The NS1 Protein of a Human Influenza Virus Inhibits Type I Interferon Production and the Induction of Antiviral Responses in Primary Human Dendritic and Respiratory Epithelial Cells

Viruses and cells.TX WT, TX 1-99, and TX 1-126 influenza viruses were generated in Peter Palese's laboratory by reverse genetics similarly to other influenza virus mutants (5, 54, 63, 66). Recombinant TX WT virus as well as TX 1-99 and TX 1-126 viruses, encoding truncated NS1 proteins of 99 and 126 amino acids, respectively, were grown in 7-day-old embryonated chicken eggs (SPAFAS; Charles River Laboratories). Influenza virus deltaNS1 was generated by reverse genetics from influenza virus PR8 as previously described (10). Influenza virus A/NY/55/04 (NY), influenza virus A/WYM/3/03 (WYM), and PR8 virus were grown in 9-day-old eggs. Influenza virus deltaNS1 was grown in 6-day-old embryonated eggs. All influenza viruses were titrated by influenza virus nucleoprotein (Flu NP) immunostaining using MDCK or Vero cells. For influenza virus titrations, 2.5 μg/ml trypsin was included in the infection medium.

MDCK, Vero, and A549 cells were grown in tissue culture medium, Dulbecco's modified Eagle's medium (DMEM; Invitrogen), supplemented with 10% fetal calf serum (HyClone), 1 mM sodium pyruvate (Invitrogen), 2 mM l-glutamine (Invitrogen), and 50 μg/ml gentamicin (Invitrogen). All cells were grown at 37°C in 7% CO₂.

Isolation and culture of human DCs.Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation (Histopaque, Sigma Aldrich) from buffy coats of healthy human donors (Mount Sinai Blood Donor Center and New York Blood Center) as previously described (15). Briefly, CD14⁺ cells were immunomagnetically purified using anti-human CD14 antibody-labeled magnetic beads and iron-based MiniMACS LS columns (Miltenyi Biotech). After elution from the columns, cells were plated (0.7 × 10⁶ cells/ml) in DC medium (RPMI medium [Invitrogen], 10% fetal calf serum [HyClone] or 4% human serum [Cambrex/Lonza], 100 U/ml penicillin, and 100 μg/ml streptomycin [Pen/Strep; (Invitrogen]) supplemented with 500 U/ml human granulocyte-macrophage colony-stimulating (Peprotech), and 1,000 U/ml human interleukin-4 (hIL-4; Peprotech) and incubated for 5 to 6 days at 37°C.

Infections and treatments of DCs.After 5 to 6 days in culture, DCs were infected with influenza viruses at a multiplicity of infection (MOI) of 0.5, 1, or 2 for 60 min in serum-free RPMI medium. Cells were then plated in DC medium (described above) at 1 × 10⁶ cells/ml maintained in culture for different time periods, depending on the experiment.

HTBE cell cultures.Cryopreserved normal human tracheo-bronchial epithelial (HTBE) cells were purchased from Cambrex and cultivated according to the manufacturer's instructions and a previously described protocol (21). Briefly, cells were passaged twice in bronchial epithelial growth medium (BEGM) containing necessary supplements and growth factors (Cambrex) and seeded onto Transwell-Clear Permeable filters (24-mm diameter and 0.4-mm pore size; Millipore) at a density of 1.4 × 10⁵ cells/filter. Before the seeding step, filters were coated with collagen I derived from human placenta (BD Biosciences). Cells were grown submerged in a 1:1 mixture of DMEM and BEGM supplemented with growth factors for 1 week. Then, medium from the apical surface was removed, and cells were maintained in an air-liquid interface for 3 weeks. When cells are maintained in an air-liquid interface for long periods of time, they differentiate into different cell types present in the mucosal surface of the respiratory tract. The differentiation status of cells was routinely monitored by immunofluorescence of cells with anti-β-tubulin antibodies (Sigma) specific for ciliated cells.

Infections of HTBE cells.HTBE cells were infected with either TX WT or TX 1-126 viruses at an MOI of 2 or mock infected for 1 h at 37°C. Prior to infection, cells were washed 10 times with growth medium. After infection cells were washed once, and medium (BEGM-DMEM) was left to cover apical surfaces of cells as well as the basal compartment of wells. At 9.5 and 25 h postinfection, medium from apical and basal compartments was collected, and cell layers were either resuspended in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis buffer for analysis by Western blotting or fixed for immunostaining. Harvested supernatants from apical surfaces were used to determine viral titers. In order to detect the presence of IFN-α/β, a bioassay was utilized as described previously (61). Briefly, medium from the basal compartment of infected HTBE cells was dialyzed against low-pH buffer (50 mM glycine, pH 2) to inactivate virus, and neutral pH was restored by subsequent dialysis against phosphate-buffered saline (PBS). Vero cells were then incubated with different dilutions of medium from infected HTBE cells for 24 h and infected with Newcastle disease virus expressing green fluorescent protein (NDV-GFP), which is known to be sensitive to the action of IFN. Twenty-four hours later, the intensity of green fluorescence was measured. As controls, Vero cells were treated with a known quantity of IFN-β (Calbiochem). To plot the data, we considered the fluorescence level of cells incubated with medium from mock-infected HTBE cells to be 100%. In other experiments, HTBE cells were infected at an MOI of 0.001 for 1 h at 37°C, virus was removed, and cells were washed once with medium and continued to be incubated in the air-liquid interface. At various time points, medium was added to apical surfaces for 30 min and harvested for virus titration.

