Cleavage and polyadenylation specificity factor (CPSF), elongation initiation factor 4GI (eif4gi),

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1 JVI Accepts, published online ahead of print on 29 April 2009 J. Virol. doi: /jvi Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. TITLE 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. RUNNING TITLE Responses to human influenza mutants in primary human cells. Kester Haye 1,4, Svetlana Bourmakina 1,4, Thomas Moran 1,4, Adolfo García-Sastre 1,2,3,4 and Ana Fernandez- Sesma 1,3,4* 1 Department of Microbiology, 2 Department of Medicine, Division of Infectious Diseases, 3 Emerging Pathogens Institute, 4 Center for Investigating Viral Immunity and Antagonism (CIVIA). Mount Sinai School of Medicine. New York, NY, 10029, USA. * Author for correspondence. Department of Microbiology, Mount Sinai School of Medicine. One Gustave L. Levy Place. New York, NY, 10029, USA. Phone (212) Fax (212) ana.sesma@mssm.edu ABBREVIATIONS Cleavage and polyadenylation specificity factor (CPSF), elongation initiation factor 4GI (eif4gi), interferon inducible protein of 56KDa (IFI56K), interferon α receptor (IFNAR), melanoma differentiationassociated gene 5 (MDA5), myxovirus resistance protein A (MxA), nuclear localization signal (NLS), non structural protein 1 (NS1), poly(a)-binding protein 1 (PABP1), protein kinase R (PKR), retinoic acid-inducible gene I (RIG-I). 1

2 ABSTRACT The NS1 protein of the influenza A virus is a potent virulence factor that inhibits type I interferon (IFN) synthesis, allowing the virus to overcome host defenses and replicate efficiently. However, limited studies have been conducted on NS1 function using human virus strains and primary human cells. We used NS1 truncated mutant influenza viruses derived from the human isolate influenza A/TX/91 (TX WT) to study the functions of NS1 in infected primary cells. Infection of primary differentiated human tracheo-bronchial epithelial (HTBE) cells with a NS1 truncated mutant demonstrated limited viral replication and enhanced type I IFN induction. Additionally, human dendritic cells (DCs) infected with human NS1 mutant viruses showed higher levels of activation and stimulated naïve T-cells better than TX WT virus infected DCs. We also compared infections of DCs with A/TX/91 and our previously characterized laboratory strain A/PR/8/34 (PR8) and its NS1 knockout (delta NS1). TX WT infected DCs displayed higher viral replication, but had decreased anti-viral gene expression at late time points and reduced naïve T-cell stimulation compared to PR8 infections, suggesting an augmented inhibition of IFN production and human DC activation. Our findings show that human-derived influenza A viruses have high capability to inhibit the anti-viral state in a human system and here we have evaluated the possible mechanism of this inhibition. Lastly, C-terminal truncations in the NS1 protein of human influenza virus are sufficient to make the virus attenuated and more immunogenic, supporting their use as live attenuated influenza vaccines in humans. 2

