Influenza A virus-encoded NS1 virulence factor protein inhibits innate immune response by targeting IKK

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1 Cellular Microbiology (2012) doi: /cmi Influenza A virus-encoded NS1 virulence factor protein inhibits innate immune response by targeting IKK Shijuan Gao, 1 Liping Song, 1 Jiandong Li, 1 Zhenzhu Zhang, 1 Haiyan Peng, 1 Wei Jiang, 1 Qingtao Wang, 1 Tiebang Kang, 2 Shuai Chen 1 and Wenlin Huang 1,2 * 1 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing , China. 2 State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou , China. Summary The IKK/NF-kB pathway is an essential signalling process initiated by the cell as a defence against viral infection like influenza virus. This pathway is therefore a prime target for viruses attempting to counteract the host response to infection. Here, we report that the influenza A virus NS1 protein specifically inhibits IKK-mediated NF-kB activation and production of the NF-kB induced antiviral genes by physically interacting with IKK through the C-terminal effector domain. The interaction between NS1 and IKKa/IKKb affects their phosphorylation function in both the cytoplasm and nucleus. In the cytoplasm, NS1 not only blocks IKKb-mediated phosphorylation and degradation of IkBa in the classical pathway but also suppresses IKKa-mediated processing of p100 to p52 in the alternative pathway, which leads to the inhibition of nuclear translocation of NF-kB and the subsequent expression of downstream NF-kB target genes. In the nucleus, NS1 impairs IKKmediated phosphorylation of histone H3 Ser 10 that is critical to induce rapid expression of NF-kB target genes. These results reveal a new mechanism by which influenza A virus NS1 protein counteracts host NF-kB-mediated antiviral response through the disruption of IKK function. In this way, NS1 diminishes antiviral responses to infection and, in turn, enhances viral pathogenesis. Received 20 March, 2012; revised 16 July, 2012; accepted 6 August, *For correspondence. hwenl@mail.sysu.edu.cn; Tel. (+86) ; Fax (+86) Introduction Influenza A virus is a significant cause of morbidity and mortality in both humans and animal species. The 2009 H1N1 flu pandemic was caused by an influenza virus of porcine origin that spread from person to person worldwide [Fraser et al., 2009; Influenza A(H1N1)v investigation teams, 2009]. The highly pathogenic H5N1 avian influenza virus still retains a considerable pandemic potential if efficient human-to-human transmission is achieved through mutation or reassortment (Webster, 2004; Rappole and Hubalek, 2006). Genetic variation in these viruses renders antiviral therapies that target virusencoded factors, including vaccines and drugs, potentially ineffective. Because host innate immune responses play crucial roles in establishing early antiviral defences, a better understanding of the interplay between viral factors and innate immune signalling may provide insights into developing more effective antiviral interventions (de Jong et al., 2006; Salomon and Webster, 2009). Influenza A virus belongs to the Orthomyxoviridae family, and its genome consists of eight negative-sense RNA segments encoding 10 or 11 viral proteins. Among these viral proteins, the multifunctional NS1 has been identified as a determinant of virulence that counters type I IFN-mediated antiviral responses (Garcia-Sastre et al., 1998; Geiss et al., 2002; Imai et al., 2010). NS1 prevents virally induced IFN-b production and IFN-mediated effector functions through two independent mechanisms. First, it suppresses virus-induced activation of the transcription factors IRF-3, AP1 and NF-kB that target the IFN-b promoter by forming a complex with viral RNAs or cellular components, such as RIG-I and TRIM25 (Bergmann et al., 2000; Wang et al., 2000; Donelan et al., 2003; Pichlmair et al., 2006; Opitz et al., 2007; Gack et al., 2009). Second, NS1 inhibits CPSF30-mediated cellular pre-mrna processing and the nucleo-cytoplasmic transport of host mrnas (Qiu and Krug, 1994; Nemeroff et al., 1998; Hale et al., 2010). Other strategies that the multifunctional protein uses to promote efficient viral replication include enhancing viral mrna translation, activating phosphoinositide 3-kinase (PI3K) to prevent premature apoptosis, and disrupting cellular PDZ domain-containing proteins (de la Luna et al., 1995; Hale et al., 2006; Obenauer et al., 2006; Ehrhardt et al., 2007; Zhirnov and Klenk, 2007; Jackson et al., 2008; Golebiewski et al., 2012 Blackwell Publishing Ltd cellular microbiology

2 2 S. Gao et al. 2011; Ayllon et al., 2012). These functions appear to be strain-specific, host-dependent, and may correlate with viral virulence. The IKK/NF-kB signalling pathway is an important cellular process that plays a key role in the induction of the innate immune response against viral infection (Ghosh et al., 1998). In mammals, there exist two major NF-kB activating signalling pathways: the classical NF-kB pathway and the alternative NF-kB pathway. The classical NF-kB pathway is activated by extracellular signals such as viruses and/or host-derived cytokines and characterized by the involvement of IKKb-induced phosphorylation and degradation of IkBa releasing p65/p50 dimers. The alternative NF-kB pathway is triggered by signalling from a subset of TNFR members and heavily dependent on NIK and IKKa-induced p100 processing to p52 (DiDonato et al., 1997; Karin and Ben-Neriah, 2000; Xiao et al., 2001). The activation of NF-kB by IKK is thought to be dependent on its cytoplasmic role in the phosphorylation of IkB or p100. However, it has recently been shown that IKKa shuttles between the cytoplasm and nucleus and that nuclear IKKa can be recruited upon cytokine exposure to NF-kB-regulated promoters. There, IKKa phosphorylates Ser 10 of histone H3, which is essential for the subsequent acetylation of Lys 14 by CBP. Thus, nuclear IKKa promotes rapid cytokine-induced NF-kB target gene expression through a mechanism that is distinct from its traditional function of phosphorylating IkB (Anest et al., 2003; Yamamoto et al., 2003). Given that IKK is a key regulator in the induction of the NF-kB-dependent host antiviral response, several viruses have developed various