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

Author summaryCytokine storm is an important point in the transition of COVID-19 from mild to severe/critical diseases. Clinical cases show that the viral loads in patients are not directly proportional to the severity of disease symptoms, which indicates that in addition to virion release, there is a unique indirect cellular communication mechanism in the development of cytokine storm. In this study, we found that accessory protein ORF8 is a secretory protein that acts as messenger for the inter-cellular communication between alveolar epithelial cells (the main targets of virus) and monocytes/macrophages (the main executors of cytokine storm). Our findings are helpful to understand the occurrence of cytokine storm caused by SARS-CoV-2, and provide a potential strategy for COVID-19 therapeutic drugs. 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. Conventionally secreted ORF8 is glycosylated and loses the ability to recognize interleukin 17 receptor A of macrophages, possibly due to the steric hindrance imposed by N-glycosylation at Asn78. However, unconventionally secreted ORF8 does not undergo glycosylation without experiencing the ER-Golgi trafficking, thereby activating the downstream NF-kappa B signaling pathway and facilitating a burst of cytokine release. Furthermore, we show that ORF8 deletion in SARS-CoV-2 attenuates inflammation and yields less lung lesions in hamsters. Our data collectively highlights a role of ORF8 protein in the development of cytokine storms during SARS-CoV-2 infection.