3 INTRODUCTION Models of influenza A infection have shown that upon initial exposure to virus, innate immune functions are activated primarily by the release of type I IFN(s) from infected cells. These critical cytokines can be released from any infected cell and trigger the synthesis of anti-viral proteins in adjacent cells that function which wards off attacking virus (52). Most pathogenic viruses including influenza virus possess antagonistic proteins that thwart the protective effect of IFN and allow establishment of infection (52). Once the innate barrier has been breached an adaptive immune response is initiated that is characterized by the production of cytotoxic T cells (CTL), T helper 1 (Th1) CD4 T cells and a neutralizing antibody response. Recovery from primary infection is mediated by CTLs which are expanded and activated in draining lymph nodes, that cycle back to the infection site and kill virus infected cells (8, 20). DCs are important in detecting influenza A infections and function as an essential link between innate immunity and adaptive immunity leading to viral clearance (41). Steady state DCs residing in tissue actively take up antigen and upon recognition of unique viral structures initiate signaling cascades that activate (mature) the DCs leading to their production of chemokines, expression of chemokine receptors (56) and production of cytokines required for leukocyte recruitment and stimulation. In addition, the maturational process leads to an increased expression of MHCII, CD86, CD80 and CD40 needed for cognate antigen presentation to stimulate adaptive immunity (41, 45). While these events are relatively similar when initiated by other types of microbes, viruses trigger the release of large amounts of type I IFN that function both as an anti-viral alarm as well as a signal that enhances the DC maturation. The co-incident timing and regulation of DC maturation and type I IFN production suggests that the entire process is regulated by common pathways (15, 37). In influenza A virus infections, the RNA helicase RIG-I is the major trigger of host antiviral responses (29, 30, 34, 36, 37), binding viral-derived dsrna and ssrna bearing uncapped 5 phosphates, which results in a conformational change that triggers the initiation of multiple antiviral signaling pathways (10, 27, 53, 64). RIG-I signals through the mitochondrial protein, IPS-1, to activate noncanonical TBK and IKKε kinases and the transcription factors c-jun, ATF, NFκB, IRF3 and IRF7, which upregulate IFN and inflammatory cytokines (31, 72). Secreted type I IFN from virus-infected cells amplifies the antiviral response by binding to membrane IFN receptors, and activating the intracellular Jak/STAT pathway that leads to the upregulation of various cellular host products such as MxA, IRF7, cytokines and chemokines that further stimulates innate antiviral responses and alerts the immune system against viral infection (18). The viral NS1 protein is a potent virulence factor for the influenza A virus, being implicated in masking viral presence and in inhibition of immunity via multiple mechanisms (14, 16, 24). NS1 binds to RIG-I/IPS-1 complexes 3

4 (22, 42, 48) and blocks downstream signaling (39, 65, 71), resulting in attenuation of type I IFN and inflammatory cytokine expression. However, NS1 can also disrupt cellular mrna processing and nuclear-cytoplasmic export by binding to the cellular mrna processing factor CPSF, down-regulating the induction of cellular genes at a posttranscriptional level (9, 32, 46, 47). These two activities are possible due to a complex regulation of NS1 cellular localization mediated by two nuclear localization signals (NLS 34-38, NLS ) and one nuclear export signal (NES ), which allow the shuffling of this protein between the cytoplasm and nucleus of the infected cell (33). The CPSF inhibitory properties of NS1 appears to be dispensable for some influenza virus strains (32, 47, 68). Other functions of NS1 that could contribute directly or indirectly to regulation of the host antiviral response include sequestration of viral dsrna from cellular sensors (7, 25, 38), disruption of nuclear pore function (58), activation of the PI 3 K/Akt pathway (13, 23, 62) and interactions with cellular proteins involved in translation (1, 5). Consistent with a prominent role of NS1 in regulating host responses to viral infection, truncation of the C- terminus of NS1 results in influenza A virus attenuation in mice, pigs, horses and macaques (5),(54, 55, 63, 66) (12, 71). Animals infected with NS1 mutant viruses show reduced pathogenesis and increased IFN expression with respect to animals infected with wild-type influenza virus, and animals immunized with NS1 mutant influenza viruses are protected from wild type influenza virus infection (55, 66, 69). This suggests that NS1 C-terminus truncated mutants could be further developed as live attenuated vaccines against influenza (28, 49, 50). Interestingly, the multiple activities of the NS1 protein result in diminishing activation of DCs, blocking cytokine expression as well as T-cell activation in our model of human DCs, thus counteracting both innate and adaptive immune responses (15). Despite several studies on the effects of the NS1 of influenza virus in viral replication and inhibition of antiviral host responses during viral infection, only limited studies have been performed using human influenza virus strains and/or human primary cells. And, though it is known that the NS1 C-terminus truncated mutants are immunogenic and serve as efficient vaccines in mice, swine, equine and non-human primate models, it is yet to be demonstrated in a human system. Using a human influenza virus strain, we examined the effects of NS1 C-terminal truncations on viral replication in human primary respiratory epithelial cells and in induction of immune responses using human DCs as a surrogate model for immune activation. In an attempt to map the regions of the NS1 protein responsible for the inhibition of DC activation observed in our previous studies we decided to generate and use recombinant influenza viruses with truncations in the NS1 protein using a human influenza virus as the backbone strain. Via reverse genetics a human isolate of H1N1 influenza A virus circulating in 1991 (influenza A/Texas/91, TX WT), and two different recombinant viruses containing NS1 C-terminal deletions (TX 1-99 and TX expressing NS1 truncated proteins of 99 and 126 amino acids, respectively) were generated. In the present study, 4