strategies to counteract its activation. These strategies include the inhibition of IKK phosphorylation by the B14 protein of the vaccinia virus (VACV), the ORFV024 protein of the parapoxvirus orf virus (ORFV) and the EBNA1 protein of the Epstein-Barr virus (EBV) (Chen et al., 2008; Diel et al., 2010; Valentine et al., 2010). Like many other viruses, influenza A virus infection leads to the activation of NF-kB pathway, resulting in the expression of a variety of antiviral genes. One of the strategies developed by the influenza A virus to counter this antiviral response involves the NS1 protein, where it was previously found to be an NF-kB antagonist that inhibits viral RNA induced-nf-kb activation by suppression of RIG-I/TRIM25-mediated sensing of viral RNA or by sequestering RNA from PKR (Wang et al., 2000; Donelan et al., 2003; Gack et al., 2009). In our study, we observed that the A/QH/12/05 (H5N1) NS1 efficiently suppressed the induced NF-kB activation in transfected cells after treatment with the innate TLR stimulator dsrna, the pro-inflammatory cytokine TNFa or IL-1b. Based on these observations, we hypothesize that the NS1 protein could inhibit multiple pathways leading to NF-kB activation and the NS1 protein might act at a position at or downstream of a common molecule these signals converge on, such as IKK. Here we show that NS1 directly interacts with and inhibits the function of IKK thereby suppresses NF-kB activation. Interestingly, NS1 not only impairs IKKb-mediated classical NF-kB signalling pathway but inhibits IKKa-mediated alternative NF-kB signalling pathway. Furthermore, IKKa-directed phosphorylation of nuclear histone H3 Ser 10 was also decreased in the presence of NS1, indicating that NS1 disrupts both the cytoplasmic and nuclear roles of IKK, each of which normally lead to the expression of NF-kB target genes. Accordingly, results of experiments from the infection of cells with a recombinant influenza A virus lacking the NS1 gene supported the finding of the inhibitory effects of the NS1 protein on the host antiviral immune responses through its interference with NF-kBdirected innate immune gene expression, which took place in an IKK-dependent manner. In this way, NS1 may diminish host antiviral response to viral infection and enhance viral pathogenesis and may provide a target for development of future antiviral therapies. Results H5N1 NS1 protein prevents the production of NF-kB-regulated innate cytokines To identify new potential antiviral genes regulated by the NS1 protein of high pathogenic avian influenza A virus H5N1 independent of viral infection, the human alveolar epithelial cell line A549 was transfected with a plasmid expressing the A/QH/12/05 (H5N1) NS1 protein, and the resulting gene expression profiles were examined using DNA microarray analysis. Several NF-kB-targeted innate immune genes, including IL-6, IL-8, TNFa, IFN-b and CCL-2 (Viemann et al., 2004; 2007; Schmolke et al., 2009), were downregulated in the presence of NS1 (data not shown), indicating that NS1 may inhibit innate immune genes downstream of the NF-kB pathway. These results were further confirmed using quantitative real-time PCR in human A549 cells and chicken embryo fibroblast cell line DF-1 with or without TNFa treatment (Fig. 1A, Fig. S1). To determine whether NF-kB was necessary for the NS1 effect on the downregulation of these cytokines, we performed IL-6 and IL-8 reporter assays using a reporter with an IL-6 or IL-8 promoter driving the expression of a luciferase gene. The binding sites of NF-kB on both the IL-6 promoter (p-il6-luc) and the IL-8 promoter (p-il8- Luc) were mutated and designated p-il6-luc (DNF-kB) and p-il8-luc (DNF-kB) respectively. Using A549 cells treated with TNFa, the luciferase activities in both p-il6- Luc (DNF-kB) and p-il8-luc (DNF-kB) dropped dramatically relative to those in p-il6-luc and p-il8-luc respectively (Fig. 1B). While the luciferase activities in

3 NS1 suppresses NF-kB activity by targeting IKK 3 (Fig. 1B). From the results of the luciferase assay, we concluded that the NF-kB binding sites are essential for the downregulation of some innate cytokines, including IL-6 and IL-8, by the NS1 protein. H5N1 NS1 protein blocks TNFa-induced activation of NF-kB by the C-terminal effector domain but not the N-terminal RNA-binding domain Fig. 1. H5N1 NS1 protein inhibits the NF-kB-mediated innate cytokines expression. A. A549 cells were transfected with pcdna or pcdna-flag-h5n1-ns1. At 30 h post transfection, the cells were treated with TNFa (15 ng ml -1 ) for 2 h or left untreated as indicated and the mrna levels of IL-6, IL-8, TNFa, IFN-b and CCL-2 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-h5n1-ns1-transfected cells without TNFa treatment that were arbitrarily set as 1. Data represent the mean fold change SD of three independent experiments. Statistical significance was determined by ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001). Expression of Flag-NS1 was monitored by Western blotting with an anti-flag antibody. B. A549 and 293T cells were co-transfected with pcdna or pcdna-h5n1-ns1 and p-x-luc or p-x-luc (DNF-kB) (where x is IL-6 or IL-8, as indicated, and DNF-kB denotes the site-specific mutation in the NF-kB binding sites) along with prl-tk as an internal control. The cells were treated with TNFa (15 ng ml -1 ) for 4 h and luciferase activity was measured. The data are shown as the mean SD from three independent experiments. Statistical significance was determined by ANOVA (**P < 0.01, ***P < 0.001, ns: no significant difference). both p-il6-luc- and p-il8-luc-transfected cells were unaffected by the empty vector control, the presence of the NS1 plasmid inhibited luciferase activity, further implicating the inhibitory function of NS1 on NF-kB-targeted innate immune genes. However, the presence of NS1 caused no significant difference in luciferase activity in either p-il6-luc- (DNF-kB) or p-il8-luc- (DNF-kB) transfected cells (Fig. 1B), suggesting that NF-kB is necessary for the inhibition of these genes by NS1. To ensure that this was not specific to the A549 epithelial cell line, this phenomenon was also detected in other epithelial cells, such as the human embryonic kidney cell line 293T We then investigated