Capture ELISA.Capture enzyme-linked immunosorbent assays (ELISAs) for IL-6, tumor necrosis factor alpha (TNF-α) (R & D systems), IFN-α, and IFN-β (Biosource) were used according to the manufacturer's instructions to quantify the cytokines and chemokines in the DC supernatants. Plates were read in an ELISA reader from Biotek instruments.

Flow cytometry.Cells were stained with phycoerythrin-linked CD86 according to the manufacturer's instructions (Beckman Coulter and BD-Pharmingen), and expression was determined by flow cytometry. For Flu NP staining, cells were fixed in 3% formaldehyde for 15 min on ice and then permeabilized in 0.1% saponin in PBS for 30 min on ice. Cells were then incubated with rabbit polyclonal anti-Flu NP for 1 h, and then goat anti-rabbit secondary antibody conjugated to Alexa 488 (Invitrogen) for 1 h. Cells were washed twice with 0.05% fetal bovine serum-PBS twice between each step. Data were analyzed using Flowjo software.

Total RNA isolation and extraction and generation of cDNA from human DCs.Samples of 1 × 10⁵to 5 × 10⁵ DCs were either infected with virus or mock infected, and RNA was isolated and DNase treated using an Absolutely RNA reverse transcription-PCR (RT-PCR) miniprep kit (Stratagene). RNA was quantified using a Nanodrop spectrophotometer (Nanodrop Technologies). The yields of RNA were approximately 50 to 100 μg/ml.

Quantitative real-time PCR.Quantitative RT-PCR (qRT-PCR) was performed according to a previously published protocol using SYBR Green in an ABI 7900 HT protocol(15, 73). Each transcript in each sample was assayed in duplicate, and the mean threshold cycle of three housekeeping genes (RSP11, β-actin, and α-tubulin) was used to calculate the relative copy number for each gene.

Allospecific stimulation of CD4 T cells by DCs.DCs either left untreated or infected with different recombinant influenza viruses were mixed together with allogeneic peripheral blood mononuclear cells from buffy coats and cultured for 4 days in 96-well plates at a ratio of 1:5 (10⁶ DCs/ml to naive T cells). Supernatants from the cocultures were tested by ELISA for IFN-γ release at different times of culture (eBioscience).

Western blotting and antibodies.Samples of 10⁶ cells were lysed in 100 μl of lysis buffer (150 mM NaCl, 25 mM Tris-HCl [pH 7.4], 2 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) or 100 μl of Phosphosafe buffer (Calbiochem). Cell lysates were centrifuged (14,000 × g for 10 min) to remove cellular debris and boiled for 5 min in SDS sample buffer containing 1 mM dithiothreitol. Protein lysates were resolved on 4 to 15% SDS-polyacrylamide gel electrophoresis gradient gels (Bio-Rad), transferred to nitrocellulose membranes, and probed with rabbit polyclonal anti-NP and anti-NS1 as previously described (63) and with mouse monoclonal anti-M1 and TX WT-specific anti-HA (Mount Sinai Hybridoma center), anti phospho-IRF3 (S396) (Cell Signaling), anti-phospho-NF-κBp65/RelA (S536) (Cell Signaling), anti phospho-c-Jun (S73) (Cell Signaling), anti-NF-κBp65/RelA (Cell Signaling), anti-c-Jun (Cell Signaling), anti-IRF3 (Cell Signaling), and anti-actin (Santa Cruz). Immunoreactive bands were detected using horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit immunoglobulin G (Bio-Rad) and enhanced chemiluminescence (Bio-Rad).

Generation of recombinant TX WT NS1 C-terminal deletion viruses.We aimed to determine whether NS1 proteins in several influenza virus strains were also associated with decreased inhibition of IFN-related gene expression and decreased levels of DC activation in human influenza virus-infected DCs. Two NS1 mutant viruses in the TX WT background were generated, TX 1-126 and TX 1-99, that encode C-terminally truncated NS1 proteins of 126 and 99 amino acids, respectively, in contrast to the 230-amino-acid-long NS1 protein of the corresponding TX WT virus (as previously described in Materials and Methods). Rescue of TX 1-126 virus from plasmids was previously reported (5). TX 1-99 virus was rescued by using a truncated NS gene similar to that of the TX 1-126 virus except that the NS1 stop codon was inserted after amino acid residue 99.

