FIG 4.

SARS-CoV-2 ORF3a-induced apoptosis and necrosis are correlated with the induction of cellular oxidative stress and innate immune proinflammatory responses in mammalian cells. Expression of SARS-CoV-2 ORF3a induces cellular growth reduction and cell death 72 hpt in human lung epithelial A549 cells (A) and human kidney epithelial 293T cells (B). (C) ORF3a induces apoptosis and necrosis 48 hpt measured by Annexin V (a), necrosis (b), and caspase-3 cleavage (c). (D) ORF3a triggers the induction of oxidative stress 48 hpt measured by the DHE straining. The ORF3a was cloned in a lentiviral constitutive expression vector (4). Scale bar = 20 μM. (E) ORF3a elevates NF-κB-mediated transcriptional activities. (F) ORF3a triggers elevated production of TNF-α, IL-6, and NF-κB in Calu-3 (a) and 293T (b) cells. Data are presented as mean ± SE from three independent experiments. Statistical differences between ORF3a and mock (indicated with #) or empty vector control (indicated with *) were evaluated. * or #, P < 0.05; ** or ##, P < 0.01; *** or ###, P < 0.001 (pair-wise t test).