the effects of the isolated NS1 protein on NF-kB activation induced by TNFa. 293T cells were transfected with pcdna-h5n1-ns1, pnf-kb-luc (a reporter construct that carries the NF-kB binding sites driving the expression of a firefly luciferase gene), and a constitutively expressed renilla luciferase reporter prl-tk as the internal control. The cells were subsequently stimulated with TNFa (15 ng ml -1 ) for 2 h. We found that the NS1 protein significantly blocked TNFa-induced activation of NF-kB in 293T cells in a dose-dependent manner (Fig. S2). NF-kB activity was also decreased by NS1 to 35%, 48% and 31% in DF-1, A549 and HeLa cells respectively (Fig. 2A). We also observed that the NS1 protein efficiently suppressed poly (I:C) and IL-1b induced NF-kB activation, which indicated that NS1 might inhibit multiple pathways leading to NF-kB activation (data not shown). The NS1 protein contains an N-terminal RNA-binding domain (1 73 aa) and a C-terminal effector domain ( aa) (Fig. 6A). The RNA-binding domain sequesters viral RNA from recognition by its cellular sensors, such as RIG-I. The effector domain mediates interactions with host-cell proteins (Chien et al., 1997; Wang et al., 2002; Bornholdt and Prasad, 2006; Hale et al., 2008a,b; Gack et al., 2009). To determine which domain is responsible for the inhibition of NF-kB activation, we transfected 293T cells with pnf-kb- Luc, prl-tk, an empty vector, the plasmids expressing the full-length NS1, the N-terminal fragment (1 73 aa), the C-terminal fragment ( aa) and the R38A/K41A NS1 mutant that is known to be deficient in dsrna-binding property. After treatment with TNFa (15 ng ml -1 ) for 2 h, a luciferase assay was performed. The full-length NS1, the R38A/K41A NS1 mutant and the C-terminal effector domain ( aa), but not the N-terminal RNA-binding domain (1 73 aa), inhibit TNFa-induced NF-kB activation (Fig. 2B and C). In agreement with these results, the wild-type NS1, the R38A/K41A NS1 mutant and the C-terminal effector domain ( aa), but not the N-terminal RNA-binding domain (1 73 aa), suppress expression of the NF-kBtargeted gene IL-6 (Fig. S3). Notably, the C-terminal effector domain of NS1 has been shown to contain CPSF30 binding sites, and the interaction with CPSF30 blocks general gene expression (Nemeroff et al., 1998; Das et al., 2008). To exclude the possibility that the inhibition of NF-kB-directed gene expression by NS1 resulted from CPSF30-mediated sup-

4 4 S. Gao et al. Fig 2. Inhibition of TNFa-induced NF-kB activation by the C-terminal effector domain but not the N-terminal RNA-binding domain of NS1. A. DF-1, A549 and HeLa cells were co-transfected with pcdna or pcdna-h5n1-ns1, pnf-kb-luc and prl-tk. At 30 h post transfection, the cells were treated with TNFa (15 ng ml -1 ) for 2 h and luciferase activity was assayed. B. 293T cells were co-transfected with pnf-kb-luc, prl-tk, and 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 (NS aa), the C-terminal effector domain (NS aa) or the C-terminal mutant lacking the binding sites for CPSF30 (NS aa CPSFm) as indicated. At 30 h post transfection, the cells were treated with TNFa and luciferase activity was assayed. NS1 ( aa CPSFm) denotes NS1 ( aa, F98A, M101A). C. Western blot analysis of lysates from (B) with an anti-flag antibody. Data in (A) and (B) represent the mean SD of three independent experiments. Statistical significance was determined by paired t-test (A) or ANOVA (B) (**P < 0.01, ns: no significant difference). pression of global gene expression, we constructed a mutant C-terminal effector domain ( CPSFm, F98A/ M101A), which contained two point mutations within the CPSF30 binding sites and thereby lack the ability to limit general gene expression (Das et al., 2008; Hale et al., 2010; Forbes et al., 2012). The results that the mutant construct failed to bind CPSF30 or limit general gene expression (Fig. S4A and B) but still possess the ability to inhibit NF-kB-mediated gene expression (Fig. 2B, Fig. S3) suggest that the inhibition of NF-kB activation by NS1 is specific rather than the global inhibition. H5N1 NS1 protein prevents TNFa-induced nuclear translocation of NF-kB To further explore the mechanism through which the NS1 protein inhibits the activation of NF-kB, we asked whether NS1 interfered with either one or both of the two NF-kB subunits, p50 and p65. We assayed the total protein levels of p50 and p65 in 293T cells with and without TNFa treatment and found that neither subunit was altered by overexpression of the NS1 protein, even when the cells were treated with TNFa (Fig. 3A and B). Given that nuclear translocation of NF-kB is a prerequisite for the promotion of downstream genes, we examined the levels of p50 and p65 in the cytoplasmic and nuclear fractions. NS1 decreased the amounts of both p50 and p65 in the nuclear fractions in cells treated with TNFa. An increase in p50 and p65 in the cytoplasmic fractions was observed, indicating that NS1 prevented the nuclear translocation of NF-kB when it was induced by TNFa (Fig. S5A and B). This was confirmed by immunofluorescence, as p65 was localized in the cytoplasm (red) and translocated to the nucleus after TNFa stimulation in GFP-transfected cells. In contrast, 70% of the NS1-GFP-transfected cells retained p65 in the cytoplasm after treatment with TNFa (Fig. 3C). Taken together, these results show that the inhibition of NF-kB activation by NS1 occurs by preventing the nuclear translocation of NF-kB. H5N1 NS1 protein inhibits TNFa-induced IkBa phosphorylation and degradation Because IkBa phosphorylation and its subsequent ubiquitin-dependent degradation are prerequisites for the release and nuclear translocation of NF-kB (Yaron et al., 1997), we investigated the effect of NS1 on IkBa phosphorylation and degradation. Cells from the highly transfectable 293T line were transfected with NS1-expressing plasmids or control plasmids. Thirty hours post transfection, the cells either were stimulated with TNFa or were left untreated, and the levels of IkBa and IkBa phosphorylation

