Journal: The Journal of biological chemistry

Article Title: SAMHD1 impairs type I interferon induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection.

doi: 10.1016/j.jbc.2023.104925

Figure Lengend Snippet: Figure 3. SAMHD1 suppresses MAVS aggregation and disrupts MAVS interaction with IKKε. A, THP-1 Ctrl and SAMHD1 KO cells were mock-infected or infected with SeV (MOI of 10) for 8 h. Crude mitochondria were isolated and subjected to SDD-AGE (top). Whole cell lysates were analyzed by SDS-PAGE (bottom). Quantification of pTBK1 (S172) and pIRF3 (S396) levels was performed by densitometry and normalized relative to total TBK1 and IRF3, respectively. B, HEK293T cells were transfected with expression plasmids encoding the indicated proteins (FLAG-MAVS, myc-IKKε, and increasing amounts of HA-SAMHD1). Cells were lysed 36 h posttransfection, and IP was performed using an anti-FLAG antibody. Indicated proteins were detected by IB. C, THP-1 Ctrl and SAMHD1 cells were mock-infected (M) or infected with SeV (MOI of 10) and harvested at the indicated time points. Cell lysates were analyzed by IB with the indicated antibodies. Quantification of pIKKε (S172) levels was performed by densitometry and expressed relative to total IKKε. A–C, representative results from three independent experiments are shown. IB, immunoblot; IKKε, inhibitor of nuclear factor kappa-B kinase epsilon; IRF, IFN regulatory factor; M, mock infection; MAVS, mitochondrial antiviral-signaling protein; MOI, multiplicity of infection; pIKKε, phospho-IKKε; SAMHD, sterile alpha motif and HD domain-containing protein; SDD-AGE, semidenaturing detergent agarose gel electrophoresis; SeV, Sendai virus; SeV NP, nucleoprotein of SeV; TBK1, TANK binding kinase 1.

Article Snippet: FLAG-tagged kinaseinactive IKKε K38A was a gift from Tom Maniatis (Addgene plasmid #26202). plentiCRISPR v2-Blasticidin was a gift from Mohan Babu (Addgene plasmid # 83480).

Techniques: Infection, Isolation, SDS Page, Transfection, Expressing, Western Blot, Sterility, Agarose Gel Electrophoresis, Virus, Binding Assay

Journal: The Journal of biological chemistry

Article Title: SAMHD1 impairs type I interferon induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection.

doi: 10.1016/j.jbc.2023.104925

Figure Lengend Snippet: Figure 4. SAMHD1 inhibits IKKε-mediated IFN-I signaling and disrupts IRF7 binding to the IKKε kinase domain. A, recombinant SAMHD1 and IKKε purified from Escherichia coli and HEK293T cells, respectively, were pulled down with an anti-SAMHD1 antibody and analyzed by IB. Representative data from two independent experiments are shown. B, HEK293T cells were cotransfected with increasing amounts of plasmid encoding HA-SAMHD1, IFN-β- luciferase reporter, renilla-TK, and myc-tagged IKKε. Dual luciferase assay was performed at 24 h posttransfection, and cell lysates were harvested for IB. Error bars represent mean ± SD. Statistical significance was determined using one-way ANOVA; ***p < 0.001 compared with the vector control in the same group. C, schematic representation of full-length (FL) human IKKε (top). Five myc-tagged C-terminal and N-terminal deletion mutants are represented by the solid bars below the FL IKKε. The aa numbers of IKKε are shown. D–F, HEK293T cells were cotransfected with plasmids encoding HA-SAMHD1 and myc- tagged IKKε FL or C-terminal deletion mutants (D), IKKε N-terminal deletion lacking the kinase domain (ΔKD) (E), or IKKε kinase-inactive mutant K38A (F). Cells were lysed, and IP was performed using anti-HA antibody at 36 h posttransfection. Nonspecific IgG was used as a negative control in IP, and the indicated proteins were detected by IB. G, HEK293T cells were cotransfected with myc-IKKε KD, FLAG-IRF7, and HA-SAMHD1. Cells were lysed, and IP was performed using anti-myc antibody at 36 h posttransfection. The indicated proteins were detected by IB. A and B and D–G, representative data from three independent experiments are shown. HLH, helix-loop-helix domain; IB, immunoblot; IFN, interferon; IKKε, inhibitor of nuclear factor kappa-B kinase epsilon; IRF, IFN regulatory factor; KD, kinase domain; LZ, leucine zipper domain; SAMHD, sterile alpha motif and HD domain-containing protein; ULD, ubiquitin-like domain; V, vector controls; WT, wild-type.

Article Snippet: FLAG-tagged kinaseinactive IKKε K38A was a gift from Tom Maniatis (Addgene plasmid #26202). plentiCRISPR v2-Blasticidin was a gift from Mohan Babu (Addgene plasmid # 83480).

Techniques: Binding Assay, Recombinant, Plasmid Preparation, Luciferase, Control, Mutagenesis, Negative Control, Western Blot, Sterility, Ubiquitin Proteomics

Journal: The Journal of biological chemistry

Article Title: SAMHD1 impairs type I interferon induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection.

doi: 10.1016/j.jbc.2023.104925

Figure Lengend Snippet: Figure 10. SAMHD1 impairs IFN-I induction through the MAVS, IKKε, and IRF7 signaling axis during viral infection. SAMHD1 inhibits MAVS- and IKKε-mediated IFN-I induction in monocytic cells. Mechanistically, SAMHD1 interacts with the CARD of MAVS and suppresses MAVS aggre- gation, resulting in reduced IKKε recruitment to MAVS and IKKε phos- phorylation (indicated with a letter P). SAMHD1 also binds to the kinase domain of IKKε and disrupts binding of IRF7 to IKKε KD. SAMHD1 sup- pression of the IRF7-mediated antiviral response depends on its interaction with the inhibitory domain of IRF7. Our results also indicated that endog- enous IRF7 in THP-1 cells is required for efficient HIV-1 infection and viral gene expression. The red T-shaped symbols indicate inhibitory functions confirmed in experiments, while the black T-shaped symbols indicate pro- posed inhibitory mechanisms. This figure was created with BioRender.com. CARD, caspase-recruitment domain; HIV, human immunodeficiency virus; IFN, interferon; IKKε, inhibitor of nuclear factor kappa-B kinase epsilon; IRF, IFN regulatory factor; KD, kinase domain; MAVS, mitochondrial antiviral- signaling protein; SAMHD, sterile alpha motif and HD domain-containing protein.

Article Snippet: FLAG-tagged kinaseinactive IKKε K38A was a gift from Tom Maniatis (Addgene plasmid #26202). plentiCRISPR v2-Blasticidin was a gift from Mohan Babu (Addgene plasmid # 83480).

Techniques: Infection, Binding Assay, Gene Expression, Virus, Sterility