A novel mechanism for the inhibition of interferon regulatory factor-3-dependent gene expression by human respiratory syncytial virus NS1 protein - PubMed (original) (raw)

A novel mechanism for the inhibition of interferon regulatory factor-3-dependent gene expression by human respiratory syncytial virus NS1 protein

Junping Ren et al. J Gen Virol. 2011 Sep.

Abstract

Human respiratory syncytial virus (RSV), a leading cause of respiratory tract infections in infants, inhibits type I interferon (IFN)-dependent signalling, as well as IFN synthesis. RSV non-structural protein NS1 plays a significant role in this inhibition; however, the mechanism(s) responsible is not fully known. The transcription factor interferon regulatory factor (IRF)-3 is essential for viral-induced IFN-β synthesis. In this study, we found that NS1 protein inhibits IRF-3-dependent gene transcription in constitutively active IRF-3 overexpressing cells, demonstrating that NS1 directly targets IRF-3. Our data also demonstrate that NS1 associates with IRF-3 and its transcriptional coactivator CBP, leading to disrupted association of IRF-3 to CBP and subsequent reduced binding of IRF-3 to the IFN-β promoter without blocking viral-induced IRF-3 phosphorylation, nuclear translocation and dimerization, thereby identifying a novel molecular mechanism by which RSV inhibits IFN-β synthesis.

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Figures

Fig. 1.

Effect of NS1 protein deletion on type I IFN secretion and IRF-3-dependent gene transcription. (a) A549 cells were infected with rRSV-WT or rRSV-ΔNS1, at an m.o.i. of 3, and harvested at 6, 15 and 24 h p.i. to measure IFN-β secretion in cell supernatants by ELISA. Data shown are representative of three independent experiments. Statistical significance was analysed by ANOVA. _P_-value of less than 0.05 was considered significant. *P<0.05 and **P<0.01, relative to rRSV-WT-infected A549 cells. (b) Viral protein expression analysis of infected cells. Total cell lysates were prepared after the infections. Equal amounts of protein were subjected to 4–20 % SDS-PAGE, followed by Western blot using an antibody against RSV (AbD Serotec). The results are representative of two independent experiments. (c) A549 cells were cotransfected with a luciferase reporter plasmid IFN-β-Luc (left panel) or PRDIII-I-Luc (right panel), and the expression plasmid containing RSV NS1 protein or the control vector (CV), and infected with rRSV-WT or -ΔNS1, at an m.o.i. of 3. Cells were harvested at 15 h p.i. to measure luciferase activity. Uninfected plates served as controls. For each plate, luciferase was normalized to the β-galactosidase reporter activity. Data are representative of two independent experiments and are expressed as mean±

of normalized luciferase activity. ANOVA was used for statistical analysis. P less than 0.05 was considered significant. *P<0.05 relative to ΔNS1+CV.

Fig. 2.

NS1 protein directly affects IRF-3-dependent gene expression. 293 cells in 24-well plate were transfected with PRDIII-I-Luc, plasmids encoding either RIG-I, MAVS, IKKϵ, TBK-1 or the constitutively active form of IRF-3 (IRF-3 5D), or their control vectors (0.1–0.2 µg per well), and a plasmid expressing RSV NS1 or its CV (0.2 µg per well), as indicated at the bottom of each column. Cells were harvested 30 h post-transfection to measure luciferase activity. For each plate, luciferase was normalized to the β-galactosidase reporter activity. Data are representative of two independent experiments and are expressed as mean±

of normalized luciferase activity. Statistical significance was analysed by ANOVA. *P<0.05 relative to signal inducer in each group with CV for NS1.

Fig. 3.

