SEARCH

SEARCH BY CITATION

FilenameFormatSizeDescription
cmi12005-sup-0001-si.pdf390K

Fig. S1. H5N1 NS1 protein inhibits NF-κB-dependent innate cytokines expression in chicken fibroblast cell line DF-1. (A) DF-1 cells were transfected with pcDNA or pcDNA-Flag-H5N1-NS1. At 30 h post transfection, the cells were treated with TNFα (15 ng ml−1) for 2 h and the mRNA levels of IL-6, IL-8 and IFN-β were assessed using quantitative real-time PCR. The primer sequences used in the real-time PCR were as follows: IL-6 forward 5′-CAGGACGAGATGTGCAAGAAGT-3′ and reverse 5′-CTTGGGCAGGTTGAGGTTGT-3′; IL-8 forward 5′-CCTAACCATGAACGGCAAGC-3′ and reverse 5′-ACCAGC GTCCTACCTTGCG-3′; IFN-β forward 5′-ACAACTTCCTACAGCACAACAACTA-3′ and reverse 5′-GCCTGGAGGCGGACATG-3′; GAPDH forward 5′-CCCCAATGTCTCTGTT GTTGAC-3′ and reverse 5′-CAGCCTTCACTACCCTCTTGAT-3′. GAPDH mRNA values were used for normalization. Relative mRNA expression was calculated as n-fold of the levels of pcDNA-H5N1-NS1-transfected cells that were arbitrarily set as 1. Expression of Flag-NS1 was monitored by Western blotting with an anti-Flag antibody (B). Data represent the mean ± SD of three independent experiments. Statistical significance was determined by anova (*P < 0.05, **P < 0.01).

cmi12005-sup-0001-si.pdf390K

Fig. S2. H5N1 NS1 protein inhibits TNFα-induced NF-κB activation in a dose-dependent manner. 293T cells were co-transfected with different amounts of pcDNA-H5N1-NS1, as indicated, along with 0.05 μg of pNF-κB-Luc and 3 ng of pRL-tk. The cells were either treated with TNFα (15 ng ml−1) for 2 h or left untreated, and luciferase activity was then assayed. Data represent the mean ± SD of three independent experiments. Statistical significance was determined by anova (*P < 0.05, **P < 0.01).

cmi12005-sup-0001-si.pdf390K

Fig. S3. Inhibition of TNFα-induced IL-6 expression by the C-terminal effector domain but not the N-terminal RNA-binding domain of NS1. 293T cells were transfected with the expression plasmids for Flag-tagged H5N1-NS1, the mutant NS1 that is deficient in dsRNA binding activity (NS1 R38A/K41A), the N-terminal RNA-binding domain (NS1 1–73 aa), the C-terminal effector domain (NS1 74–225 aa), or the C-terminal mutant lacking the binding sites for CPSF30 (NS1 74–225 aa CPSFm) as indicated. At 30 h post transfection, the cells were treated with TNFα (15 ng ml−1) for 2 h, the mRNA levels of IL-6 were assessed using quantitative real-time PCR. GAPDH mRNA values were used for normalization. Relative mRNA expression was calculated as n-fold of the levels of pcDNA-transfected cells that were arbitrarily set as 1. Data represent the mean ± SD of three independent experiments. Statistical significance was determined by anova (*P < 0.05, ns: no significant difference).

cmi12005-sup-0001-si.pdf390K

Fig. S4. The mutant H5N1 NS1 lacking a binding site for CPSF30 is unable to bind CPSF30 or limit general gene expression.

A. 293T cells were transfected with pcDNA, pcDNA-H5N1-NS1, pcDNA-H5N1-NS1CPSFm (F98A/M98A) or pcDNA-H5N1-NS1(74–225 aa CPSFm). At 30 h post transfection, the cell lysates were immunoprecipitated using an anti-Flag antibody and immunoblotted using anti-CPSF30 antibody.

B. 293T cells were co-transfected with pcDNA-Flag-luciferase and pcDNA, pcDNA-H5N1-NS1, pcDNA-H5N1-NS1CPSFm (F98A/M98A) or pcDNA-H5N1-NS1(74–225 aa CPSFm). At 30 h post transfection, the cell lysates were immunoblotted with antibodies as indicated.

cmi12005-sup-0001-si.pdf390K

Fig. S5. NS1 protein inhibits TNFα-induced nuclear translocation of NF-κB.

A. 293T cells were transfected with pcDNA or pcDNA-H5N1-NS1 and treated with TNFα for 20 min or left unstimulated, as indicated. The cytoplasmic and nuclear proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology, Beijing, China) and assayed by Western blotting with anti-p50 and anti-p65 antibodies. β-Actin and RNF8 were used as protein loading controls.

