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. 2000 Dec;74(24):11566-73.
doi: 10.1128/jvi.74.24.11566-11573.2000.

Influenza A virus NS1 protein prevents activation of NF-kappaB and induction of alpha/beta interferon

X Wang  1 , M Li, H Zheng, T Muster, P Palese, A A Beg, A García-Sastre
Affiliations

Influenza A virus NS1 protein prevents activation of NF-kappaB and induction of alpha/beta interferon

X Wang et al. J Virol. 2000 Dec.

Abstract

The alpha/beta interferon (IFN-alpha/beta) system represents one of the first lines of defense against virus infections. As a result, most viruses encode IFN antagonistic factors which enhance viral replication in their hosts. We have previously shown that a recombinant influenza A virus lacking the NS1 gene (delNS1) only replicates efficiently in IFN-alpha/beta-deficient systems. Consistent with this observation, we found that infection of tissue culture cells with delNS1 virus, but not with wild-type influenza A virus, induced high levels of mRNA synthesis from IFN-alpha/beta genes, including IFN-beta. It is known that transactivation of the IFN-beta promoter depends on NF-kappaB and several other transcription factors. Interestingly, cells infected with delNS1 virus showed high levels of NF-kappaB activation compared with those infected with wild-type virus. Expression of dominant-negative inhibitors of the NF-kappaB pathway during delNS1 virus infection prevented the transactivation of the IFN-beta promoter, demonstrating a functional link between NF-kappaB activation and IFN-alpha/beta synthesis in delNS1 virus-infected cells. Moreover, expression of the NS1 protein prevented virus- and/or double-stranded RNA (dsRNA)-mediated activation of the NF-kappaB pathway and of IFN-beta synthesis. This inhibitory property of the NS1 protein of influenza A virus was dependent on its ability to bind dsRNA, supporting a model in which binding of NS1 to dsRNA generated during influenza virus infection prevents the activation of the IFN system. NS1-mediated inhibition of the NF-kappaB pathway may thus play a key role in the pathogenesis of influenza A virus.