NS1 protein prevents IRF-3 binding to IFN-β promoter. (a) NS1 does not affect RSV-induced IRF-3 phosphorylation and nuclear translocation. A549 cells were mock-infected (as a control, CN) or infected with rRSV-WT or rRSV-ΔNS1, at an m.o.i. of 3, for various lengths of time and harvested to prepare either total cell lysates or nuclear extracts as described previously (Bao et al., 2008b). Equal amounts of protein from uninfected and infected cells were analysed by Western blot using antibodies against Ser396 phospho-IRF-3 (pIRF-3) and regular IRF-3. Membranes were stripped and reprobed for β-actin or lamin b, as control for equal loading of the samples of total cell lysate or nuclear fraction, respectively. (b) IRF-3 dimerization is not enhanced by NS1 deletion. Total proteins from cell lysates of A549 cells, mock-infected or infected with recombinant viruses, were separated by native gel electrophoresis and IRF-3 dimerization was analysed by Western blot. (c) NS1 does not facilitate the degradation of TRAF3 and IKKϵ. Upper panel: the abundance of TRAF3 and IKKϵ were compared in WT- versus ΔNS1-infected cells. Membranes were stripped and reprobed for β-actin, as a control for equal loading of the samples. Lower panel: A549 cells were transfected with a plasmid encoding TRAF3 or IKKϵ, or their control vectors, and a plasmid expressing RSV NS1 or its CV, as indicated at the bottom of each column. Cells were harvested 30 h post-transfection to measure the abundance of TRAF3/IKKϵ in the presence or absence of NS1 expression. Membranes were stripped and reprobed for β-actin for equal loading control. (d) NS1 prevents the binding of IRF-3 to the IFN-β promoter. A549 cells were infected with WT or ΔNS1, at an m.o.i. of 3, for 3, 6, 15 and 24 h. ChIP assay was performed using antibody against IRF-3. Equal amounts of DNA samples before ChIP were used for PCR, as control for equal loading of the samples. (e) NS1 associates with IRF-3 in the context of RSV infection. A549 cells were mock-infected or infected with rRSV-WT or rRSV-ΔNS1, at an m.o.i. of 3, and harvested at 6 h p.i. to prepare total cell lysates. Samples were subjected to immunoprecipitation using anti-IRF-3 antibody or control isotype. The immunoprecipitated complexes were then subjected to 4–20 % SDS-PAGE followed by Western blot using anti-NS1 antibody. The membrane was then stripped and reprobed with anti-IRF-3 antibody to determine levels of immunoprecipitated IRF-3, using clean-blot IP detection reagent (HRP) from Thermo Scientific (upper panel). NS1 and IRF-3 from total cell lysates were also measured by Western blot to ensure equal IP input (lower panel). (f) NS1 disrupts the association of IRF-3 with CBP. 293 cells were transfected with plasmids encoding Flag-tagged IRF-3 and V5-tagged NS1 or their control vectors. Total cell lysates were immunoprecipitated with anti-V5 antibody followed by Western blot using anti-Flag antibody to detect Flag-tagged IRF-3. Reverse immunoprecipitation was also done, where Flag-tagged-IRF-3 was immunoprecipitated using anti-Flag antibody, and NS1 protein was then detected using anti-V5 antibody. Membranes were stripped and reprobed to check for properly immunoprecipitated NS1 or IRF-3. The association of endogenous CBP with immunoprecipitated NS1 or IRF-3 was determined by reprobing the membrane with anti-CBP antibodies (Santa Cruz). Representative results are shown from two to four separate experiments.

References

1. Bao X., Kolli D., Liu T., Shan Y., Garofalo R. P., Casola A. (2008a). Human metapneumovirus small hydrophobic protein inhibits NF-κB transcriptional activity. J Virol 82, 8224–8229 10.1128/JVI.02584-07 - DOI - PMC - PubMed
1. Bao X., Liu T., Shan Y., Li K., Garofalo R. P., Casola A. (2008b ). Human metapneumovirus glycoprotein G inhibits innate immune responses. PLoS Pathog 4, e1000077 10.1371/journal.ppat.1000077 - DOI - PMC - PubMed
1. Barnes B., Lubyova B., Pitha P. M. (2002). On the role of IRF in host defense. J Interferon Cytokine Res 22, 59–71 10.1089/107999002753452665 - DOI - PubMed
1. Barro M., Patton J. T. (2005). Rotavirus nonstructural protein 1 subverts innate immune response by inducing degradation of IFN regulatory factor 3. Proc Natl Acad Sci U S A 102, 4114–4119 10.1073/pnas.0408376102 - DOI - PMC - PubMed
1. Casola A., Burger N., Liu T., Jamaluddin M., Brasier A. R., Garofalo R. P. (2001). Oxidant tone regulates RANTES gene expression in airway epithelial cells infected with respiratory syncytial virus. Role in viral-induced interferon regulatory factor activation. J Biol Chem 276, 19715–19722 10.1074/jbc.M101526200 - DOI - PubMed

A novel mechanism for the inhibition of interferon regulatory factor-3-dependent gene expression by human respiratory syncytial virus NS1 protein - PubMed (original) (raw)