Therapeutic inhibition of critical viral functions is important for curtailing coronavirus disease-2019 (COVID-19). We sought to identify
antiviral targets through genome-wide characterization of SARS-CoV-2
proteins that are crucial for viral pathogenesis and that cause harmful cytopathic effects. All twenty-nine
viral proteins were tested in a fission yeast cell-based system using inducible gene expression. Twelve
proteins including eight non-structural
proteins (NSP1, NSP3, NSP4, NSP5, NSP6, NSP13, NSP14 and NSP15) and four accessory
proteins (ORF3a, ORF6, ORF7a and ORF7b) were identified that altered cellular proliferation and integrity, and induced cell death. Cell death correlated with the activation of cellular oxidative stress. Of the twelve
proteins, ORF3a was chosen for further study in mammalian cells. In human pulmonary and kidney epithelial cells, ORF3a induced cellular oxidative stress associated with apoptosis and
necrosis, and caused activation of pro-inflammatory response with production of the
cytokines TNF-α,
IL-6, and IFN-β1, possibly through the activation of NF-κB. To further characterize the mechanism, we tested a natural ORF3a Beta variant, Q57H, and a mutant with deletion of the highly conserved residue, ΔG188. Compared to wild type ORF3a, the ΔG188 variant yielded more robust activation of cellular oxidative stress, cell death, and innate immune response. Since cellular oxidative stress and
inflammation contribute to cell death and tissue damage linked to the severity of
COVID-19, our findings suggest that ORF3a is a promising, novel therapeutic target against
COVID-19.