Fig. 4: M^pro inactivates NEMO and induces brain endothelial cell loss mimicking COVID-19-associated brain pathology.

a, M^pro-HA inhibited the nuclear translocation of the NF-κB subunit p65 in hCMEC/D3 cells stimulated with IL-1β (0.25 µg ml⁻¹) for 30 min. Cells (N = 3 wells per group; representative of three independent experiments) were transfected with a control plasmid (Bluescript) or pCAG-M^pro-HA. Scale bar, 25 µm. b,c, M^pro-HA blocked the activation of NF-κB by IL-1β (0.25 µg ml⁻¹) in human (b) and mouse (c) brain endothelial cells. Cells were transfected with pNF-κB-Luc plus a control plasmid or pCAG-M^pro-HA. N = 5–6 wells per group. d,e, M^pro-HA induced death of hCMEC/D3 cells, especially after exposure to TNF (100 ng ml⁻¹; 4.5 h). Cells (N = 12 wells per group) were transfected with a control plasmid or pCAG-M^pro-HA. Scale bar, 100 µm. f, In hCMEC/D3 cells, M^pro degraded NEMO-2A, whereas the inactive variant p.Cys145Ala-M^pro did not. After transfecting the cells with pCAG-NEMO-2A-GFP plus pCAG-GFP as control, pCAG-M^pro-HA or pCAG-p.Cys145Ala-M^pro-HA, immunoblots of cell lysates were performed (representative of at least six experiments). g, More hCMEC/D3 cells survived after expressing the inactive variant p.Cys145Ala-M^pro-HA than after expressing M^pro-HA. All cells were transfected with pCAG-GFP in parallel. The numbers of GFP⁺ or HA⁺ cells are depicted (N = 6 wells per group). h, In contrast to M^pro, p.Cys145Ala-M^pro did not inhibit the nuclear translocation of the NF-κB subunit p65 when hCMEC/D3 cells were stimulated with IL-1β (0.25 µg ml⁻¹) for 30 min. Cells were transfected with control plasmid, pCAG-M^pro-HA or pCAG-p.Cys145Ala-M^pro-HA (N = 6 wells per group). i, Schematic of AAV-BR1 vectors to transduce brain endothelial cells in vivo. WPRE, woodchuck hepatitis posttranscriptional regulatory element. j, AAV-BR1-M^pro but not AAV-BR1-p.Cys145Ala-M^pro led to the formation of string vessels (arrowheads) in the brain of mice. Representative images were taken in the cortex. Scale bar, 20 µm. k, Quantification of string vessel length as a percentage of total vessel length (N = 9–10 mice per group). l, Total vessel length was reduced after mice received AAV-BR1-M^pro but not AAV-BR1-p.Cys145Ala-M^pro (N = 9–10 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001. Means ± s.e.m. are shown. Detailed information on the exact test statistics, sidedness and values is provided in Supplementary Table 5.

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