5 we show that the A/Texas/91 NS1 C-terminal deletion mutants are attenuated, with decreased viral replication in primary cells, but are efficient stimulants of type I IFN production, and innate and adaptive immunity. Having generated a human ex-vivo model of human influenza infection and immune response, we then compared the activation of human DCs by human influenza A/Texas/91 viruses to that previously characterized with influenza A mouse adapted laboratory strains, influenza A/PR/8/34 (PR8) and the NS1 deleted mutant of PR8 delta NS1(15). TX WT replicates at a greater level than PR8 in human DCs, but can display stronger inhibition on the expression of IFN responsive genes. These results are consistent with reduced naïve T-cell stimulation with TX WT infected DCs compared to PR8 infected DCs. Taken together, our results support our system of human primary cell lines in being an efficient surrogate model for investigating human influenza A pathogenesis. These studies also suggest that the NS1 of human influenza A has additional adaptations for heightened inhibition of establishing the anti-viral state, but truncating the C-terminus is sufficient to attenuate this more potent virulence advocating these mutants for vaccine design. Downloaded from on January 12, 2019 by guest 5

6 MATERIALS AND METHODS Viruses and cells. Recombinant A/Texas/91 (TX WT), A/Texas/91 NS (TX 1-99) and A/Texas/91 NS (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 TXWT virus as well as TX 1-99 and TX 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 delta NS1, was generated by reverse genetics from influenza A/PR/8/34 virus (PR8) as previously described (10). TXWT was grown in 7 day old eggs, respectively. Influenza A/NY/55/04 (NY), Influenza A/WYM/3/03 (WYM) and PR8 were grown in 9 day old eggs. Influenza virus deltans1 was grown in 6 day old embryonated eggs. All influenza viruses were titrated by 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 DMEM (Invitrogen) supplemented with 10% FCS (Hyclone), 1mM sodium pyruvate (NaPy) (Invitrogen), 2 mm L-glutamine (LGlu) (Invitrogen) and 50 µg/ml gentamicin (Invitrogen). All cells were grown at 37 0 C, 7% CO 2. Isolation and culture of human DCs. PBMCs 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 immuno-magnetically 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 (0.7x10 6 cells/ml) in DC medium (RPMI (Invitrogen), 10% FCS (Hyclone), or 4% human serum (Cambrex/Lonza) 100 units (U)/ml of penicillin, and 100 µg/ml streptomycin (Pen/Strep) (Invitrogen)) supplemented with 500U/ml hgm-csf (Peprotech), 1000U/ml hil-4 (Peprotech), and incubated for 5-6 days at 37 o C. Infections and treatments of DCs. After 5-6 days in culture, DCs were infected with influenza viruses at MOI=0.5, 1 or 2 for 60 minutes in serum free RPMI. Cells were then plated in DC medium (described above) at 1x10 6 cells/ml maintained in culture for different time periods depending on the experiment. Human tracheo-brochial epithelial (HTBE) cell cultures. Cryopreserved normal human bronchial epithelial cells (HTBE) were purchased from Cambrex and cultivated according manufacturer s instructions and previously described protocol (21). Briefly, cells were passaged twice in bronchial epithelial growth medium containing necessary supplements and growth factors (BEGM, Cambrex) and seeded onto Transwell-Clear Permeable filters (Millipore; 24 mm diameter, 0.4mm pores) at the density 1.4x10 5 cells/filter. Before seeding, filters were coated 6

