Journal: Cell Communication and Signaling : CCS

Article Title: Linear ubiquitination mediates coronavirus NSP14-induced NF-κB activation

doi: 10.1186/s12964-024-01949-4

Figure Lengend Snippet: NSP14 activates the NF-κB signaling pathway. a 2 × 10 5 HEK293 cells were transfected with 0.05 µg, 0.1 µg, and 0.15 µg of FLAG-tagged SARS-CoV-2 NSP14 for 24 h. Real-time PCR analysis was conducted to assess the relative IL-8 mRNA levels. Lysates were blotted with anti-FLAG and anti-α-tubulin antibodies. b Co-transfection of FLAG-tagged NSP14 genes from indicated coronaviruses along with NF-κB firefly luciferase reporter and pRL-SV40 ( Renilla luciferase as an internal control) into HEK293 cells. After 48 h, cells were harvested, and the relative reporter activity was determined by calculating the ratio of firefly luciferase to Renilla luciferase. Lysates were blotted with anti-FLAG and anti-α-tubulin antibodies. c Schematic of NSP14 domains and mutants. ExoN: 3′-to-5′ exoribonuclease; MTase: guanine-N7-methyltransferase; ZF: zinc finger. d FLAG-tagged SARS-CoV-2 wild-type NSP14 or the indicated mutant was co-transfected with the NF-κB reporter and pRL-SV40 into HEK293 cells. Lysates were blotted with anti-FLAG and anti-α-tubulin antibodies. e FLAG-tagged SARS-CoV-2 NSP14, the D243A mutant, or the D331A/G333A mutant was co-transfected with the NF-κB reporter and pRL-SV40 into HEK293 cells. Lysates were blotted with anti-FLAG and anti-α-tubulin antibodies. f HEK293 cells were transfected with FLAG-tagged SARS-CoV-2 NSP14 or the indicated zinc finger mutant for 24 h. ZF1*: C207S/C210S; ZF2*: C261S/H264A; ZF3*: C484S/H487A. Real-time PCR analysis was performed to determine the relative IL-8 mRNA levels. Lysates were blotted with anti-FLAG and anti-α-tubulin antibodies. The p -value was calculated by one-way ANOVA followed by Tukey's multiple comparisons test ( a, b, d, e, f ). g Model illustrating NSP14-mediated NF-κB activation

Article Snippet: Mutants of SARS-CoV-2 NSP14 were constructed by PCR or using a Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Cotransfection, Luciferase, Control, Activity Assay, Mutagenesis, Activation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Linear ubiquitination mediates coronavirus NSP14-induced NF-κB activation

doi: 10.1186/s12964-024-01949-4

Figure Lengend Snippet: NSP14 induces NF-κB activation through the linear ubiquitination pathway. a FLAG-tagged NSP14 or pCMV-3Tag-8 vector was transfected into HEK293 cells. Cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-ubiquitin (Ub), and anti-α-tubulin antibodies. b Strategies targeting K63-linked ubiquitination and linear ubiquitination. c Wild-type HEK293 cells, two UBC13 knockout, and two HOIP knockout HEK293 cell lines were transfected with FLAG-tagged NSP14 for 24 h. Real-time PCR was performed to determine the relative IL-8 mRNA levels. d Vector or FLAG-tagged NSP14 (NSP14-FLAG) was transfected into HOIP wild-type and knockout HEK293 cells. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-linear ubiquitin, anti-HOIP, and anti-α-tubulin antibodies. e Wild-type and OTULIN knockout HEK293 cells were transfected with FLAG-tagged NSP14 for 24 h. Real-time PCR was performed to determine the relative IL-8 mRNA levels. f Wild-type and OTULIN knockout HEK293 cells were transfected with vector or NSP14-FLAG. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-linear ubiquitin, anti-OTULIN, and anti-α-tubulin antibodies. g NSP14-FLAG was transfected with vector or HA-tagged OTULIN (OTULIN-HA) into HEK293 cells. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-linear ubiquitin, anti-HA, and anti-α-tubulin antibodies. h HEK293 cells were transfected with NSP14-FLAG and OTULIN-HA, together with the NF-κB reporter and pRL-SV40. After 48 h, cells were harvested, and the relative reporter activity was determined by calculating the ratio of firefly luciferase to Renilla luciferase. Lysates were blotted with anti-FLAG, anti-HA and anti-α-tubulin antibodies. The p -value was calculated by one-way ANOVA followed by Tukey's multiple comparisons test ( h ) and two-way ANOVA followed by Sidak's multiple comparisons test ( c, e ). i Proposed model illustrating NSP14 linear ubiquitination regulated by LUBAC and OTULIN

Article Snippet: Mutants of SARS-CoV-2 NSP14 were constructed by PCR or using a Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA).

