Fig. 6

N represses apoptosis to promote virus replication. a, b Caco-2 cells were stably infected with Lentivirus-N, pre-treated with 50 μM apoptosis inhibitor QVD-OPH or DMSO for 4 h, then infected with SARS-CoV-2-trVLP (MOI = 0.5) for 48 h. GFP expression was observed in Caco2-N cells using microscopy (a). The viral copies in cell supernatant were absolute quantification of qRT-PCR (b, bottom). Cell lysates were analyzed by immunoblotting (b, lower). c, d Caco-2 cells were stably infected with Lentivirus-N, pre-treated with 3 μM MCL-1 inhibitor S63845 or DMSO for 4 h, then infected with SARS-CoV-2-trVLP (MOI = 0.5) for 48 h. GFP expression was observed in Caco2-N cells using microscopy (c). The viral copies in cell supernatant were absolute quantification of qRT-PCR (d, bottom). Cell lysates were analyzed by immunoblotting (d, lower). e A549 cells were stably infected with Lentivirus-CT or Lentivirus-N, pre-treated with 3 μM MCL-1 inhibitor S63845 or DMSO for 4 h, then infected with influenza virus (PR8 strain, MOI = 0.1) for 48 h. The viral copies in cell supernatant were absolute quantification by qRT-PCR (e, lower). f–i C57BL/6 genetic background mice were injected with 300 μl containing 3 × 10¹¹ vg of AAV-Lung-EGFP (n = 20) or AAV-Lung-N (n = 2 0) in their tail vein, and after three weeks, pre-treated with MCL-1 specific inhibitor S64845 (12.5 mg/kg) by intraperitoneal injection for 90 min and then infected with influenza virus (PR8 strain, 1LD50) by intranasal injection, followed by repeated treatment with S64845 (12.5 mg/kg) on the fourth day after influenza virus infection (n = 8), or the same volume solvent (50% PEG300 plus 50% PBS) as a control group (n = 8). Another untreated AAV-Lung-EGFP or AAV-Lung-N infected mice were prepared as the blank group (n = 4). Six days after infection, two mice were euthanized, and their lung tissues were collected. The viral copies in lung tissues were absolute quantification of qRT-PCR (f). Histopathology analysis of the mice’s lungs (g). Scale bar, 200 μm (10×) or 50 μm (40×). The mice’s survival rates (h) and weight changes (i) were evaluated every day post-treatment. j A549 cells were stably infected with Lentivirus-CT or Lentivirus-N, then infected with Dengue virus (NGC strain, MOI = 0.5) for 48 h, Cytopathic effect (CPE) was observed in above cells using microscopy. k A549 cells were stably infected with Lentivirus-CT or Lentivirus-N, pre-treated with 3 μM MCL-1 inhibitor S63845 for 4 h. then infected with Dengue virus (NGC strain, MOI = 0.5) for another 48 h. The sub-cellular locations of virus-dsRNA (green), Flag-tagged N (red), and nucleus marker DAPI (blue) were visual with confocal microscopy. Scale bar is 50 μm. l–n A549 cells (l, n)or THP1- cells (m) were stably infected with Lentivirus-CT or Lentivirus-N, THP-1 cells were differentiated into macrophages. Pre-treated with 3 μM MCL-1 inhibitor S63845 for 4 h. then infected with Dengue virus (NGC strain, MOI = 0.5) (l, m) or ZIKV virus (PRVABC.59 strain, MOI = 0.5) (n) for another 48 h. Cell supernatant was analyzed by plaque (upper) and cell lysates by immunoblotting (lower). o A proposed model in which SARS-CoV-2 N protein suppresses apoptosis to promote virus replication through regulating MCL-1 protein. GFP/ΔN trVLP means SARS-CoV-2-GFP/ΔN trVLP (a–d). Data are representative of two independent experiments, with one representative shown. Error bars indicate the SD of each serum sample; P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), and two-tailed Student’s t test (b, d, e, f, l, m, and n). One-way ANOV analysis (h)