5 NS1 suppresses NF-kB activity by targeting IKK 5 Fig 3. H5N1 NS1 protein inhibits TNFa-induced nuclear translocation of NF-kB. A. 293T cells were transfected with pcdna or pcdna-h5n1-ns1 and then stimulated with TNFa (15 ng ml -1 ) for 1 h or left unstimulated, as indicated. Whole-cell lysates were prepared and analysed by Western blotting with anti-p50 and anti-p65 antibodies. 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 (ns: no significant difference). C. HeLa cells were transfected with pegfpn1 or pegfpn1-ns1 and treated or left untreated with TNFa for 20 min, as indicated. The cells were fixed, permeabilized and stained for the p65 subunit of NF-kB (red). Yellow arrows indicate NS1-EGFP-expressing cells. White arrows indicate NS1-EGFP-non-expressing cells. Scale bar = 20 mm. were monitored by Western blotting. As shown in Fig. 4A and B, IkBa was reduced in cells stimulated with TNFa both in the presence and in the absence of NS1 (lanes 3 and 4 and 1 and 2 respectively). However, after TNFa treatment, the level of IkBa in the NS1-expressing cells was twice as high as that of the control cells (lanes 4 and 3). After 30 min of TNFa treatment, IkBa phosphorylation was consistently about twofold less in NS1-expressing cells than in vector controls (Fig. 4C and D). This indicates that the NS1 protein is capable of inhibiting phosphorylation of IkBa, thereby increasing the stability of IkBa and allowing it to keep sequestering the NF-kB in the cytoplasm. H5N1 NS1 protein does not alter the phosphorylation of IKK but inhibits IKK-induced NF-kB activation The IkB kinase (IKK) complex contains two catalytic subunits (IKKa and IKKb) and a regulatory subunit (IKKg). The activation of IKKa and IKKb by phosphorylation is required to then phosphorylate IkB (DiDonato et al., 1997). We therefore tested whether NS1 had a role in inhibiting the phosphorylation of the IKK complex subunits. However, we found that NS1 does not have any effect on the phosphorylation of IKK in 293T cells upon stimulation with TNFa, even when NS1 expression levels increase (Fig. 4E and F). Therefore, the data indicate that NS1 inhibits IkB phosphorylation but does not alter the phosphorylation of IKK. We alternatively hypothesized that NS1 may directly act at, or downstream of IKK, rather than acting upstream of IKK. Indeed, we found that the NS1 protein dramatically reduced IkB phosphorylation in the presence of constitutively active IKK mutant IKKa (SS/EE) (Fig. 4G and H), indicating that NS1 was directly inhibiting IKK function. NS1 also resulted in a significant reduction in NF-kB activation induced by the overexpression of IKKa, IKKa (SS/ EE), IKKb or IKKb (SS/EE) (Fig. S6A C). Taken together, these results suggest that NS1 may directly impede the phosphorylation of IkB by IKK. H5N1 and WSN H1N1 NS1 protein physically interact with IKKa and IKKb both in vitro and in vivo We next investigated whether the NS1 protein physically interacts with IKK and IkB. No interactions between the NS1 protein and IkB were detectable in vitro or in vivo (data not shown). However, the NS1 protein is able to directly bind IKKa and IKKb, but not IKKg, as shown by GST pull-down assay (Fig. 5A). These interactions were further confirmed in 293T cells at their endogenous levels of IKKa and IKKb using co-immunoprecipitation (IP) (Fig. 5B and C). This indicates that the NS1 protein is able to physically interact with endogenous IKKa and IKKb. For all experiments discussed so far, the NS1 protein used was from H5N1 (A/QH/12/05). To test whether this interaction was limited to the H5N1 NS1, we examined the interaction between IKK and NS1 from a low pathogenic isolate WSN, which exhibits 86% identity with H5N1 (A/QH/12/05) NS1 protein. As shown in Fig. 5D and E, the WSN NS1 protein was also able to bind both ectopic and

6 6 S. Gao et al. Fig 4. H5N1 NS1 protein does not alter the phosphorylation of IKK but inhibits IKK-induced IkBa phosphorylation and degradation. A. 293T cells were transfected with pcdna or pcdna-h5n1-ns1 and either left untreated or treated with TNFa (15 ng ml -1 ) for 1 h. Levels of IkBa were determined by Western blotting. C. 293T cells were transfected with pcdna or pcdna-h5n1-ns1 and treated with MG-132 (20 mm) for 4 h and TNFa (15 ng ml -1 ) for the indicated lengths of time. Whole-cell lysates were Western blotted. E. 293T cells were transfected with different amounts of pcdna-h5n1-ns1 and either left untreated or treated with TNFa (15 ng ml -1 ) for 20 min, as indicated. Levels of phospho-ikka (Ser 180)/IKKb (Ser 181) and IKKa/b were determined by Western blotting. G. 293T cells were co-transfected with pcdna-h5n1-ns1 and a constitutively active IKKa mutant IKKa(S 176S 180/E 176E 180). The phosphorylation of IkBa was detected by Western blot analysis at 28 h post transfection. B, D, F and H. Quantification of relative protein band intensities from (A), (C), (E) and (G) 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). endogenous IKKa and IKKb. To examine whether endogenous IKK could interact with NS1 in influenza virusinfected cells, A549 cells were infected with the A/WSN/33 virus. The NS1 protein was detected in the IP complexes from the cell extracts using antibodies against IKKa/IKKb, indicating that the WSN NS1 protein interacts with IKKa and IKKb during influenza A virus infection (Fig. 5F). Consistent with the results of interaction studies, WSN NS1 protein inhibits constitutively active mutant IKKa (SS/EE)- mediated IkBa phosphorylation and NF-kB activation (Fig. S8B and C), TNFa-induced nuclear translocation of NF-kB (Fig. S3C) and NF-kB-dependent gene expression (Fig. S8D). These results suggested that the WSN NS1 protein inhibited the NF-kB pathway in a way similar to that of H5N1 NS1 protein. But this phenotype was not shared by the pandemic CA09 H1N1 NS1 protein (Fig. S8). Notably, CA09 NS1 inhibits basal but not TNFainduced expression of NF-kB directed genes (Fig. S8D), which suggests that CA09 NS1 has a different mechanism of inhibiting NF-kB compared with H5N1 NS1 during influenza virus infection. The C-terminal effector domain of NS1 and the N-terminal kinase domain of IKK are required for the binding To map which region of NS1 is critical for its binding with IKK, the truncated Flag-tagged NS1 and HA-tagged IKK

