Fig. 6: SARS-CoV-2 PLpro induces the proteasomal degradation of DAXX.

a DAXX degradation after infection. 293T-ACE2 cells were infected with SARS-CoV-2 at MOI 0.1. After 24 h, cells were harvested and levels of DAXX, Lamin B, HSP90, Actin, GAPDH, Tubulin, TRIM22, RIG-I and of the viral protein spike were analyzed by Western Blot. A Western Blot representative of 3 independent experiments is shown. b GRL0617 and MG132 treatments restore DAXX expression. 293T-ACE2 cells were infected with SARS-CoV-2 at an MOI of 0.1. When indicated, cells were pretreated 2 h before infection with GRL0617 (at the indicated concentrations), or with MG132 (10 µM), a proteasome inhibitor, or Masitinib (10 µM) a 3CL inhibitor. After 24 h, cells were harvested and levels of DAXX, GAPDH and of the viral protein spike were analyzed by Western Blot. A Western Blot representative of 3 independent experiments is shown. c GRL0617 treatment restores DAXX localization. 293T-ACE2 cells were infected with SARS-CoV-2 at an MOI of 0.1. 24 h post-infection, cells were labelled with Hoescht and with antibodies against dsRNA (detecting viral RNA, in green) and HA (detecting DAXX, in red). When indicated, cells were treated with 50 µM of GRL0617 at the time of infection. Scale bars correspond to 10 µm. Images are representative of 3–6 different fields from 2 independent experiments. d–f: Nsp3 induces DAXX degradation. d 293T-ACE2 cells were transfected with 1 μg of the indicated viral proteins. After 24 h, the levels of DAXX and GAPDH were analyzed by Western Blot. e 293T-ACE2 cells were transfected with the indicated amounts of Nsp3. After 24 h, the levels of DAXX and GAPDH were analyzed by Western Blot. f 293T-ACE2 cells were transfected with 1 μg of Nsp3 or of pcDNA. 6 h post transfection, cells were also, when indicated, treated with 50 µM of GRL0617. Of, 24 h after transfection, the levels of DAXX and GAPDH were analyzed by Western Blot. Western Blots representative from 2 independent experiments are shown. The quantification of band intensity for Fig. 6d–f is shown in Fig. S8b,d, e. Source data are provided as a Source Data file.