7 with collagen I derived from human placenta (BD Biosciences). Cells were grown submerged in 1:1 mixture of DMEM and BEGM medium supplemented with growth factors for one week. Then, medium from the apical surface was removed and cells were maintained in air-liquid interface for 3 weeks. When cells are maintained in air-liquid interface for long periods of time, they differentiate into different cell types present in mucosal surface of 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 viruses at MOI of 2 or mock infected for 1 hour at 37 o 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 in the basal compartment of wells. At 9.5 and 25 hours post infection media from apical and basal compartments were collected and cell layers were resuspended in SDS-PAGE 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 was dialyzed against low ph buffer (50mM glycine ph 2) to inactivate virus, and neutral ph was restored by subsequent dialysis against PBS. Vero cells were then incubated with different dilutions of medium from infected HTBE cells for 24 hours and infected with NDV-GFP, a virus that express GFP and is known to be sensitive to the action of IFN. 24 hours later, the intensity of green fluorescence was measured. As controls, Vero cells were treated with 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 was 100%. In other experiments, HTBE cells were infected at MOI of for 1 hour at 37 o C, virus was removed, cells were washed once with medium and continued to be incubated in air-liquid interface. At various time points medium was added to apical surfaces for 30 minutes and harvested for virus titration. Capture ELISA. Capture ELISA for IL-6, TNFα (R & D systems), IFNα and IFNβ (Biosource) were used according to 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 PE linked CD86 according to 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 minutes on ice, and then permeablized in 0.1% saponin in PBS for 30 minutes on ice. Cells were then incubated with rabbit polyclonal anti-flu NP for 1 hour, and then goat anti-rabbit secondary antibody conjugated to alexa 488 (invitrogen) for 1 hour. Cells were washed twice with 0.05% FBS/PBS twice between each step. Data was analyzed using Flowjo software. 7

8 Total RNA isolation. RNA extraction and generation of cdna from human DCs. Samples of 1-5x10 5 DCs either, infected with virus, not infected or treated with maturing agents were pelleted and RNA was isolated and DNase treated using the Absolutely RNA RTPCR miniprep kit (Stratagene). RNA was quantified using the Nanodrop spectrophotometer (Nanodrop Technologies). The yields of RNA were approximately of µg/ml. Quantitative real-time PCR. qrt-pcr was performed using a previously published SYBR green in an ABI7900 HT protocol (15) and (73). Each transcript in each sample was assayed in duplicate and the mean threshold cycle (CT) 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 DC. DCs either non treated or infected with different recombinant influenza viruses were mixed together with allogeneic PBMC from buffy coats and cultured for 4 days in 96 well plates at a ratio of 1:5 (10 6 DC/ml : naive T-cells). Supernatants from the co-cultures were tested by ELISA for IFNγ release at different times of culture (ebioscience). Western blots and Antibodies Samples of 10 6 cells were lysed in 100µl 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 PMSF, 10µg/ml aprotinin, and 10 µg/ml leupeptin) or 100µl of phosphosafe buffer (calbiochem). Cell lysates were centrifuged (14,000g, 10 min) to remove cellular debris and boiled for 5 min in SDS sample buffer containing 1 mm DTT. Protein lysates were resolved on 4 15% SDS-PAGE gradient gels (Bio-Rad), transferred to nitrocellulose membranes, and probed with rabbit polyclonal anti-np and anti-ns1 as previously described in (63), mouse monoclonal anti M1 and influenza A/Texas specific anti-ha (Mount Sinai Hybridoma center), anti pospho-irf3 (S396) (Cell Signaling), anti pospho- NFκBp65/Rel A (S536) (Cell Signaling), anti pospho-c-jun (S73) (Cell Signaling), anti NFκBp65/Rel A (Cell Signaling), anti c-jun (Cell Signaling), anti IRF3 (Cell Signaling), anti Actin (Santa Cruz). Immunoreactive bands were detected using HRP conjugated goat anti-mouse or anti-rabbit IgG (BioRad) and ECL (BioRad). 8