7 NS1 suppresses NF-kB activity by targeting IKK 7 Fig 5. H5N1 and H1N1 NS1 proteins physically interact with IKKa and IKKb in NS1-transfected and A/WSN/33-infected cells. A. 293T cells were transfected with HA-tagged IKKa, IKKb or IKKg. Cell lysates were then incubated with an equal amount of GST or GST-H5N1-NS1 bound to glutathione sepharose 4B beads. After washing, the bound proteins were analysed by Western blotting (WB) with an anti-ha antibody. CBB, Coomassie Brilliant Blue staining. B and C. 293T cells were transfected with pcdna or pcdna-h5n1-ns1. The cell lysates were immunoprecipitated (IP) and immunoblotted with antibodies as indicated. D. 293T cells were co-transfected with HA-tagged IKKa or IKKb and pcdna, pcdna-h5n1-ns1 or pcdna-wsn-ns1. The cell lysates were immunoprecipitated using an anti-flag antibody and immunoblotted using an anti-ha antibody. E. 293T cells were transfected with pcdna-wsn-ns1. The cell lysates were immunoprecipitated and immunoblotted with antibodies as indicated. F. A549 cells were infected with the A/WSN/33 virus at an moi of 1 for 18 h, and the cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. were coexpressed in 293T cells. The cell lysates were immunoprecipitated with an anti-flag antibody and immunoblotted with an anti-ha antibody. We found that the C-terminal effector domain, but not the N-terminal RNA-binding domain, was sufficient to bind IKKa and IKKb (Fig. 6A and B), consistent with the functional results that the C-terminal effector domain is responsible for the inhibition of NF-kB-targeted genes (Fig. 2B and Fig. S3). As far as in the IKK complex, both IKKa and IKKb subunits contain an N-terminal kinase domain, a C-terminal helix loop helix (HLH) domain and a leucine zipper (LZ) domain (DiDonato et al., 1997; Li et al., 2008). To determine which IKK domains bind to NS1, we constructed a series of deletion mutants of IKKa or IKKb, as indicated in Fig. 6C, and performed GST pull-down assays. The kinase domains, but not the LZ or HLH domains, of IKKa and IKKb were sufficient for GST-NS1 pull-down (Fig. 6D).

8 8 S. Gao et al. Fig. 6. The C-terminal effector domain of NS1 and the N-terminal kinase domain of IKK are required for the binding. A. A schematic representation of the deletion mutants of H5N1 NS1. The NS1 protein is divided into an N-terminal RNA-binding domain (1 73 aa) and a C-terminal effector domain ( aa). B. 293T cells were co-transfected with HA-tagged IKKa or IKKb and pcdna, pcdna-h5n1-ns1, NS1(1 73 aa), NS1( aa) or NS1( aa CPSFm), which denotes NS1( aa, F98A, M101A), the mutant lacking the binding sites for CPSF30. The cell lysates were immunoprecipitated using an anti-flag antibody and immunoblotted using an anti-ha antibody. C. A diagram of the deletion mutants of IKKa and IKKb. Both IKKa and IKKb contain an N-terminal kinase domain, a C-terminal helix loop helix domain (HLH) and a leucine zipper domain (LZ), as indicated. D. The regions of IKKa and IKKb that interact with NS1 were identified using GST pull-down assays. WCL: whole-cell lysates. NS1 protein interferes with alternative NF-kB signalling pathway during influenza virus infection It has been reported that the major action of IKKa is the phosphorylation of p100 which leads to the subsequent processing of p100 to p52 in the alternative NF-kB signalling pathway (Xiao et al., 2001). Interference of NS1 with IKKa promotes us to investigate whether influenza A virus inhibits alternative NF-kB signalling via its NS1. To investigate the effects of NS1 on p100 processing to p52 in the context of virus infection, we generated a recombinant A/WSN/33 virus lacking the NS1 gene (dns1). Probably due to the lack of NS1 expression, the titres of the rescued virus were very low ( PFU ml -1 ). For biosafety consideration, H5N1 virus was not used in virus infection experiments. A549 cells were infected with either wild-type A/WSN/33 or the dns1 mutant virus at an moi of 0.5 for 18 h. Cells barely display activation of alternative NF-kB pathway in response to WSN infection, while a dramatic increase of p52 levels were observed in the dns1 virus-infected cells indicating that influenza A virus interferes with alternative NF-kB pathway via its NS1 (Fig. 7A and B). Since overexpression of NIK triggers the processing of p100 to p52 (Xiao et al., 2001), we then verified whether NS1 inhibited NIK-induced processing of p100. As shown in Fig. 7D and E, NIK expression alone dramatically increased p52 levels, while coexpression of NIK and NS1 decreased p52 production compared with control cells. Consistently, in reporter assays, overexpression of NIK alone leads to activation of NF-kB, while co-transfection of NS1 inhibits NF-kB activation (Fig. 7C). For the first time, these data indicated that NS1 protein could inhibit the activation of alternative NF-kB pathway during influenza A virus infection. NS1 protein inhibits nuclear histone H3 phosphorylation in A/WSN/33 virus-infected cells Recently, IKKa has been shown to shuttle between the cytoplasm and nucleus. The nuclear IKKa is needed for the histone H3 Ser 10 phosphorylation at NF-kB-targeted pro-

9 NS1 suppresses NF-kB activity by targeting IKK 9 Fig. 7. NS1 protein interferes with alternative NF-kB signalling pathway in influenza A virus-infected cells. A. A549 cells were infected with the A/WSN/33 virus or the recombinant virus lacking the NS1 gene (dns1) at an moi of 0.5 or left uninfected. At 16 h post infection, whole-cell lysates were Western blotted. C. 293T cells were co-transfected with pnf-kb-luc, prl-tk, and various constructs, as indicated. At 28 h post transfection, the cells were harvested, and a luciferase assay was performed. The data are shown as the mean SD of three independent experiments. D. 293T cells were transfected with the indicated vectors. At 28 h post transfection, whole-cell lysates were Western blotted. B and E. Quantification of relative protein band intensities from (A) and (D) using Image J software (NIH). Depicted are the mean values of three independent experiments. Statistical significance was determined by ANOVA (**P < 0.01, ns: no significant difference). moters upon exposure to TNFa and the subsequent gene expression (Anest et al., 2003; Yamamoto et al., 2003). In this study, we observed that inoculation of WSN virus promotes the nuclear translocation of IKKa both in virus infected and uninfected cells (Fig. S7). This data indicates that the altered localization of IKKa is dependent on the cytokines secreted by the virus-infected cells and act in an autocrine or paracrine pattern. We then investigate whether the nuclear NS1 interferes with the function of nuclear IKKa. Indeed, overexpression of the WSN NS1 protein in transfected 293T cells resulted in a decrease of histone H3 Ser 10 phosphorylation compared with a control plasmid after stimulation with TNFa (Fig. 8A and B), indicating that NS1 protein also inhibits the nuclear function of IKKa. To further investigate the effects of NS1 on histone H3 Ser 10 phosphorylation in the context of virus infection, we infected 293T cells at an moi of 0.5 for 18 h with either wild-type A/WSN/33 or the dns1 mutant virus. While we observed a slight