Reduced viral protein expression in NS1 mutant virus-infected lung epithelial cell lines.In order to investigate the impact of NS1 function during infection of human respiratory epithelial cells, the primary target for influenza virus replication in humans, we first determined the levels of viral protein synthesis in the human A549 lung alveolar cell line. TX 1-99 and TX 1-126 virus-infected A549 cells showed similar levels of NP protein and reduced M1 protein levels compared to levels seen in TX WT virus-infected A549 cells (Fig. 1A). However, HA protein and the respective truncated NS1 polypeptides of the mutant viruses were poorly expressed and only barely detectable in overexposed blots compared to the clearly visible levels observed in TX WT-infected cells (Fig. 1A).

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FIG. 1.

Replication of wild-type and NS1 mutant human influenza viruses in human respiratory cells. (A) A total of 1.0 × 10⁶ A549 cells were mock infected (NI) or infected with TX WT, TX 1-99, and TX 1-126 viruses at an MOI of 2 for 10 h. Total cell lysates were made, and Western blotting was performed for viral HA, NP, M1, and NS1 proteins. NS1 WT, TX WT NS1; NS 1-126, TX 1-126 NS1; NS1 1-99, TX 1-99 NS1. Actin is shown as a loading control. Results are from a representative experiment. HTBE cells are permissive to infection with human influenza viruses and secrete type I IFN. (B) Primary HTBE cells were infected with TX WT and TX 1-126 viruses at an MOI of 2 at the times shown; cells were fixed and stained with anti-Flu NP antibodies and anti-β-tubulin antibodies conjugated with fluorescein isothiocyanate (ciliated cells). Infected cells were visualized with secondary antibodies coupled to Texas Red. (C) Levels of NP and NS1 proteins in the lysates from infected HTBE cells at 25 h postinfection as detected by Western blotting. Triplicate lanes represent the same sample loaded in triplicate as a loading control. Virus release over time was determined in HTBE cells infected with TX WT and TX 1-126 viruses at an MOI of 2 (D) or an MOI of 0.001 (E). Error bars are the standard deviations derived from triplicate samples of one experiment. (F) Release of IFN-α/β from HTBE cells infected with TX WT and TX 1-126 viruses at an MOI of 2. Antiviral activity was detected using a Vero cell bioassay using NDV-GFP virus. The GFP fluorescence was measured. The level of GFP fluorescence in Vero cells incubated with medium from mock-infected HTBE cells was considered as 100%. A positive control using recombinant IFN is shown on the right. Error bars are the standard deviations derived from triplicate samples of one experiment. Results are from a representative experiment. hpi, hours postinfection.

Replication of TX 1-126 NS1 mutant virus is attenuated in primary HTBE cells.For the next series of studies, we compared one of the NS1-truncated mutant viruses, TX 1-126, with TX WT virus in differentiated HTBE cells grown in an air-liquid interface as a surrogate system of the human upper respiratory epithelium. Upon infection at an MOI of 2, TX 1-126 virus replicated less efficiently than TX WT virus, as confirmed by different methods. Immunofluorescence of infected cells (Fig. 1B) showed equal levels of infection by both viruses at 9.5 h postinfection but a much lower number of infected cells in the case of TX 1-126 virus at 25 h. Western blotting of HTBE lysates at 25 h postinfection was consistent with the immunofluorescence data (Fig. 1C). A truncated 126-amino-acid NS1 protein in infected HTBE cells was not detected, which is indicative of low expression of the truncated NS1 in these cells. We also detected lower virus titers in apical supernatants from TX 1-126 virus-infected HTBE cells at 25 h postinfection (Fig. 1D) than in TX WT-infected HTBE cells. Differences in viral replication between TX 1-126 and TX WT virus in HTBE cells were even more apparent when cells were infected at an MOI of 0.001, which allowed for multicycle replication of the viruses. At 24 and 48 h postinfection, virus titers in the supernatants were determined, and the TX WT virus reached titers around 10⁶ at 48 h postinfection. However, TX 1-126 viruses were detected at very low levels at 24 h postinfection and not detected at all at 48 h postinfection (Fig. 1E). Attenuation of replication of this virus in human primary respiratory epithelial cells is likely caused by an impaired ability to counteract type I IFN production in the host cells due to NS1 truncation. To confirm this hypothesis, we measured IFN-α/β in the medium of HTBE cells infected with TX WT and TX 1-126 viruses using a bioassay (as described in Materials and Methods). Indeed, increased levels of IFN-α/β in the medium of HTBE cells infected with TX 1-126 virus were observed, as demonstrated by antiviral activity against NDV-GFP replication in Vero cells (Fig. 1F). This result indicates that the TX 1-126 virus is defective in blocking type I IFN production in human primary respiratory epithelial cells, which are believed to be the main targets for human influenza virus replication in vivo.

