Fig 4.

MAGED2 inhibits SARS-CoV-2 genome replication but not restricts viral entry, assembly, and release. (A and B) HeLa-ACE2 cells were transduced with sgRNA lentivirus targeting MAGED2. Whole-cell lysates were analyzed by immunoblot with MAGED2, ACE2, and β-actin antibodies. The HeLa-ACE2 cells with or without MAGED2 depletion were infected with MLV retroviral particles (Fluc as the reporter) pseduotyped with SARS-CoV-2 spike (MLV SARS-CoV-2pp). Fluc activity was measured at 48-h post-infection. 1F11 is SARS-CoV-2 neutralizing antibody as the positive control. (C) Schematic representation of SARS-CoV-2 Gluc replicon RNA genome. (D and E) Caco-2-N cells were transduced with sgRNA lentivirus targeting MAGED2. Whole-cell lysates were analyzed by immunoblot with MAGED2 and β-Actin antibodies. Then, the cells with or without MAGED2 genetic ablation were transfected with SARS-CoV-2 Gluc WT or SAA (RdRp inactive mutant) replicon RNAs, and Gluc activity was assayed at 48-h post-transfection. (F) Schematic representation of VLP production and detection. (G and H) HEK293T cells were transduced with sgRNA lentivirus targeting MAGED2. Whole-cell lysates were analyzed by immunoblot with MAGED2 and β-actin antibodies. Then, the HEK293T cells with or without MAGED2 knockout in 10 cm dish were transfected with equal amounts of plasmids (24 µg in total) encoding the SARS-CoV-2 S, E, M, and HiBiT-N proteins. After 24 h, cell culture supernatants were collected. VLPs separated by 10%–60% sucrose gradient centrifugation were measured with Nano-Glo luciferase kit. All data are representative of three independent experiments. Values are means + standard deviations (error bars) (n = 3). *, P < 0.05; **, P < 0.01; n.s., not significantly different by one-way analysis of variance.