9 RESULTS Generation of recombinant TX NS1 C-terminal deletion viruses. We aimed to determine whether NS1 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 A/Texas/91 background were generated, TX and TX 1-99, that encode C-terminal truncated NS1 proteins of 126 and 99 amino acids, respectively, in contrast to the 230 amino acid long NS1 protein of the corresponding wild type virus (as previously described in materials and methods). Rescue of TX 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 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 virus-infected A549 cells showed similar levels of NP protein, and reduced M1 protein levels to that seen in TX WT virus-infected A549 cells (Figure 1A). However, HA protein and the respective truncated NS1 polypeptides of the mutant viruses were poorly expressed, only barely detectable in overexposed blots compared to the clearly visible levels observed in TX WT infected cells (Figure 1A). Replication of TX 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 air-liquid interface, as a surrogate system of the human upper respiratory epithelium. Upon infection at MOI of 2, TX 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 hrs post infection (hpi) but a much lower number of infected cells in the case of TX virus at 25 hours. Western blotting of HTBE lysates at 25 hpi was consistent with the immunofluorescence data (Fig. 1C). Truncated 126 aa NS1 protein in infected HTBE cells was not detected, indicative of low expression of the truncated NS1 in these cells. We also detected lower virus titers in apical supernatants from TX virus-infected HTBE cells at 25 hpi (Fig. 1D) compared with TX WT infected HTBE cells. Differences in viral replication between TX and TX WT virus in HTBE cells were even more apparent when cells were infected with an MOI of 0.001, which allowed for multi-cycle replication of the viruses. At 24 and 48 hpi, virus titers in the supernatants were determined and the TX WT virus reached titers around 10 6 at 48 hpi. However, TX viruses were detected at very low levels at 24 hpi and not detected at all at 48 hpi (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 9

10 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 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 virus was observed, as demonstrated by antiviral activity against NDV-GFP replication in Vero cells (Fig. 1F). This result indicates that the TX 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 published for mouse DCs (35)) and examined viral protein expression by Western blot. Levels of NP in NS1 mutant virus-infected DCs were equal to that of wild type virus-infected DCs (Fig. 2A). But in DCs infected with TX 1-99 and TX mutant viruses, a significant reduction in the levels of HA, M1 or NS1 proteins as compared to TX WT virus-infected DCs was seen (Figs 2A and 2B). 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 4h, 8h and 16h post-infection (Fig. 2B). TX WT, TX 1-99 and TX 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 4h post-infection in TX WT-infected DCs. Again, DCs infected with TX 1-99 or TX 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 8h postinfection and the levels were increased at 16h post-infection. However, DC infection with TX 1-99 and TX showed poor expression of HA even at 16h post-infection (Fig. 2B). In summary, the C-terminus NS1 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. 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 other pro-inflammatory cytokines secretion by activation of NFκB/RelA, c-jun and IRF3 (51, 60). It has been demonstrated that phosphorylation 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 mutant viruses would be at 10

11 stimulating these processes. When phosphorylation and activation of IRF3 (at residue S396) was examined, an increase in phosphorylation of this transcription factor in TX 1-99 and TX infected DCs was observed compared to TX WT infected cdcs at 4 and 8 hours post-infection (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 transcriptionl factors (4). These results strongly suggest that the inability of the NS1 truncation mutants to inhibit IRF phosphorylation contribute to their enhanced immunogenicity, while conferring onto them an attenuated phenotype. 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 messenger RNA isolated at different times post-infection, 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, Supplementary Fig. 1), 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 proinflammatory cytokine gene expression such as TNFα, IL6, IL12p35 and MIP 1β in TX 1-99 and TX infected DCs versus TX WT infected DCs (Fig. 4A, Supplementary Fig. 1). Nevertheless, a cohort of genes (TBK, IKKe, TLR3) 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, IFNβ secretion (Fig. 3C), as well as TNFα and IL6 secretion (Fig. 4B) was augmented in DC infections with TX 1-99 and TX 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 (PAMPS) 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. Human DCs infected with human influenza virus NS1 mutants are better stimulators of adaptive immunity than DCs infected with the wild type human influenza virus A/TX/91. We next questioned if DCs infected with the NS1 C-terminus deletions can be better antigen presenting cells than those infected with the wildtype virus. When co-stimulatory surface expression was examined by flow cytometry, we observed increased surface CD86 (Fig. 5A) and CD83 and MHC class II molecules (data not shown) in NS1 mutant influenza virus infected DCs 11

