The results presented here highlight a previously unknown effect of the NS1 protein of influenza A virus: the inhibition of activation of NF-κB. Several products might contribute to the activation of NF-κB during wild-type influenza A virus infection, including dsRNA, which is presumed to be generated during virus infections. In addition, overexpression of different influenza virus proteins, such as NP, M, and HA, was shown to result in the activation of NF-κB and the transcription of NF-κB-responsive reporter genes (16
). Despite this observation involving the expression of individual influenza virus proteins, we found only a marginal activation of NF-κB in wild-type influenza virus-infected cells. Significantly, our results demonstrate that the NS1 protein of influenza A virus prevents the activation of NF-κB during virus infection. In fact, expression of the NS1 protein efficiently prevented the dsRNA-, Sendai virus-, and NDV-mediated activation of NF-κB. Moreover, infection with the influenza A virus NS1 knockout virus (delNS1) resulted in uncontrolled NF-κB activation. Downstream genes activated by NF-κB include genes involved in stimulation of T-cell proliferation, such as the interleukin 2 (29
), major histocompatibility complex class I (30
), and B7 (66
) genes, among others. In addition, NF-κB participates in the transcriptional activation of the IFN-β gene (28
), which leads to the expression of antiviral genes. Therefore, activation of NF-κB plays an important role in the inhibition of virus replication by stimulating both innate and adaptive immune responses in the host. In this study, we demonstrate that one possible mechanism by which viruses can inhibit antiviral defense mechanisms of the host is by preventing NF-κB activation.
Activation of NF-κB is mediated by phosphorylation of its inhibitor, IκB, by the IKK kinase complex (IKKα, IKKβ, and IKKγ) (12
). In turn, IKKβ can become activated by PKR (9
). It is well established that binding to dsRNA results in the activation of PKR (61
). Thus, it is possible that binding of dsRNA by the NS1 protein (26
) prevents dsRNA from activating constitutive levels of endogenous PKR, therefore preventing NF-κB activation. Our observations are in agreement with this hypothesis. Thus, mutant forms of the NS1 protein containing only the dsRNA binding domain, NS1(1–73) and NS1(1–126), are competent in preventing NF-κB activation when expressed from plasmids in transfected cells or when expressed by a recombinant influenza A virus. In contrast, a mutant NS1 affected in its dsRNA binding ability, NS1(1–73,R38A/K41A), was not able to efficiently prevent NF-κB activation. On the other hand, expression of NS1(1–73) did not affect the activation by TNF or by overexpression of p65 of an NF-κB-responsive promoter (data not shown), suggesting that the dsRNA binding domain of NS1 is specifically involved in inhibiting virus and/or dsRNA-induced NF-κB activation. These results are consistent with a model in which synthesis of NS1 prevents the dsRNA-mediated activation of PKR, inhibiting the PKR-mediated stimulation of the NF-κB pathway. Specifically, dsRNA generated during influenza virus infection might be sequestered by the NS1 protein and might not be accessible for activation of PKR. On the other hand, the NS1 protein might be targeted to interact with PKR by virtue of its dsRNA binding properties, resulting in PKR inhibition. In fact, infection with delNS1 virus or with NS1 temperature-sensitive influenza A viruses, but not with wild-type virus, resulted in PKR activation (4
). Interestingly, transfection of a plasmid expressing a kinase dominant-negative form of PKR (PKR K296R) did not prevent the activation of the IFN-β promoter in delNS1- or Sendai virus-infected cells (data not shown). These results are in agreement with recent observations demonstrating that overexpression of catalytically inactive PKR results in stimulation of IKK, most likely through protein-protein interactions (5
). Therefore, experiments using enzymatically inactive dominant-negative mutants of PKR do not rule out the possibility that PKR is responsible for NF-κB activation in delNS1 virus-infected cells. Nevertheless, we cannot exclude that an unknown dsRNA-activated kinase different from PKR is the major target of the NS1-mediated inhibition of the NF-κB pathway.
NF-κB is an essential positive regulator for the activation of the IFN-β gene (35
). Stimulation of the synthesis of IFN-β is presumed to initiate the IFN-α/β cascade (39
). Previous studies also suggested that NF-κB might be important for the activation of IFN-α genes (9
). Consistent with this, we observed that IFN-α/β synthesis was stimulated in delNS1 virus-infected cells (Fig. ). In contrast, we were unable to detect significant levels of IFN-α/β mRNA in wild-type PR8 virus-infected cells by Northern blot analysis. These results suggest that the NS1 of influenza A virus serves as a virus-encoded IFN antagonist by inhibiting the synthesis of IFN-α/β. In addition to NF-κB, other transcription factors such as AP-1, IRF3, and IRF7 have also been implicated in the activation of the IFN-β gene during viral infections (58
). Previous studies in our laboratory demonstrated that the NS1 protein also inhibits the activation of IRF-3 in virus-infected cells (54
), further supporting a critical role of the NS1 protein in the inhibition of the IFN-α/β system during influenza virus infection (Fig. ).
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 (more ...)
Coevolution of viruses and hosts has resulted in the establishment of complex interactions which modulate virus pathogenicity and host disease. The IFN-α/β system serves as a potent first line of defense against virus infections. Activation of the synthesis of IFN during viral infection results in the transcriptional activation of many host genes involved in antiviral defense mechanisms. However, most viruses have responded to this antiviral system by encoding IFN antagonists. The NS1 of influenza A virus appears to target the synthesis of IFN-α/β by virtue of its dsRNA-binding properties. In this respect, it appears to have a functional role analogous to that of the E3L protein of vaccinia virus, a DNA virus (7
). The presence of analogous proteins performing similar functions in vaccinia and influenza A viruses underscores the significance of the role of these proteins in the replication of viruses within the host.