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The Journal of General Virology logoLink to The Journal of General Virology
. 2011 Sep;92(Pt 9):2093–2104. doi: 10.1099/vir.0.032060-0

The NS1 protein of influenza A virus suppresses interferon-regulated activation of antigen-presentation and immune-proteasome pathways

Jennifer R Tisoncik 1, Rosalind Billharz 1, Svetlana Burmakina 2, Sarah E Belisle 1, Sean C Proll 1, Marcus J Korth 1, Adolfo García-Sastre 2,3,4, Michael G Katze 1,5,✉
PMCID: PMC3353386  PMID: 21593271

Abstract

The NS1 protein of influenza virus counters host antiviral defences primarily by antagonizing the type I interferon (IFN) response. Both the N-terminal dsRNA-binding domain and the C-terminal effector domain are required for optimal suppression of host responses during infection. To better understand the regulatory role of the NS1 effector domain, we used an NS1-truncated mutant virus derived from human H1N1 influenza isolate A/Texas/36/91 (Tx/91) and assessed global transcriptional profiles from two independent human lung cell-culture models. Relative to the wild-type Tx/91-induced gene expression, the NS1 mutant virus induced enhanced expression of innate immune genes, specifically NF-κB signalling-pathway genes and IFN-α and -β target genes. We queried an experimentally derived IFN gene set to gauge the proportion of IFN-responsive genes that are suppressed specifically by NS1. We show that the C-terminally truncated NS1 mutant virus is less efficient at suppressing IFN-regulated gene expression associated with activation of antigen-presentation and immune-proteasome pathways. This is the first report integrating genomic analysis from two independent human culture systems, including primary lung cells, using genetically similar H1N1 influenza viruses that differ only in the length of the NS1 protein.

Introduction

Influenza A virus poses a serious global health threat. As demonstrated by the recent swine-origin H1N1 pandemic, novel influenza A viruses can emerge in the human population, causing widespread disease. Each year there are approximately 250 000 influenza-associated hospitalizations in the USA (Thompson et al., 2004), resulting in an estimated 36 000 deaths (Thompson et al., 2003). Host innate immune responses are critical for establishing early antiviral defences. The interferon regulatory factor (IRF) 3/7 and NF-κB signalling pathways are activated in response to infection, leading to the production of type I interferon (IFN-α/β). IFN-α/β signalling through the Jak-STAT pathway results in the expression of hundreds of IFN-stimulated genes (ISGs) encoding effector proteins that reinforce an antiviral state (Darnell et al., 1994). Influenza A virus has evolved multiple strategies to counter this antiviral programme. One mechanism involves translational control of eukaryotic elongation initiation factor eIF2α (Katze et al., 1986, 1988). Influenza virus mRNAs are selectively translated, promoting viral protein synthesis over translation of host mRNAs (Garfinkel & Katze, 1992; Katze & Krug, 1990). A second mechanism by which influenza counters antiviral host defences is through P58IPK, the cellular inhibitor of eIF2α kinases PKR (IFN-induced, dsRNA-activated protein kinase R) and PERK (activated during cellular stress) (Goodman et al., 2007). A third strategy is through the multifunctional activities of the viral NS1 protein.

The viral NS1 protein blocks PKR activity by binding dsRNA (Lu et al., 1995). NS1 also modulates the IFN response (García-Sastre, 2001) and, through its interaction with E3 ubiquitin ligase TRIM25, can inhibit IFN-β production by preventing RIG-I activation (Gack et al., 2009). The NS1 protein C-terminal effector domain interacts with the nuclear proteins cleavage and polyadenylation specificity factor (CPSF; Nemeroff et al., 1998) and poly(A)-binding protein II (PABII), required for 3′-end processing of cellular pre-mRNAs (Chen et al., 1999). Interaction with CPSF imposes a post-transcriptional control mechanism that prevents transport of mature mRNAs into the cytoplasm; abolishing this interaction attenuates virus replication severely (Noah et al., 2003). Additional NS1 interactions with host factors through PDZ-binding motifs located at the distal C terminus of avian NS proteins (Obenauer et al., 2006) can promote an anti-apoptotic state (Liu et al., 2010), although it appears that this may be strain-specific and dependent on the host (Zielecki et al., 2010). It is possible that NS1 has evolved redundant mechanisms to offset host defences and that different species-specific isolates emphasize one strategy over another to counter host antiviral responses effectively.

We have shown previously through functional genomic analyses that the NS genes from the 1918 pandemic virus and influenza A/PR8/34 virus suppress host innate immune responses induced in human lung A549 epithelial cells during infection (Billharz et al., 2009; Geiss et al., 2002). Billharz and coworkers demonstrated that, in the presence of the 1918 NS gene, expression of lipid-based pro-inflammatory mediators and multiple ISGs associated with hypercytokinaemia was tightly controlled. Differential cellular gene expression related to IFN and NF-κB signalling pathways was observed after infection with A/PR8/34 and a mutant influenza virus lacking the NS1 C-terminal domain (Geiss et al., 2002), suggesting that this region plays an important role in NS1 regulation of innate immune responses. NS1 modulates adaptive immune cell stimulation (Fernandez-Sesma et al., 2006). The regulatory region of the NS1 C terminus of the human H1N1 influenza A/Texas/36/91 virus isolate (Tx/91) modulates IFN responsiveness in human dendritic cells (DCs), reducing DC activation and stimulation of naive T cells (Haye et al., 2009). The impact of the NS1 C-terminal domain in all of these pathways is likely to be a combination of its inhibition of CPSF and a reduction in expression of the remaining NS1 N-terminal domain in the absence of the C terminus (Haye et al., 2009).

