Secreted SARS-CoV-2 ORF8 modulates the cytokine expression profile of human macrophages

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still adapting to its new human host. Attention has focussed on the viral spike protein, but substantial variation has been seen in the ORF8 gene. Here, we show that SARS-CoV-2 ORF8 protein undergoes signal peptide-mediated processing through the endoplasmic reticulum and is secreted as a glycosylated, disulphide-linked dimer. The secreted protein from the prototype SARS-CoV-2 virus had no major effect on viability of a variety of cell types, or on IFN or NF-κB; signalling. However, it modulated cytokine expression from primary CSF1-derived human macrophages, most notably by decreasing IL-6 and IL-8 secretion. Furthermore, a sequence polymorphism L84S that appeared early in the pandemic associated with the Clade S lineage of virus, showed a markedly different effect, of increasing IL-6 production. We conclude that ORF8 sequence polymorphisms can potentially affect SARS-CoV-2 virulence and should therefore be monitored in sequencing-based surveillance.

The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and induces a hyper-inflammatory cytokine storm

“…Furthermore, the profile differed from LPS as a well-characterized pro-inflammatory control (Figure S5, Figure S11 b). The binding of ORF8 to monocytes was recently reported by other groups in preprinted articles (38,57). Their observations back up and support our findings and demonstrate the unique function of ORF8 in the context of inflammation.…”

SARS-CoV-2 Omicron subvariants evolved to promote further escape from MHC-I recognition

“…Our data indeed demonstrated the enhanced ability of VOCs to suppress ISG expression. Multiple functions of SARS-CoV-2 ORF8 were reported so far, which include inhibition of type I IFN, ISGs, or NF-kB signaling (Geng et al, 2021; Lei et al, 2020; Li et al, 2020) and modulation of cytokine expression from macrophages (Kriplani et al, 2021). Interestingly, several studies showed that SARS-CoV-2 ORF8 is actively secreted into the cell culture media in a signal peptide-dependent manner when it is overexpressed in vitro (Kriplani et al, 2021; Wang et al, 2020).…”

“…Multiple functions of SARS-CoV-2 ORF8 were reported so far, which include inhibition of type I IFN, ISGs, or NF-kB signaling (Geng et al, 2021; Lei et al, 2020; Li et al, 2020) and modulation of cytokine expression from macrophages (Kriplani et al, 2021). Interestingly, several studies showed that SARS-CoV-2 ORF8 is actively secreted into the cell culture media in a signal peptide-dependent manner when it is overexpressed in vitro (Kriplani et al, 2021; Wang et al, 2020). Furthermore, ORF8 peptides and anti-ORF8 antibodies can be detected abundantly in serum of patients, suggesting the relevance of the active secretion of ORF8 to actual infection in humans (Wang et al, 2020).…”

“…Given the truncation mutation likely rendering ORF8 in the Alpha variant non-functional, this raises a question as to how such mutations might be tolerated. Multiple functions beyond MHC-I downregulation are documented for SARS-CoV-2 ORF8, which include inhibition of type I IFN, ISGs, or NF-kB signaling (30)(31)(32), epigenetic modulation through histone mimicry (33) and induction of proinflammatory cytokines from macrophages and monocytes via IL-17RA (34)(35)(36)(37). Interestingly, several studies showed that SARS-CoV-2 ORF8 is actively secreted into the cell culture media in a signal peptide-dependent manner when it is overexpressed in vitro (26,34,38).…”

“…Multiple functions beyond MHC-I downregulation are documented for SARS-CoV-2 ORF8, which include inhibition of type I IFN, ISGs, or NF-kB signaling (30)(31)(32), epigenetic modulation through histone mimicry (33) and induction of proinflammatory cytokines from macrophages and monocytes via IL-17RA (34)(35)(36)(37). Interestingly, several studies showed that SARS-CoV-2 ORF8 is actively secreted into the cell culture media in a signal peptide-dependent manner when it is overexpressed in vitro (26,34,38). Furthermore, ORF8 peptides and anti-ORF8 antibodies can be detected abundantly in serum of patients, suggesting the relevance of the active secretion of ORF8 to actual infection in humans (38).…”