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Figures

FIG. 1
FIG. 1
Induction of IFN-α/β-specific mRNAs by delNS1 virus infection in MEFs. Northern blot analyses of mRNAs corresponding to IFN-α and IFN-β were performed with RNA isolated from mock-infected MEFs or MEFs infected with PR8 or delNS1 viruses at an MOI of 1 for the indicated times. A total of 10 μg of RNA was used. Northern blot detection of mRNA derived from a housekeeping gene (β-actin) is shown as a control.
FIG. 2
FIG. 2
Activation of NF-κB by delNS1 virus infection in MEFs. (A) Detection of activated NF-κB by EMSA. MEFs were untreated (UT), mock infected, or infected with PR8, delNS1, or NDV viruses at an MOI of 1 for the indicated times. Nuclear extracts were subjected to EMSA with a DNA probe specific for NF-κB. TNF-α- and dsRNA-treated cells were included as positive controls. (B) Supershift of NF-κB complexes. Anti-p65 and anti-p50 antibodies (Ab) were used to shift the NF-κB complexes in delNS1- or NDV-infected MEFs at 6 h p.i.
FIG. 3
FIG. 3
NF-κB is essential for delNS1 virus-induced IFN-β gene expression. (A) Monolayers of 293 cells were cotransfected with pIFN-CAT (encoding a CAT reporter gene under a mouse IFN-β promoter) and a plasmid expressing a superrepressor form of IκB, IκB(SA), or empty vector. One day posttransfection, cells were mock treated or transfected with 50 μg of dsRNA or infected with PR8, delNS1, or NDV viruses at an MOI of 1. One day later, CAT assays were performed with 5 μl of cell extracts. (B) Quantitative analysis of the results shown in Fig. 3A. (C) Monolayers of 293 cells were cotransfected with pIFN-CAT and a plasmid expressing a dominant-negative form of IκB kinase, IKKβ(KA), or empty vector. One day posttransfection, cells were treated as in panel A. Quantitative results are indicated. (D) 293 cells were transfected with a plasmid expressing IκB(SA) or empty vector. One day posttransfection, cells were infected with PR8, delNS1, or Sendai viruses at an MOI of 1 for the indicated times. RNAs were extracted and subjected to Northern blot analysis with probes specific for IFN-β and β-actin mRNAs.
FIG. 4
FIG. 4
The dsRNA binding domain of the NS1 protein is sufficient to prevent NF-κB activation and IFN-β induction. (A) 293 cells were transfected with a plasmid containing a reporter gene under the control of an NF-κB-responsive promoter, pκB-Luc. In addition, cells were cotransfected with plasmids expressing NS1, NS1(1–73), NS1(1–73,R38A/K41A), or empty vector. One day posttransfection, cells were transfected with 10 μg of dsRNA or infected with PR8, delNS1, or Sendai viruses at an MOI of 1. Two days posttransfection, luciferase activity was determined. In all transfections, pRL-TK-Luc, encoding a Renilla luciferase under the control of a constitutive promoter, was cotransfected, and Renilla luciferase activity was used as an internal control to normalize the results. (B) 293 cells were cotransfected with pIFN-CAT and plasmids expressing NS1, NS1(1–73), or NS1(1–73,R38A/K41A) proteins or empty vector. One day posttransfection, cells were transfected with 10 μg of dsRNA or infected with PR8 or delNS1 viruses at an MOI of 1 or infected with Sendai virus at an MOI of 10. Two days posttransfection, CAT assays were performed, and the results were quantified. (C) 293 cells were transfected with plasmids expressing NS1 or NS1(1–73) proteins or empty vector. One day posttransfection, cells were infected with delNS1 or Sendai viruses at an MOI of 1 for the indicated time points. RNA was extracted and subjected to Northern blot analysis with probes specific for IFN-β and β-actin mRNAs.
FIG. 4
FIG. 4
The dsRNA binding domain of the NS1 protein is sufficient to prevent NF-κB activation and IFN-β induction. (A) 293 cells were transfected with a plasmid containing a reporter gene under the control of an NF-κB-responsive promoter, pκB-Luc. In addition, cells were cotransfected with plasmids expressing NS1, NS1(1–73), NS1(1–73,R38A/K41A), or empty vector. One day posttransfection, cells were transfected with 10 μg of dsRNA or infected with PR8, delNS1, or Sendai viruses at an MOI of 1. Two days posttransfection, luciferase activity was determined. In all transfections, pRL-TK-Luc, encoding a Renilla luciferase under the control of a constitutive promoter, was cotransfected, and Renilla luciferase activity was used as an internal control to normalize the results. (B) 293 cells were cotransfected with pIFN-CAT and plasmids expressing NS1, NS1(1–73), or NS1(1–73,R38A/K41A) proteins or empty vector. One day posttransfection, cells were transfected with 10 μg of dsRNA or infected with PR8 or delNS1 viruses at an MOI of 1 or infected with Sendai virus at an MOI of 10. Two days posttransfection, CAT assays were performed, and the results were quantified. (C) 293 cells were transfected with plasmids expressing NS1 or NS1(1–73) proteins or empty vector. One day posttransfection, cells were infected with delNS1 or Sendai viruses at an MOI of 1 for the indicated time points. RNA was extracted and subjected to Northern blot analysis with probes specific for IFN-β and β-actin mRNAs.
FIG. 4
FIG. 4
The dsRNA binding domain of the NS1 protein is sufficient to prevent NF-κB activation and IFN-β induction. (A) 293 cells were transfected with a plasmid containing a reporter gene under the control of an NF-κB-responsive promoter, pκB-Luc. In addition, cells were cotransfected with plasmids expressing NS1, NS1(1–73), NS1(1–73,R38A/K41A), or empty vector. One day posttransfection, cells were transfected with 10 μg of dsRNA or infected with PR8, delNS1, or Sendai viruses at an MOI of 1. Two days posttransfection, luciferase activity was determined. In all transfections, pRL-TK-Luc, encoding a Renilla luciferase under the control of a constitutive promoter, was cotransfected, and Renilla luciferase activity was used as an internal control to normalize the results. (B) 293 cells were cotransfected with pIFN-CAT and plasmids expressing NS1, NS1(1–73), or NS1(1–73,R38A/K41A) proteins or empty vector. One day posttransfection, cells were transfected with 10 μg of dsRNA or infected with PR8 or delNS1 viruses at an MOI of 1 or infected with Sendai virus at an MOI of 10. Two days posttransfection, CAT assays were performed, and the results were quantified. (C) 293 cells were transfected with plasmids expressing NS1 or NS1(1–73) proteins or empty vector. One day posttransfection, cells were infected with delNS1 or Sendai viruses at an MOI of 1 for the indicated time points. RNA was extracted and subjected to Northern blot analysis with probes specific for IFN-β and β-actin mRNAs.
FIG. 5
FIG. 5
NS1(1–126) virus infection prevents the activation of NF-κB and the induction of IFN-β. (A) MDCK cells were infected with PR8 or NS1(1–126) viruses at an MOI of 1. Six hours p.i., cell extracts were made. Cell extracts (10 μl) were subjected to Western analysis with an anti-NS1 antibody. wt, wild type. (B) 293 cells were mock infected or infected with delNS1, PR8, or NS1(1–126) viruses at an MOI of 1. Six hours p.i., nuclear extracts were made and subjected to EMSA with a probe specific for activated NF-κB. (C) 293 cells were infected with either NS1(1–126) or delNS1 viruses. RNA was extracted at the indicated times and subjected to Northern blot analysis with probes specific for IFN-β and β-actin mRNAs.
FIG. 6
FIG. 6
Model for the mechanism of inhibition of IFN-β induction by the NS1 protein of influenza A virus. Influenza virus infection results in the generation of dsRNA, which in turn activates transcription factors AP-1 (ATF2/c-JUN), IRFs (IRF-3/7), and NF-κB. Cooperation between these transcription factors upon binding to the IFN-β promoter facilitates the recruitment of the RNA polymerase II machinery (enhancesome formation) and stimulates the synthesis of IFN-β mRNA. Expression of NS1 protein during influenza virus infection prevents the dsRNA-mediated activation of IRF-3 (54) and of NF-κB (this study), therefore inhibiting IFN-β production. This inhibitory effect is dependent on the ability of NS1 to bind dsRNA. Therefore, activation of ATF2/c-JUN might also be prevented during influenza A virus infection by the NS1 protein. In addition, it should be noted that PKR, a dsRNA-activated kinase which plays an important role in different IFN pathways, both as an inducer of IFN synthesis, as well as an inhibitor of translation whose levels are transcriptionally increased in response to IFN and IRF-1 activation (43, 61), has also been found to be inhibited by the NS1 protein during influenza A virus infections (4, 27). Inhibition of IRFs and NF-κB activation by the NS1 protein most likely involves inhibition of PKR and/or uncharacterized upstream kinases activated by dsRNA.
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