10 10 S. Gao et al. Fig. 8. NS1 protein inhibits histone H3 Ser 10 phosphorylation during IAV infection. A. 293T cells were transfected with pcdna or pcdna-wsn-ns1. At 30 h post transfection, the cells were treated with TNFa (15 ng ml -1 ) for 1 h. Cells were then lysed and levels of phospho-histone H3 (Ser 10) were determined by Western blotting using specific antibodies. C. 293T cells were infected with the A/WSN/33 virus or the recombinant virus lacking the NS1 gene (dns1) at an moi of 0.5 or left uninfected. At 18 h post infection, whole-cell lysates were prepared and levels of phospho-histone H3 (Ser 10) were determined by Western blotting using specific antibodies. B and D. Quantification of relative phospho-histone H3 (Ser 10) band intensities from (A) and (C) using Image J software (NIH). Depicted are the mean values of three independent experiments. Statistical significance was determined by paired t-test (B) or ANOVA (D) (*P < 0.05, **P < 0.01). decrease in histone H3 Ser 10 phosphorylation in A/WSN/33 virus-infected cells relative to the mock-treated cells, infection with dns1 virus enhanced histone H3 Ser 10 phosphorylation over mock-treated cells and induced histone H3 Ser 10 phosphorylation by 2.5-fold relative to wild-type A/WSN/33 virus infection (Fig. 8C and D), indicating that NS1 was necessary the for suppression of IKK-mediated histone H3 Ser 10 phosphorylation in the nucleus during influenza virus infection. Knock-down of IKK results in reduced increase of NF-kB-dependent innate immune gene expression upon infection with dns1 virus To evaluate the biological significance of the inhibitory effects of the NS1 protein on the NF-kB pathway, the innate immune genes IL-6, IL-8, TNFa, IFN-a and IFN-b were examined in A549 cells infected with A/WSN/33 virus or dns1 virus. As shown in Fig. 9A (and in other data not shown), the mrna levels of these innate immune genes were elevated in cells infected with A/WSN/33 or dns1 virus. The ability of the dns1 virus to induce these cytokines was stronger than that of the A/WSN/33 virus, indicating that the NS1 protein suppresses the induction of innate immune genes during influenza infection. This notion was further supported by the enzyme-linked immunosorbent assay (ELISA) assay for IL-6 and IFN-b, two representative innate immune genes (Fig. 9B). To determine the role of IKK in the induction of innate immune genes during A/WSN/33 virus infection, both IKKa and IKKb were knocked down in A549 cells infected with the A/WSN/33 or dns1 virus. The knock-down of IKK reduces the levels of innate immune genes in both A/WSN/33- and dns1-infected cells, indicating that IKKs play a critical role in the induction of innate immune genes during virus infection. However, IKK knock-down leads to no significant difference in gene expression between cells infected with A/WSN/33 virus or dns1 virus, suggesting that NS1 suppresses the induction of innate immune genes through the IKK/NF-kB pathway during influenza virus infection. Due to the significantly inhibitory effect of antiviral gene on viral replication (Fig. S9), inhibition of NF-kB targeted antiviral gene expression may contribute to efficient virus replication. Discussion The IKK/NF-kB pathway is an essential signalling process initiated by the cell as a defence against viral infection like influenza virus (Ludwig et al., 2003; Ludwig and Planz, 2008). Disruption of NF-kB is one of the important evasion tactics used by the influenza virus to evade the host s

11 NS1 suppresses NF-kB activity by targeting IKK 11 Fig 9. Knock-down of IKK results in reduced increase of NF-kB-dependent innate immune gene expression upon infection with dns1 virus. A549 cells were transfected with si-ikka/si-ikkb or control sirna for 48 h. They were then infected with the A/WSN/33 virus or the dns1 virus at an moi of 0.5 for 18 h. Both mock transfection and mock infection were performed in parallel. The supernatants were collected to assess IL-6 and IFN-b concentrations using ELISA. Data represent the mean SD of three independent experiments (B). Cells were lysed, and total RNA was isolated for the analysis of the mrna levels of IL-6, TNFa and IFN-b using quantitative real-time PCR. GAPDH mrna values were used for normalization. Relative mrna expression was calculated as n-fold of the levels of mock-infected cells that were arbitrarily set as 1. Data represent the mean fold change SD of three independent experiments (A). Whole-cell lysates were also prepared and analysed by Western blotting (C). immune surveillance. Among influenza viral proteins, NS1 is the main NF-kB antagonist because infection with dns1, an influenza A virus lacking the NS1 gene, resulted in uncontrolled NF-kB activation (Wang et al., 2000). This shows that NS1-mediated inhibition of NF-kB activation and subsequent downregulation of NF-kB target genes may play a key role in the pathogenesis of influenzaavirus. Several molecular mechanisms have been described for the interference of NS1 with NF-kB signalling including suppression of RIG-I signal transduction by interaction with TRIM25 (Gack et al., 2009). Our study describes a novel mechanism by how the influenza A virus NS1 disrupts NF-kB signalling through interaction with IKKa/b. Interestingly, this inhibition involves not only IKKb-mediated classical NF-kB pathway but also IKKa-mediated alternative NF-kB pathway. Two molecular mechanisms have previously been found to contribute to influenza virus-triggered NF-kB activation: the accumulation of viral RNA species triggers RIG-I- and PKR-mediated NF-kB activation (Ludwig et al., 2003), and the overexpression of viral proteins HA, NP and M1 stimulates antioxidant-sensitive NF-kB activation (Flory et al., 2000). The viral RNA-induced NF-kB activation has been identified to be suppressed by NS1 through targeting RIG-I and/or PKR (Guo et al., 2007; Opitz et al., 2007; Gack et al., 2009). In our experiment, we observed that dsrna-, TNFa- or IL-1b-induced NF-kB activation can be suppressed by NS1 in the transfected cells, indi-