To unravel host pathways impacted by NS1 control of host transcriptional responses, we infected primary tracheobronchial epithelial (HTBE) cells and lung A549 epithelial cells with wild-type Tx/91, encoding an NS1 protein of 230 aa, and a mutant virus derivative expressing only the first 126 aa of the NS1 protein. Until now, transcriptional profiles determined by microarray from H1N1 influenza virus-infected HTBE cells have not been investigated. Here, we integrate global gene-expression patterns from two independent human lung culture infection models with an experimentally derived IFN-response network and demonstrate that IFN-activated antigen-presentation and immune-proteasome pathways are suppressed by NS1.

Results

Wild-type and NS1-mutant viruses replicate in A549 and HTBE human lung cells

We first established A549 and HTBE cell infection models, measuring viral NS1 mRNA and protein expression to evaluate wild-type Tx/91 and NS1-mutant (Tx/91 NS1 : 1–126) replication efficiencies. There were reduced levels of NS1 transcript for Tx/91 NS1 : 1–126 relative to Tx/91 in A549 and HTBE cells at each time point (Fig. 1a). Whilst the NS1 protein was identified readily in Tx/91-infected A549 cells, protein levels were reduced markedly to non-detectable levels in Tx/91 NS1 : 1–126-infected cells (Fig. 1b). The truncated NS1 protein has an intact dsRNA-binding domain that can be detected with rabbit polyclonal anti-NS1 antibody (Wang et al., 2000). Previous studies have shown that C-terminal truncation of the NS1 protein results in reduced NS1 protein levels compared with full-length NS1 in infected cell lysates (Haye et al., 2009; Solórzano et al., 2005). Immunofluorescence analysis of viral NP demonstrated efficient infection of both A549 and HTBE cells, although Tx/91 infection appeared to be more robust at 24 h post-infection (p.i.) in A549 cells (Fig. 1c).

Fig. 1.

Fig. 1.

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Tx/91 and Tx/91 NS1 : 1–126 infection of A549 and HTBE human lung cells. (a) NS1 transcript levels from A549 and HTBE cells infected with Tx/91 (empty bars) and Tx/91 NS1 : 1–126 (filled bars). A549 cells were infected at an m.o.i. of 2 and HTBE cells were infected at an m.o.i. of 20. Mean log10(ratio) values were calculated by using the equation 2-ΔΔCt. 18S rRNA was used as an endogenous control. (b) Viral NP and NS1 protein expression in A549 cells. Cells were infected at an m.o.i. of 2 and cell lysates were collected at 6 and 24 h p.i. (c) Immunofluorescence staining for viral NP protein in Tx/91- and Tx/91 NS1 : 1–126-infected A549 and HTBE cells. A549 cells were infected at an m.o.i. of 2 and HTBE cells were infected at an m.o.i. of 20. In infected A549 cells, FITC fluorescence corresponds to viral NP protein. Images shown for A549 infections are at 40× magnification. Detection of viral NP protein in HTBE cells is visualized with Texas red-conjugated secondary antibody; FITC fluorescence corresponds to ciliated cells. Images shown for HTBE infections are at 63× magnification.

NS1 is necessary to control production of antiviral cytokines

To measure the truncation effect of the NS1 gene on cytokine production, we used a Newcastle disease virus (NDV) growth-inhibition bioassay. Vero cells pretreated with supernatants collected from A549 cells infected with Tx/91 or Tx/91 NS1 : 1–126 viruses were infected with IFN-sensitive NDV–GFP (Park et al., 2003) and GFP fluorescence was measured. This served as an indirect measure of influenza-induced antiviral cytokine production in A549 cells. Vero cells exposed to supernatants from Tx/91-infected A549 cells exhibited high GFP fluorescence following NDV–GFP infection (Fig. 2a). In contrast, at low dilutions, pre-treatment with Tx/91 NS1 : 1–126-infected A549 supernatants resulted in low levels of GFP expression. GFP fluorescence increased with increasing dilutions of Tx/91 NS1 : 1–126-infected A549 supernatants. A similar trend was observed when a commercial preparation of IFN-β was added to Vero cells prior to NDV–GFP infection. These data suggest that Tx/91 and Tx/91 NS1 : 1–126 infections resulted in differential production of antiviral cytokines, supporting the notion that NS1 is required to suppress IFN synthesis and secretion, and confirming our previous observations (Haye et al., 2009).

Fig. 2.

Fig. 2.

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Production of antiviral cytokines is regulated by NS1. (a) A549 cells were infected with Tx/91 and Tx/91 NS1 : 1–126 viruses at an m.o.i. of 2 and, at 24 h p.i., supernatants were collected and used for an NDV–GFP growth-inhibition bioassay. Percentage GFP expression is normalized to mock. Vero cells pretreated with IFN served as a positive control. (b) Hierarchical clustering of gene expression for genes derived from the ipa canonical pathway ‘activation of IRF by pathogen-recognition receptors’. Expression is shown as the log10(ratio) for infection conditions relative to mock from A549 cells at 24 h p.i. Red indicates that the gene expression is higher than the uninfected reference. Enhanced gene-expression changes for Tx/91 NS1 : 1–126 relative to Tx/91 are grouped categorically, with grey denoting 5–1.5-fold changes in gene expression and blue denoting 10–5-fold changes in gene expression. Clustering was performed using hierarchical UPGMA (unweighted average) with Euclidean distance similarity measure in Spotfire DecisionSite. (c) IFN-β, IL-6 and NFKBIA mRNAs were analysed by qRT-PCR from infected A549 cells at 24 h p.i. Mean log10(ratio) values of biological replicates (n = 3) for each infection condition referenced to mock are shown. 18S rRNA was used as an endogenous control.

