Fig 2: Human NPCs express IL17RA and respond to exogenous IL-17a by modulating the activity of ERK1/2 and mTORC1 signaling pathways. (A) Relative transcript levels of IL17RA in iPSC-derived NPCs of control (n = 4) and ASD (n = 7) subjects. (B) Relative protein levels of IL17RA in iPSC-derived NPCs of control (n = 5) and ASD (n = 7) subjects. ß-actin was used as a loading control and representative immunoblot images are shown. Human NPCs endogenously express IL17RA. (C) Relative protein levels of IL17RA in iPSC-derived NPCs of control (n = 2–3) and ASD (n = 5) subjects after treatment with either vehicle (–) or rhIL-17a (10 and 50 ng/ml) for 48 h. The data are expressed as fold change in IL17RA expression relative to vehicle-treated samples. ß-actin was used as a loading control and representative immunoblot images are shown. No significant differences in the protein levels of IL17RA were observed between rhIL-17a-treated and untreated human NPCs. (D) Time course analysis of pERK1/2 expression in iPSC-derived NPCs of control (n = 4) and ASD (n = 6) subjects after treatment with either vehicle (–) or rhIL-17a (50 ng/ml). Total-ERK was used as a loading control and the data are expressed as fold change in normalized pERK1/2 expression relative to vehicle-treated samples. Representative immunoblot images are shown. (E) Time course analysis of pRPS6 expression in iPSC-derived NPCs of control (n = 3) and ASD (n = 4) subjects after treatment with either vehicle (–) or rhIL-17a (50 ng/ml). ß-actin was used as a loading control and the data are expressed as fold change in normalized pRPS6 expression relative to vehicle-treated. Representative immunoblot images are shown. Exogenous IL-17a led to a significant increase in pERK1/2 levels and significant decrease in pRPS6 levels in human NPCs. *p = 0.05, **p = 0.01, and ****p = 0.0001. (F) Time course analysis of pNF-kB65 expression in iPSC-derived astrocytes (used as positive control of pNF-kB expression) and in iPSC-derived NPCs of control (n = 2) and ASD (n = 1) subjects after treatment with either vehicle (–) or rhIL-17a (50 ng/ml). Total-NF-kB and ß-actin were used as a loading controls and representative immunoblot images are shown. While iPSC-derived astrocytes express pNF-kB65, no detectable expression of this protein was observed in human NPCs.

Fig 3: ORF8 binding enhances the heterodimerization of hIL-17RA/C. (A) Purified ORF8-Flag interacts with hIL-17RA and hIL-17RC. At 24 h posttransfection of the IL-17 receptor construct, 293T cells were subsequently incubated for 30 min with mock-treated or purified ORF8-Flag, followed by staining with anti-Flag antibody for flow cytometry. (B) Purified ORF8-Flag protein interacts with endogenous IL-17RA and IL-17RC in THP-1 cells. THP-1 cells were incubated with purified ORF8-Flag for 2 h. Cell lysates were immunoprecipitated with anti-Flag, followed by immunoblotting with the indicated antibodies. (C) Effects of IL-17RA/B/C deficiency on the ORF8-induced phosphorylation of p65 and I?Ba. IL-17RA, IL-17RB, or IL-17RC knockout (KO) THP-1 cells were generated by the CRISPR-Cas9 method. IL-17RA KO, IL-17RB KO, IL-17RC KO, or control THP-1 cells were mock treated or treated with ORF8 for the indicated times, followed by immunoblotting with the indicated antibodies. (D) Effects of IL-17RA/B/C deficiency on ORF8-mediated cytokine gene expression. IL-17RA KO, IL-17RB KO, IL-17RC KO, or control THP-1 cells were mock treated or treated with ORF8 for 16 h, followed by qRT-PCR to measure the mRNA levels of indicated genes. (E) Proximity ligation assay (PLA) of IL-17RA/C heterodimerization in NIH 3T3 cells. At 24 h posttransfection with HA-hIL-17RA and Myc-hIL-17RC, NIH 3T3 cells were treated with hIL-17A or purified ORF8 for 30 min, followed by PLA. P values were calculated by two-way ANOVA with Dunnett’s posttest in panel D or one-way ANOVA with Tukey’s posttest in panel E. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

Fig 4: ORF8 natural variants for hIL-17R binding and inflammatory signaling activity. (A) S24, V62, and L84 residues in SARS-CoV-2 ORF8 structure. (B) Binding activity of ORF8 natural variants to THP-1 cells. THP-1 cells were incubated with mock treatment or ORF8 WT or variants for 2 h, followed by flow cytometry with anti-Flag antibody. (C, D) Binding activity of ORF8 natural variants to hIL-17RA (C) or hIL-17RC (D). At 24 h posttransfection with hIL-17RA (C) or hIL-17RC (D) expression plasmids, 293T cells were subsequently incubated for 30 min with mock treatment or purified ORF8 WT or variants, followed by flow cytometry with anti-Flag antibody. (E) Signaling activity of ORF8 variants to induce inflammatory cytokine gene expressions. THP-1 cells were mock treated or treated with ORF8 WT or variants for 16 h, followed by RT-qPCR to measure the mRNA levels of indicated genes, with horizontal lines indicating median values of the mRNA levels of indicated genes in mock-treated THP1 cells observed. (F) L84S variant shows the loss of IL-17RA/RC heterodimerization. At 24 h posttransfection with HA-hIL-17RA and Myc-hIL-17RC, NIH 3T3 cells were treated with the ORF8 WT or L84S variant for 30 min, followed by PLA. (G) Predicted structural representation of the Y42, Y106, and L84 residues at the binding interface between ORF8 and IL-17RA. P values were calculated by one-way ANOVA with Dunnett’s posttest in panels E and F. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.