12 12 S. Gao et al. cating that the NS1 protein may possess the ability to inhibit multiple pathways leading to NF-kB activation and the NS1 protein might act at a position at or downstream of a common molecule these signals converge on. Investigating the mechanism by which NS1 interferes with NF-kB signalling, we found that forced expression of NS1 did not alter IKK phosphorylation but did inhibit IKK downstream functions of phosphorylation and degradation of IkBa. Moreover, NS1 inhibited the NF-kB activation driven by the constitutively active IKK mutant IKK (SS/EE), suggesting that IKK is a site of action of NS1. Finally, we determined that NS1 interacts with both IKKa and IKKb in vitro and in vivo in epithelial cells after viral infection. These results are consistent with a model in which the direct binding of NS1 to IKKa and IKKb impairs the phosphorylation and degradation of IkBa, resulting in the inhibition of the nuclear translocation of NF-kB and, ultimately, NF-kB-directed innate immune gene expression. This model was supported by the data showing that a mutant influenza A/WSN/33 virus lacking the NS1 gene was unable to suppress expression of NF-kB-mediated innate immune genes like the WT virus, and the knockdown of IKK lead to no significant difference in the gene expression between cells infected with the dns1 and WT viruses. It is well known that the classical NF-kB pathway is significantly activated upon influenza virus infections resulting in the expression of a variety of antiviral cytokines and the proapoptotic ligands that are beneficial for virus replication by promoting the nuclear export of vrnp (Wurzer et al., 2003; 2004; Schmolke et al., 2009). However, to our knowledge, the effects of influenza viruses on the alternative NF-kB pathway have not been reported yet. In the present study, the results indicated that strong activation of alternative NF-kB pathway was induced by the mutant virus lacking NS1 protein, while almost no activation of alternative NF-kB pathway was observed in wild-type WSN virus-infected cells. Interference of NS1 with IKKa explains this phenomenon. Since the major action of alternative NF-kB pathway is the regulation of adaptive immunity, blockage of alternative NF-kB pathway via its NS1 might be a novel mechanism by which influenza virus suppresses the specific immune response. Further studies are then required to detect the molecules that trigger the alternative NF-kB pathway and understand the biological consequences of this interference. Recent reports have described a chromatin-associated function for IKKa. For example, in response to TNFa, IKKa is recruited to NF-kB-responsive promoters and mediates the phosphorylation of Ser 10 of histone H3, thereby facilitating the rapid expression of NF-kB-regulated genes (Anest et al., 2003; Yamamoto et al., 2003). In our experiment, we found that NS1 suppressed the phosphorylation of histone H3 (Ser 10) in both transfected and infected cells, it is conceivable that this inhibitory effect leads to the downregulation of the rapid IKK-mediated expression of NF-kB target genes, thereby facilitating viral replication. In this way, upon influenza virus infection, the NS1 protein functions as an NF-kB inhibitor through interference of both virus-induced cytoplasmic IKK activity and cytokineinduced nuclear IKK activity. This is the first report to propose that viral proteins interfere with nuclear IKK activity in a way that affects the expression of innate immune genes. In fact, nuclear IKK-mediated phosphorylation of histone H3 (Ser 10) acts in many kinds of biological processes, including chromosome condensation at the onset of mitosis and cytokine-induced activation of specific genes (Cheung et al., 2000; Strahl and Allis, 2000). Whether NS1 enhances viral spread and replication by regulating histone phosphorylation-mediated biological processes remains to be determined. Co-immunoprecipitation assays were conducted to identify the interaction domains of NS1 and IKK. It was observed that the C-terminal effector domain, but not the N-terminal RNA-binding domain, of NS1, was sufficient for the binding. In agreement with this, the results from luciferase assay and real-time PCR suggested that the C-terminal effector domain was the functional domain. Further characterization of the specific binding motifs within NS1 and the engineering of recombinant viruses expressing the mutant NS1 lacking IKK binding will be helpful for the evaluation of the important role of the NS1-mediated IKK inhibition in influenza A virus virulence. Unfortunately, we failed to narrow down the detailed binding sites. To investigate whether the interference with IKK is shared by NS1 proteins of other virus origins, we also examined NS1 from pandemic CA/09 strain. H5N1 NS1, WSN NS1, but not CA09 NS1 interferes with IKK suggesting that sequence variations in the NS1 proteins of different virus strains influence the protein s ability to bind IKK which may correlate with viral pathogenesis. Although NS1 has been identified here and elsewhere as a major suppressor of NF-kB, still a significant activity of NF-kB and high levels of NF-kB-dependent gene are observed during influenza virus infection (Fig. S10A and B) (Kobasa et al., 2007; Wang et al., 2008; Schmolke et al., 2009). Moreover, activation of NF-kB pathway is to some extent beneficial for virus replication (Nimmerjahn et al., 2004; Wurzer et al., 2004; Pinto et al., 2011). In fact, it seems that influenza virus has acquired the capability to redirect NF-kB activity for its own efficient replication. First, the NF-kB-dependent expression of proapoptotic factors promotes export of caspase-mediated viral ribonucleoprotein (vrnp) (Wurzer et al., 2004; Ehrhardt et al., 2007). Second, the accumulation of viral 5 -triphosphate RNA can induce the NF-kB-dependent induction of SOCS-3 expression, which in turn blocks

