Fig 1. 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 ΔORF8 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.