Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species

doi: 10.1080/22221751.2020.1870414

Figure Lengend Snippet: Principle of the screen of protease activity of SARS-CoV-2 PLpro and 3CLpro. (A) Schematic of the organization of the genome of SARS-CoV-2, focusing on the non-structural proteins Nsp1-16. As depicted, two proteases are encoded in ORF1a: NSP3 or papain-like protease (PLpro) and NSP5, or 3C-like protease (3CLpro). PLpro is responsible for three proteolytic cleavages, while 3CLpro cuts the large polyprotein at eleven different sites. (B) Results obtained for the family of IRF proteins ; the additional band obtained for IRF3 in the presence of PLpro indicates cleavage (C) Overview of the proteins tested in this study and the proteolytic events detected: out of the 71 proteins tested, PLpro cleaves only IRF3 (indicated in blue) and 3CLpro cleaves NLRP12 and TAB1 (as shown in red).

Article Snippet: The following commercial antibodies were used: anti-IRF3 (ab68481) and anti-ACE2 (ab108252) from Abcam, anti-NLRP12 (PA5-21027) from Invitrogen, anti-TAB1 (#3226) from Cell Signaling, anti-SARS-CoV-2 N (GTX632269) from Genetex, anti-COXIV (11242-1-AP) from Proteintech.

Techniques: Activity Assay

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species

doi: 10.1080/22221751.2020.1870414

Figure Lengend Snippet: IRF3, TAB1 and NLRP12 are decreased upon infection with SARS-CoV-2 in 293T-ACE2 cells, in two independent laboratories. (A–C) 293T-ACE2 cells were infected with SARS-CoV-2 and analysed for viral and host protein levels 72 h post-infection. (D–I) Independently, stable 293T-ACE2 were infected with icSARS-CoV-2 mNeonGreen) and the levels of IRF3, TAB1 And NLRP12 were visualized by Western Blotting at 6 h (D), 24 h (E) and 48 h (F).(G–I): Protein levels measured by densitometry for IRF3 (G), TAB1 (H) and NLRP12 (I) and plotted for the Mock, poly(I:C) and SARS-CoV-2 infection at 6 h, 24 h and 48 h.

Article Snippet: The following commercial antibodies were used: anti-IRF3 (ab68481) and anti-ACE2 (ab108252) from Abcam, anti-NLRP12 (PA5-21027) from Invitrogen, anti-TAB1 (#3226) from Cell Signaling, anti-SARS-CoV-2 N (GTX632269) from Genetex, anti-COXIV (11242-1-AP) from Proteintech.

Techniques: Infection, Western Blot

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species

doi: 10.1080/22221751.2020.1870414

Figure Lengend Snippet: Analysis of the protein sequences across species for IRF3 and NLRP12 cleavage sites. (*) see Materials and Methods for protein sequences for Cotton rats ( Sigmodon hispidus ) and Minks ( Neovison vison ).

Article Snippet: The following commercial antibodies were used: anti-IRF3 (ab68481) and anti-ACE2 (ab108252) from Abcam, anti-NLRP12 (PA5-21027) from Invitrogen, anti-TAB1 (#3226) from Cell Signaling, anti-SARS-CoV-2 N (GTX632269) from Genetex, anti-COXIV (11242-1-AP) from Proteintech.

Techniques:

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species

doi: 10.1080/22221751.2020.1870414

Figure Lengend Snippet: Cleavage of NLRP12 by 3CLpro of SARS-CoV-2. (A): SDS-page analysis of the cleavage of NLRP12 protein, with a C-terminal GFP tag. The protein was expressed alone or in the presence of increasing concentrations of the SARS-CoV-2 protease 3CLpro. (B) Logo analysis of the cleavage site predicted for 3CLpro, from the polyprotein cleavage of SARS-CoV-2. (C) Representation of the domains found in NLRP12 and the position of the cleavage sites.(D) Alignment of the amino acids for human NALPs (NLRPs), demonstrating that only NLRP12 would be predicted to be cleaved as observed. Below, alignment of mouse NLRP12, showing that the first cleavage site is conserved, but the second site presents an A→V mutation that would disrupt cleavage. (E) SDS-page analysis of human and mouse NLRP12, with different tag orientations, to demonstrate the differences between species. The banding patterns obtained in the presence of 3CLpro are consistent with the predicted sizes. Using the mouse NLRP12 constructs, only one cleaved fragment is observed in the N-term and C-term constructs. (F) Representation of NLRP12 structure (derived from the structure of NLRP3 from PDB 6NPY). (top): The cleavage sequence LFQG (site 1, at residue 238) is highlighted in red; the site is presented in a flexible loop and seems fully exposed for cleavage by 3CLpro. (middle): The second cleavage site, (LQA) found at residue 938, it presented in a flexible loop and would be accessible at the tip of an alpha helix in the LR repeats. (bottom): in this view, both cleavage sites are visible.

Article Snippet: The following commercial antibodies were used: anti-IRF3 (ab68481) and anti-ACE2 (ab108252) from Abcam, anti-NLRP12 (PA5-21027) from Invitrogen, anti-TAB1 (#3226) from Cell Signaling, anti-SARS-CoV-2 N (GTX632269) from Genetex, anti-COXIV (11242-1-AP) from Proteintech.

Techniques: SDS Page, Mutagenesis, Construct, Derivative Assay, Sequencing

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species

doi: 10.1080/22221751.2020.1870414

Figure Lengend Snippet: PLpro and 3CLpro of SARS-CoV-2 interfere with innate immune response by directly cleaving IRF3, TAB1 and NLRP12. (1) In blue: PLpro (Nsp3) inhibits IFNβ production by cleaving IRF3. (2) in red: 3CLpro could interfere with production of pro-inflammatory cytokines at two levels: cleavage of TAB1 would inhibit activation of NF-κB via TAK1 (2a), while cleavage of NLRP12 could release its inhibitory effect on NF-κB (2b). In addition, the cleavage of NLRP12 by 3CLpro could perturb the NLRP3 inflammasome assembly (2c), especially as one of the cleavage sites would release a PYD domain from NLRP12. This could trigger the cleavage of pro-Caspase-1 and enhance the release of IL-1β.

Article Snippet: The following commercial antibodies were used: anti-IRF3 (ab68481) and anti-ACE2 (ab108252) from Abcam, anti-NLRP12 (PA5-21027) from Invitrogen, anti-TAB1 (#3226) from Cell Signaling, anti-SARS-CoV-2 N (GTX632269) from Genetex, anti-COXIV (11242-1-AP) from Proteintech.

Techniques: Activation Assay