Techniques: Activation Assay, Plasmid Preparation, Transfection, Immunoprecipitation, Knock-Out, Real-time Polymerase Chain Reaction, Activity Assay, Luciferase

Journal: Cell Communication and Signaling : CCS

Article Title: Linear ubiquitination mediates coronavirus NSP14-induced NF-κB activation

doi: 10.1186/s12964-024-01949-4

Figure Lengend Snippet: HOIP interacts with and ubiquitinates NSP14. a NSP14-FLAG was transfected into HOIL-1 wild-type and knockout A549 cells. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-HOIP, anti-HOIL-1, and anti-α-tubulin antibodies. b NSP14-FLAG was transfected into HOIP wild-type and knockout A549 cells. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-HOIP, anti-HOIL-1, and anti-α-tubulin antibodies. c Purified bacterial recombinant FLAG-tagged HOIP N563 was incubated with either MBP or MBP-tagged NSP14 (NSP14-MBP) at 4 °C for 16 h. Subsequently, MBP pull-down assays were carried out, followed by Western blotting with anti-MBP and anti-FLAG antibodies. d FLAG-tagged NSP14 or the indicated domain was co-transfected with HA-tagged HOIP (HOIP-HA) into HEK293 cells. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG and anti-HA antibodies. e Schematics of NSP14 mutants. f Co-transfection of FLAG-tagged NSP14 or the indicated mutants with HOIP-HA into HEK293 cells. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-HA, and anti-α-tubulin antibodies. g FLAG-tagged NSP14 or the indicated ZF mutant was co-transfected with HOIP-HA into HEK293 cells. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-HA, and anti-α-tubulin antibodies

Article Snippet: Mutants of SARS-CoV-2 NSP14 were constructed by PCR or using a Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA).

Techniques: Transfection, Knock-Out, Immunoprecipitation, Purification, Recombinant, Incubation, Western Blot, Cotransfection, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: Linear ubiquitination mediates coronavirus NSP14-induced NF-κB activation

doi: 10.1186/s12964-024-01949-4

Figure Lengend Snippet: HOIP ubiquitinates NSP14 at multiple sites. a Conserved lysines in NSP14 proteins from six coronaviruses, including SARS-CoV, SARS-CoV-2, MERS, HCoV OC43, HCoV 229E, and TGEV. b HEK293 cells were transfected with FLAG-tagged NSP14 or the indicated mutant, along with NF-κB reporter and pRL-SV40. After 48 h, cells were harvested, and the relative reporter activity was determined by the ratio of firefly luciferase to Renilla luciferase. Lysates were blotted with anti-FLAG and anti-α-tubulin antibodies. c FLAG-tagged NSP14 or NSP14 K/R was co-transfected with the NF-κB reporter and pRL-SV40 into HEK293 cells. After 48 h, cells were harvested, and the relative reporter activity was determined by the ratio of firefly luciferase to Renilla luciferase. d NSP14-FLAG or NSP14 K/R -FLAG was transfected into HEK293 cells. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-linear ubiquitin, and anti-α-tubulin antibodies. e FLAG-tagged NSP14 or NSP14. K/R was co-transfected with vector or HA-tagged HOIP into HEK293 cells for 24 h. Real-time PCR analysis was performed to determine the relative IL-8 mRNA levels. The p -value was calculated by one-way ANOVA followed by Tukey's multiple comparisons test ( b, c, e )

Article Snippet: Mutants of SARS-CoV-2 NSP14 were constructed by PCR or using a Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA).