To assess NS1-mediated control of host transcriptional responses, we used microarrays to profile global gene-expression changes from Tx/91- and Tx/91 NS1 : 1–126-infected A549 (12 and 24 h p.i.) and HTBE (9.5 and 25 h p.i.) cells. Differentially expressed genes – defined as having a ≥1.5-fold change in expression – were analysed by using Ingenuity Pathways Analysis (ipa; Ingenuity Systems) to determine cellular pathways impacted by Tx/91 and Tx/91 NS1 : 1–126 viruses. As expected, IFN signalling was revealed as the most significant pathway regulated differentially across infection in both A549 and HTBE cells (Table 1). NF-κB signalling-pathway genes and several IRF and/or NF-κB target genes, including IFN-α, IFN-β and IL-6, were more highly upregulated in A549 cells infected with Tx/91 NS1 : 1–126 than in cells infected with the wild-type virus (Fig. 2b). Direct comparison of gene-expression changes differentiating the two virus groups showed >5-fold upregulation for Tx/91 NS1 : 1–126-induced IFNA8, IFNA10, IFNA5, IL-6, IFNA4 and IFN-β gene expression relative to Tx/91 (Fig. 2b, blue shading). IFN-β, IL-6 and NFKBIA transcript levels in infected A549 cells were also measured by quantitative (q)RT-PCR, confirming that Tx/91 NS1 : 1–126 infection resulted in greater expression of IFN-β and IL-6 mRNAs (Fig. 2c).

Table 1. Canonical pathways distinguishing Tx/91 and Tx/91 NS1 : 1–126 viruses in A549 and HTBE cells at each time point.

Top canonical pathways were determined using ipa of genes that were expressed differentially for each infection condition per time point relative to time-matched mock-infected cells (fold change≥1.5). Benjamini–Hochberg (B-H) multiple testing correction P-value was used to rank the significance associated with each pathway.

Cell type Time p.i. (h) Canonical pathway B-H P-value
A549 12 Interferon signalling 6.57×10−13
Role of pattern-recognition receptors in recognition of bacteria and viruses 4.04×10−9
Activation of IRF by cytosolic pattern-recognition receptors 1.31×10−6
Acute phase response signalling 1.26×10−5
Role of hypercytokinaemia/hyperchemokinaemia in the pathogenesis of influenza 1.65×10−5
24 Interferon signalling 1.87×10−6
Activation of IRF by cytosolic pattern-recognition receptors 1.98×10−6
Acute phase response signalling 1.27×10−5
Role of RIG-I-like receptors in antiviral innate immunity 1.27×10−5
Type I diabetes mellitus signalling 1.37×10−4
HTBE 9.5 Interferon signalling 2.30×10−11
Role of pattern-recognition receptors in recognition of bacteria and viruses 3.15×10−5
Activation of IRF by cytosolic pattern-recognition receptors 7.20×10−5
Prolactin signalling 1.65×10−3
Pathogenesis of multiple sclerosis 1.91×10−3
25 Interferon signalling 6.42×10−6
Activation of IRF by cytosolic pattern-recognition receptors 1.62×10−5
Aryl hydrocarbon receptor signalling 1.62×10−5
Pyrimidine metabolism 8.54×10−5
Propanoate metabolism 1.54×10−4
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Upregulation of antiviral cytokine gene expression is mediated by the NF-κB signalling pathway

As NS1 plays a central role in the suppression of host responses during virus infection, we focused on cellular pathways that uniquely differentiated the NS1 mutant virus from Tx/91, creating the ratio of expression in silico between the two virus groups. Through a network analysis, we identified pathways that were more highly induced by Tx/91 NS1 : 1–126, and highlighted gene-expression differences that were common between the A549 and HTBE culture models. At 12 h p.i., NF-κB-mediated expression of chemokine and cytokine genes was more highly induced in A549 cells infected with Tx/91 NS1 : 1–126 compared with cells infected with Tx/91 (Fig. 3a). IFN-β, CXCL10 and GPR109B (associated with G-protein-coupled receptor signalling) had the highest expression, upregulated 8-fold or greater by Tx/91 NS1 : 1–126 relative to Tx/91-induced gene expression. Next, we investigated whether the network nodes were upregulated differentially by Tx/91 NS1 : 1–126 relative to Tx/91 in infected HTBE cells at 9.5 h p.i. Genes that were upregulated by Tx/91 NS1 : 1–126 in both A549 and HTBE cells are outlined in blue in Fig. 3(a). Analysis of IFN-inducible IFIT2, IFIT3 and MX1 mRNAs by qRT-PCR showed that Tx/91 NS1 : 1–126 infection resulted in greater induction of these genes than Tx/91 infection in HTBE cells (Fig. 3b).

Fig. 3.

Fig. 3.

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Upregulation of antiviral cytokine gene expression is mediated by the NF-κB signalling pathway. (a) Cellular network analysis of induced genes differentiating Tx/91 NS1 : 1–126 and Tx/91 virus groups in A549 cells at 12 h p.i. Top ipa biological functions represented include ‘infection mechanism’, ‘infectious disease’ and ‘cell-to-cell signalling and interaction’. Gene expression from Tx/91 NS1 : 1–126-infected A549 cells relative to Tx/91-induced expression changes are shown. Red indicates upregulated gene expression. Nodes that are upregulated in A549 and HTBE cells by Tx/91 NS1 : 1–126 (referenced to Tx/91) are outlined in blue. (b) IFIT1, IFIT3 and MX1 mRNAs from infected HTBE cells were analysed by qRT-PCR at 25 h p.i. Mean log10(ratio) values of biological replicates (n = 3) for each infection condition referenced to mock are shown. 18S rRNA was used as an endogenous control.