B. Quantification of relative p65 and p50 band intensities from (A) using Image J software (NIH). Depicted are the mean values of three independent experiments. Statistical significance was determined by anova (*P < 0.05, **P < 0.01, ns: no significant difference).

C. A549 cells were transfected with Flag-WSN-NS1 and treated or left untreated with TNFα for 20 min. The cells were fixed, permeabilized and stained for the p65 subunit of NF-κB (red) and Flag-NS1 (Green). Yellow arrows indicate NS1-expressing cells. White arrows indicate NS1-non-expressing cells. Scale bar = 20 μm.

cmi12005-sup-0001-si.pdf390K

Fig. S6. H5N1 NS1 protein inhibits IKK-induced NF-κB activation. 293T cells (A and B) and DF-1 cells (C) were co-transfected with pNF-κB-Luc, pRL-tk, and various constructs, as indicated. At 28 h post transfection, the cells were harvested, and a luciferase assay was performed. IKKα(S176S180/E176E180) and IKKβ(S177S181/E177E181) denote constitutively active IKK mutants. The data are shown as the mean ± SD of three independent experiments. Statistical significance was determined by anova (**P < 0.01).

cmi12005-sup-0001-si.pdf390K

Fig. S7. Localization of NS1 and IKKα in influenza A virus-infected A549 cells. A549 cells were infected with the A/WSN/33 virus at an moi of 1 or left uninfected. At 18 h post infection, the cells were fixed, permeabilized and stained for the NS1 (green) and IKKα (red). Scale bar = 20 μm.

cmi12005-sup-0001-si.pdf390K

Fig. S8. CA09 NS1 does not bind to IKK or inhibit IKK-mediated NF-κB activation.

A. 293T cells were co-transfected with HA-IKKα and pcDNA, pcDNA-H5N1-NS1 or pcDNA-CA09-NS1. At 30 h post transfection, the cell lysates were immunoprecipitated (IP) and immunoblotted with antibodies as indicated.

B. 293T cells were co-transfected with a constitutively active IKKα mutant IKKα(S176S180/E176E180) and the constructs as indicated. The phosphorylation of IκBα was detected by Western blot analysis at 28 h post transfection.

C. 293T cells were co-transfected with pNF-κB-Luc, pRL-tk, and various constructs, as indicated. At 28 h post transfection, the cells were harvested, and a luciferase assay was performed. IKKα(S176S180/E176E180) denote constitutively active IKK mutants.

D. 293T cells were transfected with H5N1 NS1, WSN NS1 or CA09 NS1. At 30 h post transfection, the cells were treated with TNFα (15 ng ml−1) for 2 h or left untreated and the mRNA levels of IL-8 and IFN-β were assessed using quantitative real-time PCR. GAPDH mRNA values were used for normalization. Relative mRNA expression was calculated as n-fold of the levels of pcDNA-transfected cells that were arbitrarily set as 1.

Data in (C) and (D) represent the mean fold change ± SD of three independent experiments. Statistical significance was determined by anova (*P < 0.05; **P < 0.01; ns: no significant).

cmi12005-sup-0001-si.pdf390K

Fig. S9. TNFα inhibits WSN influenza virus replication in A549 cells. A549 cells were maintained in serum-free medium overnight and treated with different doses of TNFα as indicated for 1 h. The cells were then infected with WSN virus at an moi of 0.5. Virus titres were measured at 18 h post infection. Data represent the mean ± SD of three independent experiments.

cmi12005-sup-0001-si.pdf390K

Fig. S10. Time-course of WSN influenza virus-induced NF-κB activation.

A. A549 cells were infected with WSN virus (moi = 5) for the indicated time points. The mRNA levels of IFN-β and IL-8 were assessed using quantitative real-time PCR. GAPDH mRNA values were used for normalization. Relative mRNA expression was calculated as n-fold of the levels of cells infected with WSN at 0 h that were arbitrarily set as 1. Data represent the mean fold change ± SD of three independent experiments.

B. A549 cells were infected with WSN virus (moi = 5) for the indicated time points. Cell lysates were prepared and subjected to Western blot analyses with specific antibodies against phospho-histone H3 (Ser 10), phospho-IκBα and IκBα. Equal protein load was verified in histone H3 Western blots. Ongoing viral replication was monitored by accumulation of NS1 protein with specific antibody.

C. Cell lysates from (B) were also immunoprecipitated (IP) with IgG or IKKα/β antibody and immunoblotted with an anti-NS1 antibody.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.