Journal: Emerging Microbes & Infections

Article Title: Middle East respiratory syndrome coronavirus M protein suppresses type I interferon expression through the inhibition of TBK1-dependent phosphorylation of IRF3

doi: 10.1038/emi.2016.33

Figure Lengend Snippet: MERS-CoV M protein suppresses IRF3 activation but not NF-κB signaling. An expression plasmid for dominant-active RIG-I N mutant containing the N-terminal domain of RIG-I alone was used as a potent IFN inducer and co-transfected with an M expression plasmid and a firefly luciferase plasmid driven by tandem copies of ( A ) IRF3-binding elements (IRF3-luc) or ( B ) κB elements (κB-luc) as well as the control Renilla luciferase plasmid. A canonical NF-κB inhibitor IκB-α (IκB) was included as a positive control for the inhibition of NF-κB activity. Cells were collected at 40 h post transfection for dual-luciferase reporter assay. Bars represent the mean of three biological replicates ( n =3) and error bars indicate their s.d. The statistical significance between selected samples was evaluated using a two-tailed Student's t -test for unpaired samples with equal variance and P -value ( P ) was indicated. retinoic acid-inducible gene-I, RIG-I.

Article Snippet: Rabbit anti-IRF3 and anti-phospho-IRF3 (Ser 386) antibodies were purchased from IBL-America (Minneapolis, MN, USA).

Techniques: Activation Assay, Expressing, Plasmid Preparation, Mutagenesis, Transfection, Luciferase, Binding Assay, Control, Positive Control, Inhibition, Activity Assay, Reporter Assay, Two Tailed Test

Journal: Emerging Microbes & Infections

Article Title: Middle East respiratory syndrome coronavirus M protein suppresses type I interferon expression through the inhibition of TBK1-dependent phosphorylation of IRF3

doi: 10.1038/emi.2016.33

Figure Lengend Snippet: The inhibitory effect of the MERS-CoV M protein occurs upstream of IRF3 activation. ( A – E ) HEK-293 cells were transfected with increasing doses of the MERS M-V5 expression plasmid, a firefly luciferase reporter plasmid driven by IFN-β promoter (IFNβ-luc) and a control Renilla luciferase reporter plasmid, together with different expression plasmids of RLR pathway activators, RIG-I N in A, mitochondrial adapter MAVS in B, IRF3 kinases TBK1 and IKK-ɛ in C and D as well as a dominant-active IRF3 mutant (IRF3 5D) in E. Cells were collected after 40 h for dual-luciferase reporter assay. Bars represent the mean of three biological replicates ( n =3) and error bars indicate their s.d. The statistical significance between selected samples was evaluated using a two-tailed Student's t -test for unpaired samples with equal variance and P -value ( P ) was indicated. Retinoic acid-inducible gene-I, RIG-I; RIG-I-like receptor, RLR.

Article Snippet: Rabbit anti-IRF3 and anti-phospho-IRF3 (Ser 386) antibodies were purchased from IBL-America (Minneapolis, MN, USA).

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Control, Mutagenesis, Reporter Assay, Two Tailed Test

Journal: Emerging Microbes & Infections

Article Title: Middle East respiratory syndrome coronavirus M protein suppresses type I interferon expression through the inhibition of TBK1-dependent phosphorylation of IRF3

doi: 10.1038/emi.2016.33

Figure Lengend Snippet: MERS-CoV M protein binds with TRAF3 adapter and perturbs TRAF3–TBK1 interaction. ( A ) HEK-293 cells were transfected with plasmids expressing MERS M-V5 protein and different FLAG-tagged wild-type RLR pathway activators (FLAG-RIG-I, -MAVS and -TRAF3). An empty vector was used as a negative control in lane 1. Cells were collected after 40 h for a co-immunoprecipitation experiment with anti-FLAG antibody. The bound fraction of immunoprecipitates (IP) as well as total cell lysate (as input) were analyzed by western blotting (WB) with anti-FLAG and anti-V5 antibodies. ( B ) HEK-293 cells were transfected with different combinations of expression plasmids for MERS M-V5, FLAG-tagged TRAF3 and HA-tagged TBK1, and collected after 40 h for co-immunoprecipitation experiment with an anti-FLAG antibody to assay for TBK1 recruitment using an anti-HA antibody. ( C , D ) HEK-293 cells were transfected with different combinations of expression plasmids for wild-type IRF3, RIG-I N, and MERS M-V5. After 40 h, IRF3 dimerization from cell lysates was visualized by non-denaturing native PAGE followed by western blotting with anti-IRF3 antibodies ( C ). IRF3 phosphorylation was also probed with anti-phopho-IRF3 antibodies ( D ). The expression level of the ectopically expressed proteins was also individually verified in denaturing SDS-PAGE with respective antibodies. The relative band intensity (*/#) of co-immunoprecipitated TBK1 in B, dimeric IRF3 in C or phospho-IRF3 (p-IRF3) in D for each sample was measured using ImageJ software. Retinoic acid-inducible gene-I, RIG-I; RIG-I-like receptor, RLR.

Article Snippet: Rabbit anti-IRF3 and anti-phospho-IRF3 (Ser 386) antibodies were purchased from IBL-America (Minneapolis, MN, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Negative Control, Immunoprecipitation, Western Blot, Clear Native PAGE, Phospho-proteomics, SDS Page, Software