, and virus production were carried out in Vero E6 cells. (B) Schematic diagram of SARS-CoV-2 infection model. Calu-3 epithelial cells were infected with SARS-CoV-2 at a MOI of 0.01. After 12 hours, cell culture supernatant was centrifuged and divided into two parts for western blotting and ELISA, respectively. (C) The secretion of ORF8 in (B) was detected by western blotting and ELISA. (D) Jurkat cells or Calu-3 epithelial cells were infected with HIV-1 or SARS-CoV-2. After 12 hours, the secretion of ORF8, Nef and 3CL pro was detected by western blotting. (E) Schematic diagram of time-dependent ORF8 secretion upon SARS-CoV-2 infection. Twelve hours after SARS-CoV-2 infection, cell culture medium was replaced, and the amount of ORF8 protein in the supernatant was detected by ELISA every 2 hours. (F-H) Schematic diagram of ORF8-deleted SARS-CoV-2 variant (F). ORF8 coding sequence was deleted from the cDNA of F7 fragment. SARS-CoV-2 ?ORF8 variant was used to infect Calu-3 cells, THP-1 DM cells, and the co-culture system. The secretion of cytokines and chemokines related to cytokine storm was detected by ELISA (G, H). (I) Calu-3 Ace2+/+, Calu-3 Ace2-/-, THP-1 DM Il17ra+/+ and THP-1 DM Il17ra-/- cells were used to form four kinds of culture systems. The secretion of cytokines and chemokines in different cell culture systems was detected by ELISA. Representative images from n = 3 biological replicates are shown (C, D). Data are shown as the mean ± s.e.m. of n = 6 biological replicates (C, E, G-I). Unpaired two-tailed Student t test (C) and one-way ANOVA followed by Bonferroni post hoc test (G, I) were used for data analysis. *, p < 0.05, **, p < 0.01.

Fig 3: Unconventional secretion of ORF8 induces cytokine storm.(A) Schematic diagram of unconventional secretion model upon SARS-CoV-2 infection. Brefeldin A (3 µg/mL) or Monensin (2 µM) was used to pretreat Calu-3 cells for 2 hours, followed by SARS-CoV-2 infection, or Flag-tagged ORF8 plasmid transfection. After 12 hours, the supernatant was collected. (B-F) The supernatant collected in (A) was divided into three parts. One part was used for ELISA to examine the secretion of ORF8 protein (B); the second part was used to stimulate THP-1 DM cells (C, D); the third part was used to purify ORF8 protein and then stimulate THP-1 DM cells at a final concentration of 10 ng/mL (E, F). After 12 hours, the release of cytokines and chemokines was detected by ELISA (C-F). (G) Brefeldin A or Monensin was used to pretreat Calu-3 cells for 2 hours, followed by SARS-CoV-2 infection. ORF8 protein was purified 12 hours after infection and then PNGase F (1,000 units/µg protein), O-Glycosidase (4,000 units/µg protein) or a2–3, 6, 8, 9 Neuraminidase A (4 units/µg glycoprotein) was added to release glycans. Western blotting was used to detect the glycosylation of ORF8 protein. (H, I) Calu-3 cells were infected with SARS-CoV-2 (H) or transfected with ORF8-Flag plasmid (I). Tunicamycin (2µg/mL) was added into Calu-3 cells for 2 hours to prevent N-linked glycosylation; PNGase Fwas used to remove the N-linked glycosylation in purified ORF8 protein. After deglycosylation assays, purified ORF8 protein was used to stimulate THP-1 DM cells at a final concentration of 10 ng/mL. The release of cytokines and chemokines was detected by ELISA at 12 hours post stimulation. Representative images from n = 3 biological replicates are shown (G). Data are shown as the mean ± s.e.m. of n = 6 biological replicates (B-F, H, I). One-way ANOVA followed by Bonferroni post hoc test (B-F, H, I) was used for data analysis. *, p < 0.05, **, p < 0.01. Abbreviations: n.s., not significant.

Fig 4: 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 5: 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.