Characterization of viral protein expression in wild-type and NS1 mutant influenza virus-infected DCs.We then infected DCs, which support replication of influenza viruses but do not produce infectious particles (human data not shown, but for mouse DCs see reference35) and examined viral protein expression by Western blotting. Levels of NP in NS1 mutant virus-infected DCs were equal to that of TX WT virus-infected DCs (Fig. 2A). But in DCs infected with the TX 1-99 and TX 1-126 mutant viruses, a significant reduction in the levels of HA, M1, or NS1 proteins compared to TX WT virus-infected DCs was seen (Fig. 2A and B). Thus, truncations in the NS1 protein strongly impact the expression levels of other viral proteins in infected human DCs. To examine the kinetics of viral protein synthesis in human DCs, we performed a time course experiment examining viral protein levels at 4 h, 8 h, and 16 h postinfection (Fig. 2B). TX WT, TX 1-99, and TX 1-126 viruses showed similar rates of expression for NP protein at all time points tested (Fig. 2B). NS1 and M1 proteins were detected as early as 4 h postinfection in TX WT-infected DCs. Again, DCs infected with TX 1-99 or TX 1-126 produced drastically reduced levels of M1 and nearly undetectable amounts of their respective NS1 truncated proteins (Fig. 2B). When expression of the late viral protein HA was examined in DCs infected with TX WT, this protein was detectable as early as 8 h postinfection, and the levels were increased at 16 h postinfection. However, DC infection with TX 1-99 and TX 1-126 showed poor expression of HA even at 16 h postinfection (Fig. 2B). In summary, the NS1 C-terminal deletion mutants are compromised in their ability to express late viral proteins (such as HA and M1) while expressing normal levels of early viral proteins (such as NP) in human DCs. While several viral proteins are affected by the presence of an intact NS1, the truncation mutants are able to produce early proteins in human DCs, which could still be presented to T cells by the infected DCs, thus contributing to the initiation of adaptive immunity against the virus in vivo.

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FIG. 2.

Viral protein expression in influenza virus-infected human DCs. (A) Western blot of protein lysates from infected DCs. A total of 1.0 × 10⁶ human DCs were infected with TX WT, TX 1-99, and TX 1-126 viruses at an MOI of 0.5 or 1 for 18 h. Western blots are shown for viral NP and NS1 proteins. (B) Kinetics of viral protein expression in infected DCs. A total of 2.0 × 10⁶ human DCs were infected with TX WT, TX 1-99, and TX 1-126 viruses at an MOI of 2 for 4, 8, and 16 h. Total cell lysates were made, and Western blotting was performed for viral HA, NP, M1, and NS1 proteins. Levels of cellular actin are shown as a loading control. Results are representative of two different donors. NI, not infected; hpi, hours postinfection; NS1 WT, TX WT NS1; NS 1-126, TX 1-126 NS1; NS1 1-99, TX 1-99 NS1.

Human DCs infected with human influenza virus NS1 mutants show increased activation of IRF3.Activation of RIG-I leads to induction of type I IFN and secretion of other proinflammatory cytokines by activation of NF-κB/RelA, c-Jun, and IRF3 (51, 60). It has been demonstrated that phosphorylation of IRF3 at S396 corresponds to its activation (59). Since the RIG-I pathway and DC activation can be inhibited by the influenza virus protein NS1 (15, 42, 48), we sought to determine how effective TX 1-99 and TX 1-126 mutant viruses would be at stimulating these processes. When phosphorylation and activation of IRF3 (at residue S396) were examined, an increase in phosphorylation of this transcription factor in TX 1-99- and TX 1-126-infected DCs was observed compared to TX WT-infected conventional DCs at 4 and 8 h postinfection (Fig. 3A). These results are consistent with the proposed role of the carboxy-terminal region of the NS1 protein in promoting NS1 dimerization and enhancing its ability to prevent the activation of transcriptional factors (4). These results strongly suggest that the inability of the NS1 truncation mutants to inhibit IRF phosphorylation contributes to their enhanced immunogenicity while conferring on them an attenuated phenotype.

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FIG. 3.

Increased type I IFN and IFN-responsive gene expression in influenza virus-infected human DCs correlates with increased IRF3 activation. (A) Human DCs were mock infected (NI) or infected with TX WT or with NS1 C-terminal deletion mutant virus TX 1-99 or TX 1-126 at an MOI of 2 for 4 and 8 h. Total cell lysates were made, and Western blotting was performed for phosphorylated IRF3 and total IRF3. (B) Human DCs were mock infected (NI) or infected with TX WT or with NS1 C-terminal deletion mutant virus TX 1-99 or TX 1-126 at an MOI of 2 for 2, 4, 6, 8, and 16 h. Total RNA was isolated from 5 × 10⁵ cells, and qRT-PCR was performed for IFN-α and IFN-β mRNA. (C) A total of 2 × 10⁶ human DCs were mock infected (NI) or infected with TX WT or with NS1 C-terminal deletion mutant virus TX 1-99 or TX 1-126 at an MOI of 0.5 or 2 for 16 h. Supernatants were then analyzed by ELISA for IFN-β. (D) Human DCs were mock infected (NI) or infected with TX WT or with NS1 C-terminal deletion mutant virus TX 1-99 or TX 1-126 at an MOI of 2 for 2, 4, 6, 8, and 16 h, and qRT-PCR was performed for IFN-responsive genes IP10, IFI56K, and ISG54 as previously described.