Ubiquitin and proteasome genes are upregulated differentially by Tx/91 and Tx/91 NS1 : 1–126 viruses

The most significant cellular network at 24 h p.i. indicated a role for ubiquitin and the immune-proteasome signalling pathway during influenza virus infection of A549 cells. Similar to what was observed at 12 h p.i., NF-κB signalling was an integral part of the cellular network (Fig. 4a). NFKB2, a member of the Rel/NF-κB transcription factor complex, is connected directly to ubiquitin and 20S proteasome subunit PSMA6 by direct protein–protein interactions. The UBD gene encoding ubiquitin D was the most highly expressed node in the network, upregulated 14.6-fold in Tx/91 NS1 : 1–126-infected A549 cells relative to cells infected with Tx/91. The proteasome is a multicatalytic complex and we found that Tx/91 NS1 : 1–126 infection led to enhanced expression of several IFN-inducible proteasome-subunit genes, such as PSMB10 (MECL1) and PSMB9 (LMP2), proteasome components PSMA2 and PSMA4 (C3 and C9, respectively) and PSMA6, encoding proteasome subunit iota (Fig. 4a). The majority of genes depicted in the network diagram were induced in Tx/91 NS1 : 1–126-infected HTBE cells at 25 h p.i. (Fig. 4a, blue outline). The exception was TRIM25, which was expressed differentially between the two virus groups in HTBE cells, but was not regulated differentially in A549 cells. For an alternative visual representation, we clustered log10(ratio) gene expression for Tx/91 NS1 : 1–126-induced genes relative to Tx/91 for the genes represented in the network from both infection systems (Fig. 4b). In general, genes from Tx/91 NS1 : 1–126-infected HTBE cells were more highly induced than those from infected A549 cells.

Fig. 4.

Fig. 4.

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Ubiquitin and proteasome genes are upregulated differentially by Tx/91 and Tx/91 NS1 : 1–126 viruses. (a) Cellular network analysis of induced genes differentiating Tx/91 NS1 : 1–126 and Tx/91 virus groups in A549 cells at 24 h p.i. Top ipa biological functions represented include ‘inflammatory response’, ‘lymphoid tissue structure and development’ and ‘tissue development’. Expression values from Tx/91 NS1 : 1–126-infected A549 cells relative to Tx/91-infected cells are shown. Red indicates upregulated gene expression. Nodes that are upregulated in A549 and HTBE cells by Tx/91 NS1 : 1–126 (referenced to Tx/91) are outlined in blue. (b) Hierarchical clustering of gene-expression changes for nodes depicted in the network diagram. Expression for each group is shown as the log10(ratio) of expression for Tx/91 NS1 : 1–126-induced expression relative to Tx/91 in A549 cells at 24 h p.i. and in HTBE cells at 25 h p.i. Clustering was performed using hierarchical UPGMA (unweighted average) with Euclidean distance similarity measure in Spotfire DecisionSite.

IFN-stimulated immune-proteasome and antigen-presentation pathways are regulated by NS1

To determine the proportion of ISGs regulated differentially by Tx/91 and Tx/91 NS1 : 1–126 viruses, we queried an experimental IFN gene set generated by treating A549 cells with individual cytokines IFN-α, IFN-β and IFN-γ. This allowed us to compare biologically relevant IFN-responsive genes in the context of the infection model being investigated. Differentially expressed genes for all treatment groups were combined into a single collective IFN gene set and compared with Tx/91 NS1 : 1–126-enhanced gene expression relative to Tx/91-enhanced expression from infected A549 cells at 24 h p.i. (Fig. 5a). The 210 genes that were commonly upregulated were selected for pathway analysis in ipa, and molecules that showed direct functional relationships were examined further.

Fig. 5.

Fig. 5.

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Antigen-presentation and apoptosis pathways are highly upregulated by Tx/91 NS1 : 1–126. (a) Functional analysis of differentially expressed genes from IFN treatment and virus infection conditions. The Venn diagram shows induced genes changing ≥1.5-fold for Tx/91 NS1 : 1–126 relative to Tx/91 intersected with genes changing ≥1.5-fold for IFN-treated A549 cells relative to mock. (b) The 210 genes commonly differentially regulated were analysed in ipa and direct functional relationships between pathway nodes were examined. Nodes shaded yellow represent antigen-presentation genes, those shaded blue represent apoptosis- and complement-pathway genes, and nodes shaded green represent innate immune signalling genes.

Several distinct biological pathways emerged from the analysis, with IRF1 residing at the centre of the interaction map (Fig. 5b). The results from the functional analysis correlate IFN-responsive genes associated with antigen presentation (yellow nodes), complement and apoptosis (blue nodes) and innate immune signalling (green nodes) programmes with enhanced gene expression in Tx/91 NS1 : 1–126-infected A549 cells. IRF1 is shown to regulate PSMB9 and PSMB10 ISGs directly, signifying IRF1-mediated activation of immune-proteasome signalling (yellow-labelled nodes). Fold-change gene expression for virus infection and IFN-treatment conditions for the represented network molecules is listed in Table 2.

Table 2. Upregulated genes common between IFN-α and Tx/91 NS1 : 1–126 conditions in A549 cells.

Upregulated genes (≥1.5-fold) common between IFN-α treatment and Tx/91 NS1 : 1–126 infection in A549 cells at 24 h p.i. were uploaded into ipa and direct relationships between nodes were examined.