13 NS1 suppresses NF-kB activity by targeting IKK 13 type I IFN responses early in the viral replication cycle (Pauli et al., 2008). Third, NF-kB has been shown to directly interfere with promoter regions of IFN stimulated genes and leads to impaired antiviral responses (Wei et al., 2006). Finally, NF-kB differentially regulates viral RNA synthesis (Kumar et al., 2008). Due to the viral dependence on NF-kB function, influenza viruses have developed different strategies to induce NF-kB activation such as accumulation of viral RNA species, and expression of viral proteins (HA, NP and M1), while only express NS1 as the NF-kB antagonist to evade the host immune surveillance (Garcia-Sastre et al., 1998; Flory et al., 2000; Wang et al., 2000; Ludwig et al., 2003). Taking into account the dynamic process of viral replication, time-course experiments analysing NF-kB activation and NS1 intensities may help to understand the virussupportive and virus-antagonizing functions of NF-kB redirected by influenza virus. We show here that WSN infection induced NF-kB activation starting at 4 h post infection (hpi), prior to NS1 expression, which was detected at 6 hpi. The binding of NS1 with IKK was the later event of virus replication that occurs at 8 hpi (Fig. S10B and C). The early activation of NF-kB is essential for vrna synthesis (Kumar et al., 2008), which in turn activates NF-kB pathway. Therefore, the NF-kB activation in the early phase cannot be completely suppressed by NS1. In addition, by paracrine pattern, influenza virus-induced cytokines facilitate nuclear localization of IKKa in uninfected cells and contribute to amplified NF-kB-targeted gene expression, which cannot be inhibited by NS1. Together, NS1 might be a pivotal regulator in maintaining the balance between suppressing an antiviral response at a tolerable limit and keeping the sufficient activation of NF-kB for virus replication. The strain-dependent sequence differences of NS1 proteins may significantly influence this balance and consequently contribute to viral virulence. Taken together this is the first study to suggest that the influenza A virus NS1 protein suppresses NF-kBmediated antiviral response via interference with both the cytoplasmic and nuclear functions of IKK. This function of NS1 may benefit the regulation of the delicate balance between the replication process and host defence programmes induced by the NF-kB pathway and may therefore provide an attractive target for the development of antiviral therapies. Experimental procedures Cell culture, viruses and antibodies A549 cells (human type II alveolar epithelial cell line), 293T cells (human embryonic kidney cell line), HeLa cells (human epithelial carcinoma cell line), Vero cells (African green monkey kidney epithelial cell line), MDCK cells (Madin-Darby canine kidney cell line) and DF-1 cells (chicken embryo fibroblast cell line) were cultured in Dulbecco s modified Eagle s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Paisley, UK). Influenza virus A/WSN/33 (H1N1) was generated using reverse genetics and propagated in 10-day-old embryonated eggs (Neumann et al., 1999; Song et al., 2010). An influenza A/WSN/33 virus lacking the NS1 gene (dns1 virus-like particles, VLPs) was generated using plasmid-based reverse genetics. Briefly, Vero or 293T cells were transfected with 1 mg of each of the following vrna expression plasmids: ppoli-pb2, ppoli -PB1, ppoli-pa, ppoli-ha, ppoli-np, ppoli-na, ppoli-m and ppoli-ns2; and 1 mg of each of the following protein expression plasmids: pcdna-pb2, pcdna-pb1, pcdna-pa, pcaggs-np and pcdna-ns1 (Garcia-Sastre et al., 1998; Neumann et al., 1999). Twenty hours after transfection, the medium was replaced with DMEM supplemented with 1% fetal bovine serum (Bourmakina and Garcia-Sastre, 2005). Three days later, the supernatants were collected and inoculated into NS1-expressing MDCK cells. After h, rescue of the dns1 VLPs was confirmed using haemagglutination, and the viral NS segments were amplified using RT-PCR for sequence confirmation. Viral titre was measured by indirect immunofluorescence microscopy with an anti-np antibody in the MDCK cells (Bourmakina and Garcia-Sastre, 2005; Gack et al., 2009). The mouse anti-flag (M2; F3165) antibody was purchased from Sigma (USA). Mouse anti-ha (sc-7392), anti-p65 (sc-8008), anti-p-ikba (sc-8404), anti-ikka (sc ), anti-ikkb (sc ), anti-influenza A NP (sc ), anti-influenza A NS1 (sc ) and anti-b-actin (C4; sc-47778) antibodies and rabbit anti-p50 (NLS; sc-114), anti-ikba (C-21; sc-371), anti- RNF8 (X-21; sc ) and anti-ikka/b (sc-7607) antibodies were provided by Santa Cruz Biotechnology (CA, USA). Rabbit anti-p-ikka(ser 180)/IKKb(Ser 181) (No. 2681), anti-histone 3 (No. 9715), anti-p-histone 3(Ser 10) (No. 9701S) and anti-nfkb2 p100/p52 (No. 4882) antibodies were purchased from Cell Signaling Technology. Rabbit anti-ikka (3285-1) was purchased from Epitomics (USA). TRITC-conjugated anti-mouse IgG antibody was purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China). Plasmids The NS1 gene of the H5N1 virus (A/Bar-headed Goose/Qinghai/ 12/05) was amplified from the pgem-t-ns1 plasmid (kindly provided by George F. Gao, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China) using PCR. The PCR product was cloned into the prokaryotic expression vector pgex4t-1, the eukaryotic expression vector pcdna3.0-flag and pegfp-n1. The truncated constructs of H5N1 NS1 were created using PCR with specific primers and cloned into pcdna3.0-flag. The pcdna-h5n1-ns1(r38a/k41a) mutant was generated by site-directed mutagenesis(r38 A, K41 A). The pcdna-h5n1- NS1( CPSFm) was made from pcdna-h5n1-ns1(74 225) by site-directed mutagenesis(f98 A, M101 A). H1N1 NS1 cdna was derived from strain A/WSN/33 and subcloned into pcdna3.0-flag. CA09 NS1 cdna (kindly provided by George F. Gao, Institute of Microbiology, Chinese Academy of Sciences) was subcloned into pcdna3.0-flag. The pgl3-p-il6-luc plasmid and its site-specific mutant pgl3-p-il6-luc(dnf-kb) (with the NF-kB consensus sequence of 5 -GGGATTTCC-3 from