Human DCs infected with human influenza virus NS1 mutants demonstrate increased upregulation of type I IFN, IFN-responsive genes, and proinflammatory genes.To further examine the level of DC activation stimulated by these NS1 mutant influenza viruses, we infected DCs and performed qRT-PCR on total mRNA isolated at different times postinfection, measuring upregulation of multiple genes involved in DC maturation, including type I IFN and selected proinflammatory cytokines and chemokines. When DCs were infected with the mutant viruses TX 1-99 and TX 1-126, there was increased expression of type I IFN genes (Fig. 3B; see also Fig. S1 in the supplemental material), as well as IFN-regulated genes such as IP10 and ISG54 (Fig. 3D) compared to that in TX WT virus-infected DCs. Similarly, there was an increase in expression of proinflammatory cytokine genes such as TNF-α, IL-6, IL-12p35, and MIP-1β in TX 1-99- and TX 1-126-infected DCs versus TX WT-infected DCs (Fig. 4A; see also Fig. S1 in the supplemental material). Nevertheless, a cohort of genes (TANK-binding kinase, IKKε, and Toll-like receptor 3) were not affected during infection of DCs with either of the viruses (data not shown). Next, DC supernatants were analyzed by ELISA for secreted proteins. Corroborating the findings from our qRT-PCR experiments, IFN-β secretion (Fig. 3C), as well as TNF-α and IL-6 secretion (Fig. 4B), was augmented in DC infections with TX 1-99 and TX 1-126 viruses compared to TX WT virus infection. This secretion of type I IFN and proinflammatory gene products suggests that the mutant viruses are ineffective in blocking pathogen-associated molecular patterns that trigger signaling pathways leading to inflammatory responses and an IFN-mediated antiviral state. The results from the DCs infected with the NS1 mutant influenza virus directly correlate with the low levels of expression of the NS1 proteins in DCs infected by those viruses (Fig. 2). These data highlight the immunogenicity of the NS1 truncated mutants as potential vaccine candidates since they induce potent activation of human DCs.

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FIG. 4.

(A) Proinflammatory cytokine genes in influenza virus-infected human DCs. Human DCs were mock infected (NI) or infected with TX WT or with NS1 C-terminal deletion mutant virus TX 1-99 or TX 1-126 at an MOI of 2 for 2, 4, 6, 8, and 16 h. Total RNA was isolated from 5 × 10⁵ cells, and qRT-PCR was performed for TNF-α, IL-6, IL-12p35, and MIP-1β mRNAs. (B) Type I IFN and proinflammatory cytokine protein secretion in influenza virus-infected human DCs. A total of 2 × 10⁶ human DCs were mock infected (NI) or infected with TX WT or with NS1 C-terminal deletion mutant virus TX 1-99 or TX 1-126 at an MOI of 0.5 or 2 for 16 h. Supernatants were then analyzed by ELISA for TNF-α and IL-6. Error bars are the standard deviations derived from triplicate samples. This experiment is representative of three different donors.

Human DCs infected with human influenza virus NS1 mutants are better stimulators of adaptive immunity than DCs infected with the human influenza virus TX WT.We next questioned if DCs infected with viruses carrying the C-terminal deletions in NS1 can be better antigen-presenting cells than those infected with the wild-type virus. When costimulatory surface expression was examined by flow cytometry, we observed increased surface CD86 (Fig. 5A) and CD83 and MHC-II molecules (data not shown) in NS1 mutant influenza virus-infected DCs compared to TX WT-infected DCs, suggesting enhanced T-cell stimulatory function by those DCs. We next assessed the activation of infected DCs by analyzing the expression of Flu NP protein and CD86 in DCs. Figure 5B shows that mock-infected DCs had poor staining for Flu NP, but about 10% of cells expressed CD86 at high levels (CD86^hi). At 16 h postinfection at an MOI of 1, TX WT infections of DCs were 58.6% Flu NP positive (Flu NP⁺) (Fig. 5B), but only ∼23% of total cells were CD86^hi (Fig. 5B; see also Fig. S2A in the supplemental material), with 20.2% of total cells being dual Flu NP⁺ CD86^hi. When TX 1-99- and TX 1-126-infected DCs were analyzed, Flu NP⁺ cells were 64.1% and 67.5% of total cells, respectively (Fig. 5B; see also Fig. S2B in the supplemental material), and CD86^hi cells were 47.7% and 53%, respectively, with 39.4% Flu NP⁺ CD86^hi cells in TX 1-99 infections and 45.5% Flu NP⁺ CD86^hi cells in TX 1-126 infections (Fig. 5B; see also Fig. S2A in the supplemental material). Also, there were higher Flu NP-negative (Flu NP⁻) CD86^hi cells in TX 1-99 and TX 1-126 infections (8.3% and 7.6%, respectively) than in TX WT infections (2.5%). These results demonstrate that DCs infected with the truncated mutants have higher number of double NP⁺ CD86⁺ cells than DCs infected with the wild-type virus, suggesting that the mutant viruses overall will induce better immune antiviral responses than the wild-type virus, and this activation is primarily due to the actual infection.

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FIG. 5.