Techniques: Transfection, Mutagenesis, Activity Assay, Luciferase, Immunoprecipitation, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Cell Communication and Signaling : CCS

Article Title: Linear ubiquitination mediates coronavirus NSP14-induced NF-κB activation

doi: 10.1186/s12964-024-01949-4

Figure Lengend Snippet: NSP14-mediated NF-κB activation through the IKK complex. a - c Transfection of either vector or NSP14-FLAG into wild-type, IKKα knockout ( a ), IKKβ knockout ( b ), or NEMO knockout ( c ) HEK293 cells for 24 h. Real-time PCR analysis was conducted to determine the relative IL-8 mRNA levels. d Vector or NSP14-FLAG was transfected into NEMO wild-type and knockout HEK293 cells. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-IKKα, anti-NEMO, and anti-α-tubulin antibodies. e Vector, NSP14-FLAG, or NSP14 K/R -FLAG was transfected into HEK293 cells. After 48 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and blotted with anti-FLAG, anti-NEMO, and anti-α-tubulin antibodies. f NSP14-FLAG or NSP14 K/R -FLAG was transfected with Myc-tagged IKKα or IKKβ into HEK293 cells. After 48 h, cell lysates were blotted with anti-FLAG, anti-Myc, anti-p-IKK (Ser176/180), and anti-α-tubulin antibodies. g FLAG-tagged NSP14 or NSP14 K/R was transfected with Myc-tagged IKKα or IKKβ into HEK293 cells for 24 h. Real-time PCR analysis was performed to determine the relative IL-8 mRNA levels. Lysates were blotted with anti-FLAG, anti-Myc, and anti-α-tubulin antibodies. The p -value was calculated by one-way ANOVA followed by Tukey's multiple comparisons test ( g ) or two-way ANOVA followed by Sidak's multiple comparisons test ( a, b, c ). h Model illustrating NSP14 linear polyubiquitin-mediated NEMO recruitment and IKK activation

Article Snippet: Mutants of SARS-CoV-2 NSP14 were constructed by PCR or using a Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Knock-Out, Real-time Polymerase Chain Reaction, Immunoprecipitation

Journal: Cell Communication and Signaling : CCS

Article Title: Linear ubiquitination mediates coronavirus NSP14-induced NF-κB activation

doi: 10.1186/s12964-024-01949-4

Figure Lengend Snippet: HOIP deficiency impairs HCoV OC43-induced linear ubiquitination and proinflammatory cytokine expression. a HOIP wild-type and knockout A549 cells were infected with 1 MOI of HCoV OC43 for designated times. Lysates were blotted with anti-FLAG, anti-p-IKK (Ser176/180), anti-IKK, anti-linear ubiquitin, anti-OC43 N, and anti-α-tubulin antibodies. Band densitometry was calculated using Image J. The ratio of phosphorylated IKK to total IKK in each lane was indicated. b HOIP wild-type and knockout A549 cells were infected with 1 MOI of HCoV OC43 for 16 h. Real-time PCR was conducted to determine the relative mRNA levels of proinflammatory factors and ISGs. c Schematic representation of NSP14 and RLR pathways involved in coronavirus infection. Targets of HOIPIN-8 and IKK-16 are indicated. d A549 cells were treated with DMSO or 30 μM HOIPIN-8 for 2 h and then infected with 1 MOI of HCoV OC43 for designated times. Lysates were blotted with anti-FLAG, anti-p-IKK (Ser176/180), anti-IKK, anti-linear ubiquitin, anti-OC43 N, and anti-α-tubulin antibodies. e A549 cells were treated with DMSO or 10 μM IKK-16 for 2 h and then infected with HCoV OC43 for designated times. Lysates were blotted with anti-FLAG, anti-p-IKK (Ser176/180), anti-IKK, anti-linear ubiquitin, anti-OC43 N, and anti-α-tubulin antibodies. f A549 cells were treated with DMSO or 30 μM HOIPIN-8 for 2 h and then infected with 1 MOI of HCoV OC43 for 16 h. RNA sequencing was performed, and the normalized mean count of proinflammatory factors and ISGs is shown in the heatmap. g A549 cells were treated with DMSO or 30 μM HOIPIN-8 for 2 h and then infected with 1 MOI of HCoV OC43 for 16 h. Real-time PCR was performed to determine the relative mRNA levels of proinflammatory factors and ISGs. h A549 cells were treated with DMSO or 10 μM IKK-16 for 2 h and then infected with 1 MOI of HCoV OC43 for 16 h. Real-time PCR was performed to determine the relative mRNA levels of proinflammatory factors and ISGs. The p -value was calculated by two-way ANOVA followed by Sidak's multiple comparisons test ( b, g, h )

Article Snippet: Mutants of SARS-CoV-2 NSP14 were constructed by PCR or using a Q5® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA).

Techniques: Expressing, Knock-Out, Infection, Real-time Polymerase Chain Reaction, RNA Sequencing Assay