Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection

Abstract

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. The unconventionally secreted ORF8 recognizes the IL17RA receptor of macrophages and induces cytokine release. However, conventionally secreted ORF8 cannot bind to IL17RA due to N-linked glycosylation. Furthermore, we found that Yip1 interacting factor homolog B (YIF1B) is a channel protein that translocates unglycosylated ORF8 into vesicles for unconventional secretion. Blocking the unconventional secretion of ORF8 via a YIF1B knockout in hACE2 mice attenuates inflammation and yields delayed mortality following SARS-CoV-2 challenge.

SARS-CoV-2 accessory protein ORF8 is secreted extracellularly as a glycoprotein homodimer

“…These clinical evidence indicates that the orf8 gene in SARS-CoV-2 does not fully correlate with viral pathogenicity and disease severity. Interestingly, Lin et al have reported that the unglycosylated ORF8 secreted via an unconventional pathway is responsible for the COVID-19 cytokine storm through IL-17 receptor-based signaling ( 31 , 56 ). However, their proposed mechanism seems contradictory to the recent clinical evidence.…”

Regulation of virion production by the ORF8 signal peptide across SARS-CoV-2 variants

“…Our work confirms findings from previous studies, including our own, that described an interaction between Spike and ORF8, a reduction in Spike pseudovirus particle productions, but also, importantly, extends our knowledge by showing ORF8 targeting viral particle productions and causing Golgi fragmentations using live virus, replicon, and VLP. While we contribute significant knowledge in VLP, replicon and virus-infected cells, our study also opens several new areas of study.. Our model is that in SARS-CoV-2, which has intact ORF8 or produces signal peptides of ORF8, viral particle production is dampened, possibly as a way to reduce immunosurveillance of infected cells (14,16,30) or or as an unwanted effect of ORF8's other functions benefitting viral replication (8,9,18) (Fig 5E). In ORF8-deficient SARS-CoV-2, viral particle production is not disrupted, leading to increased viral particle formation (Fig 5E).…”

“…ORF8 signal peptide may be behaving in a similar way as NS1. Like NS1, ORF8 can be secreted by conventional and unconventional secretory pathways (14,33).…”

“…Structural proteins during this transit assemble into a viral particle and leave the cell as virions, while ORF8 has been shown to form dimers, adopting an immunoglobulin-like structure with an N-terminal 15-amino acid signal peptide that allows entry into the ER lumen (16,18,19). Once secreted, ORF8 elicits proinflammatory reactions through multiple host protein interactions, including binding to the IL-17RA (12,14,20). Here, we propose that its transit through the secretory pathway in the presence of the viral structural proteins, specifically Spike, allows ORF8 to regulate viral particle production directly.…”

“…A characteristic feature of ORF8 is that it is secreted from infected cells and is readily detected in COVID-19 patient sera (1,10). As such, the protein has pro-inflammatory properties and binds for example to the IL17 receptor A (IL17RA), which might harm the host and thus suppress viral replication in the long-term (5,(11)(12)(13)(14)(15). Overall, the role of ORF8 during viral replication is pleiotropic and not yet fully understood.…”