Levels of activation and T-cell priming by influenza virus-infected human DCs. (A) A total of 5 × 10⁶ human DCs were mock infected (NI) or infected with TX WT or with NS1 C-terminal deletion mutant virus TX 1-99 or TX 1-126 at an MOI of 0.5 for 16 h. Cells were then stained with anti-CD86-fluorescein isothiocyanate for assessing CD86 expression by fluorescence-activated cell sorting analysis. (B) A total of 1 × 10⁶ human DCs were mock infected (NI) or infected with TX WT, TX 1-99, or TX 1-126 at an MOI of 1 for 16 h. Cells were then stained with rabbit anti-Flu NP (and then goat anti-rabbit Alexa 488) as well as anti-CD86-phycoerythrin for assessing CD86 upregulation on infected DCs by fluorescence-activated cell sorting analysis. (C) Stimulation of T cells by influenza virus-infected human DCs. A total of 2.0 × 10⁴ human DCs mock infected (NI) or infected with TX WT or with NS1 C-terminal deletion mutant virus TX 1-99 or TX 1-126 at an MOI of 0.5 were incubated with 1.0 × 10⁵ naïve CD4 T cells for 3 days. Supernatants from the cocultures were then analyzed by ELISA for IFN-γ. Results are representative of more than three independent donors.

We then tested the ability of virus-infected DCs to stimulate T cells in an allogeneic coculture assay. To assess this, DCs were infected and incubated with naïve allogeneic CD4 T cells for 3 days at a DC-to-CD4 T-cell ratio of 1:5. The cell cocultures were analyzed for T-cell proliferation and cytokines indicative of Th1 (IFN-γ) and Th2 (IL-5 and IL-10) responses. No significant differences were detected in either T-cell proliferation or in IL-5 and IL-10 secretion in T-cell cultures with DCs infected with TX WT or the NS1 deletion mutant viruses (data not shown). However, the T-cell cultures containing DCs infected with mutant NS1 influenza virus showed increased levels of IFN-γ compared to those containing DCs infected with TX WT (Fig. 5C). Thus, the increased DC activation from TX 1-99 and TX 1-126 virus infections resulted in increased induction of IFN-γ production by T cells. These results suggest not only increased T-cell activation when NS1 function is impaired but also increased Th1 polarity in adaptive immune responses, which is known to be optimal for the clearance of influenza virus from the host (45). Our data show that the truncated NS1 mutants were more efficient at priming T cells toward a Th1 polarity than the wild-type virus with intact NS1.

Reduced IFN-responsive gene upregulation in human DCs infected with human influenza A viruses.We have previously shown that the laboratory strain of influenza virus PR8 does not induce significant levels of type I IFN in cultured human DCs, whereas a mutant PR8 virus with the NS1 gene deleted (deltaNS1 virus) is a good stimulant of IFN production in human DCs (15). The NS1 from PR8 influenza virus did not exhibit a global inhibitory effect on DCs as shown by microarray, qRT-PCR, and ELISA but, rather, a specific inhibitory effect on genes involved in DC activation (15). Conversely, the NS1 protein from the human isolate of influenza virus TX WT has been shown to induce a strong shutoff of host protein expression in infected cell lines (32). From these results, we inferred that human influenza A virus strains may display a greater inhibitory effect on human DC activation than the PR8 mouse-adapted strain. To test this hypothesis, human cultured DCs were infected with PR8 and TX WT as well as other human influenza A virus strains used in the 2005/2006 trivalent influenza A vaccine (WYM and NY) (see Materials and Methods) and analyzed by qRT-PCR for type I IFN as well as IFN-induced genes at 16 h postinfection. As previously described, PR8 infection of human DCs induced minimal levels of IFN-α and IFN-β but showed induction of IFN-responsive genes RIG-I, IP10, STAT1, MxA, and IFI56K (IFN-inducible protein of 56 kDa), most likely due to the residual type I IFN production observed (Fig. 6). DC infection with WYM, NY, and TX WT strains also resulted in minimal type I IFN, but the levels of IFN-responsive genes were reduced compared to PR8 infection of human DCs (Fig. 6). These observations support the idea of a stronger inhibitory property of human influenza A viruses in human DC activation and type I IFN induction by DCs than of the mouse-adapted PR8 influenza A virus laboratory strain. All the human strains of influenza virus tested in our system showed similar levels of inhibition of IFN-inducible genes and other genes involved in DC activation while the laboratory strain PR8 showed weaker inhibition of those genes, possibly as a result of multiple passages and mouse adaptation.

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FIG. 6.

Human isolates of influenza A virus are potent inhibitors of type I IFN responses in human DCs. Samples of 1 × 10⁶ cells were mock infected (NI) or infected with WYM, NY, TX WT, and PR8 viruses at an MOI of 0.5 for 16 h. Total RNA was isolated from samples and analyzed by qRT-PCR for the expression of the indicated genes. Error bars are the standard deviations of triplicate samples. Results are representative of at least three different donors.