Functional grouping of signature genes Entrez gene ID Fold change
Tx/91 NS1 : 1–126* IFN-α†
Antigen-presentation and immune-proteasome pathways
 Beta-2-microglobulin (B2M) 567 5.52 2.12
 MHC class I, A 3105 4.47 1.85
 MHC class I, B 3106 7.92 1.82
 MHC class I, C 3107 7.18 2.02
 MHC class I, E 3133 5.14 1.73
 MHC class I, F 3134 9.18 1.85
 Proteasome subunit, beta type, 10 (PSMB10) 5699 2.08 2.16
 Proteasome subunit, beta type, 9, large multifunctional peptidase 2 (PSMB9) 5698 5.37 1.81
 Proteasome activator subunit, PA28 beta (PSME2) 5721 3.04 1.71
 Inhibitor of kappa light polypeptide gene enhancer in B cells, kinase epsilon (IKBKE) 9641 1.66 2.10
 Transporter 1, ATP-binding cassette, subfamily B (TAP1) 6890 4.50 1.75
 Transporter 2, ATP-binding cassette, subfamily B (TAP2) 6891 2.00 1.50
 Mucin 1, cell surface-associated (MUC1) 4582 1.60 1.91
 Intercellular adhesion molecule 1 (ICAM1) 3383
 Chemokine (C-X-C motif) ligand 2 (CXCL2) 2920
Complement pathway
 Complement component 1, r subcomponent (C1R) 715 5.50 2.37
 Complement component 1, s subcomponent (C1S) 716 3.37 2.23
 Complement component 3 (C3) 718 2.44 4.33
 Complement factor B (CFB) 629 35.33 2.59
 Complement factor H (CFH) 3075 4.41 1.69
 Complement factor H-related 3 (CFHR3) 10878 4.13 1.99
 Complement factor I (CFI) 3426 4.19 2.04
Apoptosis pathway
 Caspase recruitment domain family, member 16 (CARD16) 114769 12.47 3.29
 Caspase 1, apoptosis-related cysteine peptidase (CASP1) 834 42.69 2.93
 Caspase 10, apoptosis-related cysteine peptidase (CASP10) 843 1.85 1.65
 Caspase 5, apoptosis-related cysteine peptidase (CASP5) 838 2.49 1.66
 Tumour necrosis factor (ligand) superfamily member 10 (TNFSF10) 8743 7.83 1.79
Innate immune responses
 IFN regulatory factor 1 (IRF1) 3659 1.62 3.83
 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 (DDX58) 23586 27.87 1.76
 DEXH (Asp-Glu-X-His) box polypeptide 58 (DHX58) 79132 22.07 2.15
 Guanylate-binding protein 2, IFN-inducible (GBP2) 2634 3.30 1.90
 IFN-induced protein with tetratricopeptide repeats 2 (IFIT2) 3433 42.59 2.05
 IFN-induced protein with tetratricopeptide repeats 3 (IFIT3) 3437 100.00 1.52
 Nuclear factor (erythroid-derived 2), IFN-inducible (NFE2) 4778 1.78 1.95
 Tripartite motif-containing 22 (TRIM22) 10346 86.13 1.57
 Retinoic acid receptor responder (tazarotene-induced) 3 (RARRES3) 5920 9.72 1.51
 Interleukin-12A (natural killer cell stimulator factor 1), cytotoxic lymphocyte maturation factor 1, p35 (IL12A) 3592 2.15 3.88
 Mitogen-activated protein kinase kinase kinase 8 (MAP3K8) 1326 1.54 2.11
 Transglutaminase 1 (K polypeptide epidermal type I, protein-glutamine-gamma-glutamyltransferase) (TGM1) 7051 1.72 2.34
 Tumour necrosis factor (ligand) superfamily, member 13b (TNFSF13B) 10673 6.07 2.12
 CD40 molecule, TNF receptor superfamily member 5 (CD40) 958 1.61 2.17
 Endoplasmic reticulum aminopeptidase 1 (ERAP1) 51752 3.15 1.59
 Cathepsin S (CTSS) 1520 3.37 2.30
 Cadherin 1, type 1, E-cadherin (epithelial) (CDH1) 999 1.65 1.80
 GATA-binding protein 3 (GATA3) 2625 1.67 1.57
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*

Fold change in gene expression is referenced against Tx/91-infected cells.

†

Fold change in gene expression is referenced against mock-infected cells.

IFN-γ activates MHC class I antigen presentation and stimulates reconfiguration of the constitutive proteasome to the immune proteasome. We used hierarchical clustering to evaluate differentially expressed genes related to antigen presentation and immune-proteasome activation in virus-infected cells (Fig. 6a). In both A549 and HTBE cells, Tx/91 NS1 : 1–126 induced greater gene expression than Tx/91. In A549 cells, Tx/91 infection resulted in enhanced gene expression; however, a similar induction was not observed in Tx/91-infected HTBE cells. This indicates that there may be virus-induced differences in pathway activation between the two cell types. A549 cells treated with IFN-γ or infected with Tx/91 NS1 : 1–126 showed similarly enhanced gene-expression levels for antigen-presentation and immune-proteasome pathway genes (Fig. 6b). The Tx/91 virus resulted in induced gene expression, albeit to a lesser extent. The majority of genes were upregulated <5-fold, with the exception of PSMB8, TAP1 and PSMB9. Taken together, these data suggest that IFN-responsive antigen-presentation and immune-proteasome pathways are upregulated during influenza virus infection and that NS1 may play a role in modulating these host innate immune responses.

Fig. 6.

Fig. 6.

