Zhang Jiantao, Li Qi, Cruz Cosme Ruth S, Gerzanich Volodymyr, Tang Qiyi, Simard J Marc, Zhao Richard Y
Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201.
Institute of Global Health, University of Maryland School of Medicine, Baltimore, MD 21201.
bioRxiv. 2021 Dec 14:2021.11.23.469747. doi: 10.1101/2021.11.23.469747.
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.
对关键病毒功能进行治疗性抑制对于遏制2019冠状病毒病（COVID-19）至关重要。我们试图通过对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）蛋白质进行全基因组表征来确定抗病毒靶点，这些蛋白质对于病毒发病机制至关重要，并会导致有害的细胞病变效应。使用诱导型基因表达在基于裂殖酵母细胞的系统中对所有29种病毒蛋白进行了测试。鉴定出12种蛋白质，包括8种非结构蛋白（NSP1、NSP3、NSP4、NSP5、NSP6、NSP13、NSP14和NSP15）和4种辅助蛋白（ORF3a、ORF6、ORF7a和ORF7b），它们改变了细胞增殖和完整性，并诱导细胞死亡。细胞死亡与细胞氧化应激的激活相关。在这12种蛋白质中，选择ORF3a在哺乳动物细胞中进行进一步研究。在人肺和肾上皮细胞中，ORF3a诱导与细胞凋亡和坏死相关的细胞氧化应激，并通过激活核因子κB（NF-κB）导致促炎反应激活，产生细胞因子肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和干扰素-β1（IFN-β1）。为了进一步表征其机制，我们测试了天然的ORF3a Beta变体Q57H和缺失高度保守残基的突变体ΔG188。与野生型ORF3a相比，ΔG188变体对细胞氧化应激、细胞死亡和先天免疫反应的激活更强。由于细胞氧化应激和炎症导致与COVID-19严重程度相关的细胞死亡和组织损伤，我们的研究结果表明ORF3a是一个有前景的新型抗COVID-19治疗靶点。