Viruses from the TX WT background display different kinetics in viral replication and DC activation from PR8 viruses in human DCs.Using TX WT as a representative human influenza A virus to elucidate the differences in PR8 infections of human DCs, infected DCs were analyzed for viral transcription and protein synthesis. qRT-PCR for viral NP and NS1 at 1, 4, 8, and 16 h postinfection (Fig. 7A) and Western blotting for viral NP and NS1 proteins at 4 and 8 h postinfection (Fig. 7B) show that these viral genes were more highly expressed in TX WT infections than in PR8 virus-infected DCs. To determine if the absence of NS1 protein would affect viral replication during infection, human DCs were infected with respective NS1 mutants from either background (TX 1-99 for TX WT and deltaNS1 for PR8) in the same experiment and assessed by qRT-PCR and Western blotting. TX 1-99 viral NP gene expression levels were comparable to TX WT at the mRNA and protein levels (Fig. 7A and 2B). TX 1-99 viral NS1 gene expression was different from TX WT, with lower levels of transcription seen at earlier time points postinfection but with higher levels at 16 h postinfection. Nevertheless, expression of the TX 1-99 NS1 protein was barely detectable (Fig. 7B and 2B). When viral replication in deltaNS1 DC infections was analyzed, NP expression mimicked that of PR8 (Fig. 7A and B), and as expected, there was no NS1 mRNA or protein detected. As previously shown by other investigators using permissive cell lines (57), deltaNS1-infected DCs displayed aberrant viral late gene expression, i.e., reduced HA and M1 viral protein production (data not shown). Taken together, this result supports the idea that the NS1 protein is required for proper viral gene expression in human DCs.

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FIG. 7.

Replication of TX WT viruses and PR8 viruses in human DCs. Human DCs were mock infected (NI) or infected with PR8, deltaNS1, TX WT, and TX 1-99 at an MOI of 1 for 4 and 8 h. (A) Total RNA was isolated from 1 × 10⁵ cells, and qRT-PCR was performed for viral NP and NS1 mRNA. (B) Total cell lysates were made, and Western blotting was performed for viral NP and NS1 proteins. Levels of cellular actin are shown as loading controls. Results are representative of two different donors. NS1 WT, TX WT NS1; NS 1-99, TX 1-99 NS1.

TX1-99 and deltaNS1 exhibit different kinetics in human DC activation.The ability of NS1 mutants to stimulate DC activation was first assessed by examining the phosphorylation of transcription factors RelA, IRF3, and c-Jun known to be important for induction of type I IFN (51, 60), which is linked to DC activation (36). Human DCs were infected with PR8, deltaNS1, TX WT, and TX 1-99 viruses at an MOI of 1 and harvested at different times postinfection, and respective lysates were analyzed by Western blotting for RelA phosphorylation at S536, IRF3 phosphorylation at S396, and c-Jun phosphorylation at S73 (Fig. 8A). As determined before (Fig. 3A; also data not shown), RelA, IRF3, and c-Jun phosphorylation at 4 and 8 h postinfection was greater in human DCs infected with TX 1-99 than in DCs infected with TX WT (Fig. 8A). Data shown in Fig. 8A indicate that transcription factor activation in human DCs after PR8 and deltaNS1 infections displayed a similar trend of increased phosphorylation in the absence of NS1 (Fig. 8A). Interestingly, when transcription factor activation by TX 1-99 and deltaNS1 were compared, similar amounts of RelA and IRF3 phosphorylation at 4 and 8 h after TX 1-99 and deltaNS1 infections were seen. However, levels of c-Jun phosphorylation in TX 1-99 infections were greater at 4 and 8 h than in deltaNS1 infections. Furthermore, it was observed, as in previous studies (32), that the NS1 of influenza TX WT allowed minimal activation of transcription factors, more so than PR8 infections. These results suggest faster kinetics of human DC activation in TX 1-99 infections and corroborate previous evidence that NS1 of TX WT does allow the same activation of the type I IFN induction pathway as the NS1 from PR8 (32).

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FIG. 8.

Type I IFN induction and signaling in infected human DCs. Human DCs were mock infected (NI) or infected with PR8, deltaNS1, TX WT, and TX 1-99 at an MOI of 1 for 4 and 8 h. (A) Total cell lysates were made, and Western blotting was performed for phosphorylated RelA, IRF3, and c-Jun as well as total RelA, IRF3, and c-Jun. (B) Supernatants were analyzed by ELISA for IFN-α at 4, 8, and 12 h postinfection. (C) Human DCs infected at an MOI of 1 for 1, 4, and 8 h and harvested for total lysate for Western blotting of phosphorylated STAT1, total STAT1, and actin as a loading control. (D) mRNA was isolated from infected human DCs, and qRT-PCR was performed for IFN-responsive genes IP10 and MxA as previously described. hpi, hours postinfection.