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Immune-proteasome pathway genes show differential regulation in the two model systems. (a) Hierarchical clustering of differentially expressed antigen-presentation and immune-proteasome genes are represented for A549 and HTBE cells at 24 and 25 h p.i., respectively. Gene expression from each cell type is shown as the log10(ratio) for Tx/91 NS1 : 1–126- and Tx/91-induced transcriptional changes relative to time-matched mock-infected cells. Red indicates that the gene expression is higher than the uninfected reference; black indicates that gene expression is unchanging from the reference sample. Clustering was performed using hierarchical UPGMA (unweighted average) with Euclidean distance similarity measure using an average value ordering function in Spotfire DecisionSite. (b) Fold-change expression for IFN-γ treatment (black bars), Tx/91 (blue bars) and Tx/91 NS1 : 1–126 (grey bars) infection conditions for genes depicted in the heat map. Fold-change gene expression is relative to time-matched mock-infected A549 cells.

Discussion

This is the first study to use primary human lung epithelial cells in a genomic analysis of H1N1 influenza A virus-regulated host transcriptional responses. In particular, we sought to evaluate IFN-responsive pathways impacted by viral NS1, integrating gene-expression data from independent infection culture models and querying an IFN-response gene network to gauge the proportion of genes induced differentially by human isolate Tx/91 and an NS1-mutant virus. The NS1-mutant virus is genetically similar to Tx/91, but encodes only the first 126 aa of the NS1 protein. Enhanced expression of antigen-presentation and immune-proteasome pathway genes was observed for Tx/91 NS1 : 1–126-infected cells relative to cells infected with the wild-type Tx/91 virus. Our findings indicate that NS1 suppresses IFN-stimulated MHC class I antigen presentation and immune-proteasome activation during influenza virus infection.

Type I IFN production is critical to host innate immune defences. Influenza virus induces RIG-I signalling and activation of NF-κB, which is required for assembly of the IFN-β enhancesome and IFN-β gene expression (Thanos & Maniatis, 1995). There are several means by which NS1 counters host antiviral defence mechanisms. NS1 prevents RIG-I activation by (i) targeting E3 ubiquitin ligase TRIM25 (Gack et al., 2009), (ii) binding and sequestering dsRNA through its N-terminal RNA-binding domain (Chien et al., 1997, 2004; Guo et al., 2007; Liu et al., 1997; Mibayashi et al., 2007; Opitz et al., 2007; Pichlmair et al., 2006), and (iii) possibly through direct protein–protein interaction (Mibayashi et al., 2007; Pichlmair et al., 2006). From our analysis, we found that NF-κB-mediated gene expression of IFN-β and chemokine genes encoding MIP1α and RANTES was upregulated to a greater extent in response to Tx/91 NS1 : 1–126 compared with wild-type virus. Hence, mutation in the NS gene leads to enhanced antiviral gene expression of NF-κB-regulated genes during influenza virus infection. This reinforces our previous genomic findings from PR8 NS1 : 1–126-infected A549 cells (Geiss et al., 2002), further supporting the hypothesis of NS1 prevention of NF-κB activation (Wang et al., 2000).

NS1 inhibition of antiviral responses is strain-specific and it is likely that certain viruses have evolved distinct mechanisms to counter host defences. For instance, PR8 NS1 is a potent inhibitor of IRF3 activation, whilst Tx/91 NS1 only partially reduces IRF3 dimerization (Kochs et al., 2007). The CPSF-binding domain located in the NS1 C-terminal effector domain is implicated in general inhibition of cellular gene expression (Nemeroff et al., 1998); molecular determinants destabilizing the interaction of NS1 with CPSF30 can result in inefficient control of host responses, as demonstrated with the recent H1N1 pandemic influenza virus (Hale et al., 2010). Truncation of the C terminus has two major consequences for the regulatory functions of NS1. First, the NS1 mutant is expressed poorly in virus-infected cells. Previous studies have reported low NS1 : 1–126 protein expression for Tx/91 H1N1 (Haye et al., 2009) and H3N2 (Solórzano et al., 2005) mutant viruses. It is known that the antiviral response is more profound in the absence of the NS1 protein (Geiss et al., 2002). Due to low NS1 protein levels, Tx/91 NS1 : 1–126-induced host transcriptional responses would probably mirror gene-expression profiles of a Tx/91 NS1-deletion virus. Second, the truncated NS1 lacks the CPSF-binding domain. The distal C termini of some NS proteins contain PDZ-ligand motifs (Obenauer et al., 2006), which may perturb a broad range of cellular pathways through their signalling activities (Jackson et al., 2008), although the significance of NS1 interactions with PDZ-binding proteins is still being elucidated. The observed effects in this study are probably due to a combination of low NS1 expression and the lack of the C-terminal effector domain.

We have shown that Tx/91 NS1 : 1–126 infection of A549 and HTBE cells results in a robust upregulation of ISGs related to antigen-presentation and immune-proteasome pathways, including IFN-inducible HLA class I molecules and immune-proteasome subunits MECL1/β2i (PSMB10) and LMP2/β1i (PSMB9). The immune proteasome promotes degradation of protein aggregates (Seifert et al., 2010), and activation of these pathways during H1N1 influenza infection may accelerate T-cell responses by enhancing presentation of virus-derived epitopes by MHC class I molecules (de Graaf et al., 2011). LMP2/7-deficient mice, however, effectively mount CD8+ T-cell responses to lymphocytic choriomeningitis virus infection (Nussbaum et al., 2005). Thus, whilst notable changes in expression for these pathway genes are observed in our system, the contribution of the immune proteasome toward host antiviral defences warrants further investigation. Nevertheless, the appearance of the immune proteasome in Tx/91 NS1 : 1–126-infected cells suggests that NS1 may suppress reconfiguration of the proteasome during infection, possibly through IRF1 activity.

