Background
ORF8 is an accessory protein encoded by SARS-CoV-2, containing an immunoglobulin-like domain of approximately 20 kDa, and is secreted extracellularly by infected cells. Serum levels of ORF8 have been shown to correlate with disease severity in COVID-19 patients, though its exact function and target cells remain unknown. We hypothesized that extracellular ORF8 influences COVID-19 pathogenesis through its effects on immune cells and investigated this mechanism.
Methods
We demonstrated that secreted ORF8 targets macrophages and alters their identity, inflammatory profile, and susceptibility to SARS-CoV-2 infection, thereby enhancing lung epithelial cell infection. This was shown using our primary macrophage SARS-CoV-2 infection models (SARS-CoV-2 virus-like particles and replication-competent ORF8+/− viruses) and proteomics analysis on ORF8-stimulated macrophages.
Results
Exposure to extracellular ORF8 enhances ACE2 expression on primary macrophages thereby promoting virion entry and macrophage pyroptosis. ORF8-exposed proinflammatory M1 macrophages exhibit a significant shift in protein expression toward an M2-like phenotype associated with immunosuppression. Importantly, efficient infection of macrophages depends on ORF8 expression, and co-culture of macrophages with primary human alveolar type 2 (AT2) cells reverses the negative effect of ORF8 on AT2 infection, enabling full viral replication. In animal experiments using mouse-adapted viruses, the addition of extracellular ORF8 enhances viral replication and increases IL-1β production.
Conclusions
This study highlights the role of extracellular ORF8 in modulating immunity and its contribution to the spread of infection. Our results demonstrate a paracrine function of ORF8 in targeting macrophages and promoting a proviral circuit between macrophages and lung epithelial cells in COVID-19 pathogenesis.
Table or Figure
