Despite extensive research, the specific factor associated with SARS-CoV-2 infection that mediates the life-threatening inflammatory cytokine response in patients with severe Covid-19 remains unidentified. Herein we demonstrate that the virus-encoded Open Reading Frame 8 (ORF8) protein is abundantly secreted as a glycoprotein in vitro and in patients with newly diagnosed Covid-19. ORF8 specifically binds to the NOD-like receptor family pyrin domain-containing 3 (NLRP3) in CD14+/CD16+ monocytes to induce an inflammasomal cytokine response. The levels of ORF8 protein in the blood correlate with disease mortality in patients with acute infection, and the disease trajectory in patients with severe Covid-19. Furthermore, in vitro the ORF8-induced inflammasome response can be readily inhibited by the select NLRP3 inhibitor MCC950. Our results identify the pathogenic cause and mechanism of severe disease, and a potential new treatment of severe Covid-19.
Mammalian cell lines are important expression systems for large proteins and protein complexes, particularly when the acquisition of post‐translational modifications in the protein's native environment is desired. However, low or variable transfection efficiencies are challenges that must be overcome to use such an expression system. Expression of recombinant proteins as a fluorescent protein fusion enables real‐time monitoring of protein expression, and also provides an affinity handle for one‐step protein purification using a suitable affinity reagent. Here, we describe a panel of anti‐GFP and anti‐mCherry nanobody affinity matrices and their efficacy for purification of GFP/YFP or mCherry fusion proteins. We define the molecular basis by which they bind their target proteins using X‐ray crystallography. From these analyses, we define an optimal pair of nanobodies for purification of recombinant protein tagged with GFP/YFP or mCherry, and demonstrate these nanobody‐sepharose supports are stable to many rounds of cleaning and extended incubation in denaturing conditions. Finally, we demonstrate the utility of the mCherry‐tag system by using it to purify recombinant human topoisomerase 2α expressed in HEK293F cells. The mCherry‐tag and GFP/YFP‐tag expression systems can be utilized for recombinant protein expression individually or in tandem for mammalian protein expression systems where real‐time monitoring of protein expression levels and a high‐efficiency purification step is needed.
Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can result in a cytokine storm that is associated with poor outcomes in patients. The activation of a NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome following SARS-CoV-2 infection has been shown to play a major role in inflammatory immune responses. In this study, we show that open reading frame 8 (ORF8) protein is abundantly secreted as a glycoprotein in vitro. The glycosylated ORF8 stimulates human CD14+/CD16+ monocytes to cause upregulation of proinflammatory cytokine production and cell surface marker expression within 24 hours. The data suggests that stimulation of human monocytes by ORF8 is not receptor mediated. Rather, ORF8 is likely phagocytosed by human monocytes. ORF8 then stimulates the monocytes by independently binding to the NACHT and LRR domains of the NLRP3 protein. Pharmacologic inhibition of NLRP3 significantly diminished the production of pro-inflammatory cytokines from ORF8-stimulated human monocytes. Finally, this study shows that the ORF8 protein is also secreted in patients with newly diagnosed coronavirus disease 2019 (COVID-19). Levels of ORF8 in the blood of patients diagnosed with COVID-19 correlated with disease mortality and trajectory of disease. ORF8 stimulation of monocytes causes pro-inflammatory cytokine production that leads to the development of severe COVID-19.
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