The differences in transcription factor activation were also reflected in type I IFN production by DCs after infection with the TX WT and TX 1-99 influenza viruses (Fig. 8B). TX 1-99 infection initially resulted in the fastest secretion of IFN-α at 4 h (29.8 ± 0.96 pg/ml) and 8 h (89.5 ± 1.15 pg/ml) postinfection, but these levels were overtaken by IFN-α secretion from human DCs infected with deltaNS1 after 8 h postinfection with 2,785 ± 33.8 pg/ml and with TX 1-99 at 274 ± 1.46 pg/ml at 12 h (Fig. 8B). Also corroborating previous data for type I IFN transcription in infected human DCs (Fig. 6), TX WT infections caused slightly more type I IFN secretion than PR8 infections: TX WT infection levels were 16.8 ± 0.29 pg/ml at 4 h, 15.0 ± 0.09 pg/ml at 8 h, and 23.7 ± 3.16 pg/ml at 12 h, and PR8 infection levels were below the detection limit at 4 and 8 h but at 23.0 ± 0.37 pg/ml at 12 h.

We then examined the kinetics of STAT1 Y701 phosphorylation as a marker of type I IFN signaling. STAT1 phosphorylation in TX 1-99-infected human DCs was seen as early as 1 h postinfection and also at 4 and 8 h postinfection (Fig. 8C), whereas STAT1 phosphorylation was detected only from 8 h postinfection with Delta NS1, again supporting the observation of faster kinetics of DC activation and establishment of an antiviral state in TX 1-99 infections. Surprisingly, STAT1 phosphorylation in TX WT infections of human DCs was comparable to that seen in human DCs infected with TX 1-99, and in both cases STAT1 phosphorylation was markedly increased over the minimal level in PR8 infections of human DCs (Fig. 8C). Here, we show not only that type I IFN signaling in human DCs is highly sensitive, with immediate STAT1 phosphorylation from relatively low IFN-α levels (as in TX WT infections) (Fig. 6A and B and 8B and C), but also that TX WT and PR8 show different potentials of stimulating type I IFN signaling in human DCs.

As an end product of type I IFN signaling and STAT1 phosphorylation, the induction of type I IFN-responsive genes MxA and IP10 was assessed by qRT-PCR (Fig. 8D). Corresponding to the transcription factor activation, IFN-α secretion and STAT1 activation, there was a rapid induction of MxA, IP10 (Fig. 8D), RIG-I, and IRF7 (data not shown) in TX 1-99 infections of human DCs. MxA and IP10 induction in deltaNS1 infections lagged behind that of TX 1-99 infections (Fig. 8D). Remarkably, though the STAT1 phosphorylation in TX WT-infected human DCs was similar to that in TX 1-99-infected DCs (Fig. 8C), the rate of MxA and IP10 induction was reduced, and induction even became blunted toward later time points (Fig. 8D). When MxA and IP10 induction levels in TX WT infections of human DCs were compared to those in PR8 infections, gene expression was greater at earlier time points. However, at later time points (8 h onward) (Fig. 8D), PR8 expression levels of IFN-inducible genes exceeded those from TX WT infections as previously illustrated (Fig. 6A and B). This suggests that although TX WT is “leaky” in its inhibition of type I IFN induction and pathway activation, its ability to limit IFN-responsive gene induction and the establishment of an antiviral state is stronger than the antiviral induction capability of PR8 virus.

Influenza PR8 virus-infected DCs are better stimulators of naïve CD4 T cells than human influenza TX WT virus-infected DCs.Having established that the human strain TX WT is a better inhibitor of the induction of the antiviral state than the mouse-adapted laboratory strain PR8 in human DCs, we sought to investigate what impact this would have on infected DCs to stimulate the adaptive immune response. Human DCs infected with PR8 or TX WT at an MOI of 1 were cocultured with naïve T cells for 2 and 4 days and assessed for IFN-γ expression. Cocultures with human DCs infected with PR8 virus had strikingly greater amounts of IFN-γ secretion than cocultures with TX WT-infected DCs (Fig. 9A). We then compared the PR8 background viruses to the TX WT background viruses in DC-mediated activation of naïve T cells. As shown in Fig. 9B, PR8 induces more IFN-γ production in DC and naïve CD4 cocultures than TX WT while deltaNS1 induces levels of IFN-γ in DCs comparable to those of TX 1-99 and TX 1-126. We show here for the first time that a wild-type human isolate of influenza A is a better inhibitor of human DC stimulation of adaptive immunity than a wild-type strain of mouse-adapted influenza A virus. These data support the idea that TX WT influenza virus is a better inhibitor of immune responses than PR8, both at the innate and adaptive arms of immunity.

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FIG. 9.

Difference in PR8- and TX WT-mediated T cell activation. (A) A total of 2.0 × 10⁴ human DCs mock infected (NI) or infected with PR8 or TX WT at an MOI of 1 were incubated with 1.0 × 10⁵naïve CD4 T cells for 2 and 4 days. Supernatants from the cocultures were then analyzed by ELISA for IFN-γ. Error bars are the standard deviations of triplicate samples. Results are representative of similar experiments with three different donors. (B) DCs were mock infected (NI) or infected with PR8, deltaNS1, TX WT, TX 1-99, or TX 1-126 at an MOI of 0.5 and incubated with naïve CD4 T cells for 2, 4, and 6 days. Supernatants from the cocultures were then analyzed by ELISA for IFN-γ. Error bars are the standard deviations of triplicate samples. Results are representative of similar experiments with three different donors.