Regulatory IFN-γ activation-site elements of immune-proteasome subunits, including LMP2, are recognized by IRF1 and require IFN-γ for induced gene expression (Chatterjee-Kishore et al., 1998). IRF1-mediated transcription probably results in this IFN-γ signature in Tx/91 NS1 : 1–126-infected cells, suggesting that NS1 is required for suppression of IRF1-mediated transcription. Adenovirus E1A has been shown to downregulate LMP2 transcription by interfering with IRF1 activity (Chatterjee-Kishore et al., 2000). Whilst more targeted mechanistic studies would be required to elucidate the exact cellular interactions by which NS1 achieves this, we can speculate that NS1 may interfere directly with IRF1 promoter activity to suppress immune-proteasome activation. These potential targets of IRF1 activity may be investigated with ChIP-chip technology, which has been used to characterize a role for IRF1 in DNA damage (Frontini et al., 2009).

In human epithelial cells infected with the Tx/91 NS1 : 1–126 virus, the remarkable activation of antiviral genes, and of NF-κB- and IRF-regulated immune factors involved in antigen presentation and immune activation, suggests that C-terminally truncated NS1 influenza viruses might be potent stimulators of innate and adaptive immune responses in vivo. In fact, we found previously that Tx/91 NS1 : 1–126 is a strong stimulant of adaptive immune responses, inducing high levels of IFN-γ in human DCs and increased stimulation of T cells (Haye et al., 2009). Tx/91 NS1 : 1–126 is also attenuated and highly immunogenic in macaques (Baskin et al., 2007). Vaccination of pig-tailed macaques with the attenuated NS1 mutant virus protected animals when challenged with A/Texas/36/91, supporting its efficacy as a live-attenuated vaccine. Animals that received the Tx/91 NS1 : 1–126 vaccine showed lower levels of virus replication in the lungs, milder signs of illness and higher production of influenza virus-specific CD4+ T cells than animals infected with the wild-type Tx/91 virus. Thus, our present study provides a mechanistic explanation for the potent immunogenicity of influenza viruses lacking the C-terminal region of the NS1 gene.

Methods

Cell maintenance and virus preparation.

A549 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10 % FBS and 50 U penicillin G ml−1, 50 µg streptomycin sulfate ml−1 (Gibco) at 37 °C. HTBE cells were cultivated on 24 mm diameter Transwell polyester-clear filters (0.4 µm pores; Corning) coated with human placental collagen type I (Sigma) for 3 weeks in air–liquid interface, as described previously (Steel et al., 2008). The apical cell surface was washed with PBS twice a week to remove accumulated mucus. The recombinant human H1N1 A/Texas/36/91 virus (Tx/91) and an NS1-truncated mutant virus derived from Tx/91 that encodes the first 126 aa of the NS1 protein (Tx/91 NS1 : 1–126) were grown in 7-day-old embryonated chicken eggs at 32 °C for 3 days (SPAFAS; Charles River Laboratories), as described previously (Haye et al., 2009). Allantoic fluid was collected and virus titres were determined by plaque assay and immunofluorescence staining of NP in infected Madin–Darby canine kidney or Vero cells.

HTBE and A549 cell culture infections.

Prior to infection, HTBE cells were washed ten times with growth medium (bronchial epithelial growth medium/DMEM, 1 : 1 and supplements) and infected with 4×106 p.f.u. Tx/91 or Tx/91 NS1 : 1–126 viruses per filter (m.o.i. of 20) for 1 h at 37 °C. Three replicate wells were infected for each condition per time point. Allantoic fluid was used for mock treatments. Following infection, cells were washed once and incubated in growth medium covering the apical surface and feeding medium contacting the basal layer. At 9.5 and 25 h p.i., cells were either fixed for immunofluorescence or resuspended in TRIzol reagent (Invitrogen) for total RNA extraction. A549 cells were infected with Tx/91 or Tx/91 NS1 : 1–126 viruses at an m.o.i. of 2 for 1 h at 4 °C. Following infection, cells were washed and complete growth medium was added. Three replicate wells were infected for each condition per time point. Allantoic fluid was used for mock treatments. Cells were lysed in solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5 % sarcosyl, 0.1 M β-mercaptoethanol) at 12 and 24 h p.i. Total RNA was extracted and processed for microarray.

Immunofluorescence staining for viral NP in A549 and HTBE cells.

A549 cells were grown on glass coverslides for immunofluorescence analysis. At 12 and 24 h p.i., growth medium was removed and cells were washed twice with PBS and 3 % paraformaldehyde in PBS added for 20 min. Following fixation, cells were permeabilized with 1× PBS containing 0.5 % NP-40 and 0.01 % NaN3, and then blocked in 1× PBS [0.5 % NP-40, 0.01 % NaN3 containing 10 % FBS] for 30 min. FITC-conjugated mouse monoclonal anti-NP antibody (1 : 200) (Biodesign International) was incubated with cells overnight at 4 °C. Cells were washed three times in 1× PBS containing 1 % BSA and coverslips were mounted onto glass slides by using ProLong antifade (Molecular Probes). Slides were imaged by using a Nikon Eclipse E600 microscope.

HTBE cells grown on Transwell filters were infected as described above and, at 9.5 and 25 h p.i., fixed in 4 % formaldehyde in PBS for 30 min, washed three times with PBS and incubated in 0.5 % Triton X-100 for 30 min. Following permeabilization, cells were washed three times with PBS and blocked in 5 % goat serum for 20 min. Polyclonal rabbit anti-NP (1 : 500) and monoclonal anti-α-tubulin–FITC (1 : 100) antibodies (Sigma) diluted in 5 % goat serum were incubated with cells, and then secondary antibodies (1 : 250-diluted anti-rabbit–Texas red) were used, together with 1 : 1000 DAPI, to stain cellular nuclei. Cells were mounted on glass slides using ProLong antifade (Molecular Probes) and imaged by using a Zeiss LSM 510 confocal microscope.

Immunoblot analysis of A549 cells.

A549 cells grown in 24-well plates were infected at an m.o.i. of 2 and, at 6 and 24 h p.i., medium was removed and cells were lysed in 100 µl SDS-PAGE loading buffer. Cellular lysates were boiled for 5 min, resolved on SDS-PAGE gels (4–20 %; Bio-Rad) and transferred to nitrocellulose membranes. Membranes were incubated with polyclonal anti-NP antibody (1 : 500) and then polyclonal anti-NS1 antibody (1 : 500) after stripping the membrane. Secondary HRP-conjugated goat anti-rabbit antibody was added and proteins were detected by enhanced chemiluminescence.

Expression microarray analysis.

Total RNA from three replicate wells for each infection condition per time point was pooled in equivalent amounts and analysed by microarray. As a reference, RNA from time-matched, mock-infected replicates was pooled and hybridized with pooled RNAs for each infection condition. For all microarray experiments, Cy3- and Cy5-labelled cRNA probes were generated by using an Agilent Low RNA Input Linear Amplification kit. Microarray slide hybridization was performed with Agilent human 22K microarrays according to the manufacturer’s instructions. Each microarray experiment was done with four technical replicates by reverse dye hybridization for experimental and reference samples. Gene-expression data were uploaded into the Rosetta Resolver System (Rosetta Biosoftware) for analyses and Spotfire DecisionSite 9.1 for heatmap construction. All primary expression microarray data are available at http://viromics.washington.edu/, in accordance with the proposed minimum information about a microarray experiment (MIAME) standards (Brazma et al., 2001).

Differentially expressed genes were uploaded into ipa (Ingenuity Systems; http://www.ingenuity.com) for functional analysis. This software examines RNA-expression data in the context of known biological functions and pathways, mapping each gene identifier in a dataset to its corresponding molecule in the Ingenuity Pathways Knowledge Base (ipkb). For all analyses, ipa-generated P-values were adjusted by using Benjamini–Hochberg multiple testing correction. The focus molecules within ipa-generated diagrams represent nodes, and the edges that define the biological relationship between two nodes are represented as a line. All edges are supported by at least one published reference or from canonical information stored in the ipkb. An arrow pointing between nodes signifies regulation and an arrow pointing back to the same node indicates self-regulation.

Generation of an IFN-responsive gene network in A549 cells.

A549 cells were treated individually with IFN-α (10 ng ml−1; Sigma I4276), IFN-β (500 U ml−1; Sigma I4151) and IFN-γ (10 ng ml−1; Sigma I32265). At 12 and 24 h post-treatment, cells were harvested (n = 3 per condition) and total RNA was extracted and analysed by microarray. Probe labelling and microarray slide hybridization for pooled replicates for each treatment per time point were performed using a Human Genome CGH Microarray (G4447A; Agilent Technologies).

Real-time qRT-PCR.

Real-time qRT-PCR was performed to validate infections and transcriptional findings determined by microarray. Total RNAs were treated with DNase (Ambion, Inc.) and cDNAs were generated by using a QuantiTect reverse transcription kit (Qiagen). Custom-designed TaqMan gene-expression assays for influenza NS1 target sequence were ordered from Applied Biosystems. TaqMan experiments were performed on the ABI 7500 Real-Time PCR System platform and each sample was run in quadruplicate. rRNA (18S) was used to normalize quantification of each target within A549 and HTBE cells. Quantification of normalized targets was performed using the 2−ΔΔCt calculation (Livak & Schmittgen, 2001).

NDV–GFP growth-inhibition bioassay.

To evaluate the truncation effect of the NS gene on cytokine production, we used an NDV growth-inhibition bioassay as described previously (Park et al., 2003; Quinlivan et al., 2005; Solórzano et al., 2005). A549 cells grown in 24-well plates were infected with Tx/91 or Tx/91 NS1 : 1–126 viruses at an m.o.i. of 2. At 24 h p.i., supernatants were collected, dialysed against low pH (50 mM glycine, pH 2) and then 2-fold dilutions of supernatants were added to Vero cells grown in a 96-well black microtitre plate (Costar). The next day, pretreated Vero cells were infected with NDV–GFP (m.o.i. of 100). As a positive control, dilutions of recombinant human IFN-β (333 U) were added to Vero cells. Twenty-four hours later, fluorescence was measured by using a FLUOstar OPTIMA plate reader (BMG Labtechnologies).

Acknowledgements

We thank Sara Kelly and Jean Chang for technical assistance associated with gene-expression profiling and Richard Cadagan for excellent technical assistance. This study was supported by Public Health Service grant P01 AI058113 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, to M. G. K. and A. G.-S. This study was also partly supported by NIAID grants R01AI046954, U01AI070469 and U19AI083025, and by CRIP, an NIAID-funded Center of Excellence for Influenza Research and Surveillance (HHSN266200700010C), to A. G.-S. A. G.-S. owns equity in and receives financial compensation for providing consulting services and for serving on the advisory board of Vivaldi BioScience, a biotechnology company that develops influenza vaccines based on NS1-modification technology. Mount Sinai School of Medicine has received compensation from Vivaldi in return for a licence to certain technology, the value of which may be affected by the outcome of this study.

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