12 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 Influenza A NP protein and CD86 in DCs. Figure 5B shows that mock-infected DCs had poor staining for FluNP but about 10% of cells were CD86hi. At 16 hours post-infection at an MOI of 1, TX WT infections of DCs were 58.6% FluNP+ (Fig. 5B) but only ~23% of total cells were CD86hi (Fig. 5B, Supplementary Fig. 2A), with 20.2% of total cells being dual Flu NP+ CD86hi. When TX 1-99 and TX infected DCs were analyzed, Flu NP+ cells were 64.1 % and 67.5% of total cells respectively (Fig. 5B, Supplementary Fig. 2B), and CD86hi cells were 47.7% and 53% respectively with 39.4% FluNP+ CD86hi cells in TX 1-99 infections and 45.5% FluNP+ CD86hi cells in TX infections (Fig. 5B, Supplementary Fig. 2A). Also, there were higher Flu NP- CD86hi cells in TX 1-99 and TX infections (8.3% and 7.6% respectively) compared with TX WT infections (2.5%). These results demonstrate that DCs infected with the truncated mutants have higher number of double NP positive and CD86 positive cells than DCs infected with the wildtype virus, suggesting that the mutant viruses overall will induce better immune antiviral responses than the wildtype virus, and this activation is primarily due to the actual infection. We then tested the ability of virus-infected DCs to stimulate T cells in an allogeneic co-culture assay. To assess this, DCs were infected and incubated with naïve allogeneic CD4 T-cells for 3 days at a DC:CD4 T cell ratio of 1:5. The cell co-cultures were analyzed for T-cell proliferation, and cytokines indicative of Th1 (IFNγ) and Th2 (IL5 and IL10) responses. No significant differences were detected neither in T cell proliferation, nor in IL5 and IL10 secretion in T-cell cultures with DCs infected with TX wildtype or NS1 deletion mutant viruses (data not shown). However, the T cell cultures containing DCs infected with mutant NS1 influenza virus showed increased levels of IFNγ when compared to those containing DCs infected with TX WT (Fig. 5C). Thus, the increased DC activation from TX 1-99 and TX virus infections resulted in increased induction of IFNγ production by T- cells. These results not only suggest 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 shows that the truncated NS1 mutants were more efficient at priming Tcells towards 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 A/PR/8/34 (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) 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 12

13 A/Texas/91 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 strains may display a greater inhibitory effect on human DC activation when compared to the PR8 mouse adapted strain. To test this hypothesis, human cultured DCs were infected with PR8, TX WT as well as other human influenza A virus strains used in the 2005/2006 trivalent influenza A vaccine (Influenza A/WYM, and A/NY) (see materials and methods) and analyzed by qrt-pcr for type I IFN, as well as IFN induced genes 16 hours post-infection. As previously described, PR8 infection of human DCs induced minimal levels of IFNα and IFNβ, but showed induction of IFN responsive genes RIG-I, IP10, STAT1, MxA and IFI56K most likely due to the residual type I IFN production observed (Fig. 6). DC infection with WYM, NY and TXWT 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 IFN induction by DCs when compared to 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. Viruses from Influenza A/TX/91 background display different kinetics in viral replication and DC activation when compared to A/PR8/34 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 hours post infection (Fig. 7A), and western blots for viral NP and NS1 proteins at 4 and 8 hours post infection (Fig. 7B) show that these viral genes were more highly expressed in TX WT infections with respect to 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/91 and deltans1 for A/PR/8) in the same experiment and assessed by qrt- PCR and western blot. TX 1-99 viral NP gene expression levels were comparable to that of TX WT at the mrna and protein level (Fig. 7A, 2B). TX 1-99 viral NS1 gene expression was different from TX WT, with lower levels of transcription seen at earlier time points post-infection, but with greater levels at 16 hours point infection. Nevertheless, expression of the TX 1-99 NS1 protein was barely detectable (Fig. 7B, 2B). When viral replication in delta NS1 DC infections was analyzed, NP expression mimicked that of PR8 (Fig. 7A, 7B), and as expected, there was no NS1 mrna or protein detected. As previously shown by others using permissive cell lines (57), deltans1- infected DCs displayed aberrant viral late gene expression i.e. reduced HA and M1 viral protein production (data 13

14 not shown). Taken together, this result supports that the NS1 protein is required for proper viral gene expression in human DCs. 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 Rel A, 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 virus at a MOI 1, harvested at different times postinfection and respective lysates were analyzed by western blot for Rel A phosphorylation at S536, IRF3 phosphorylation at S396 and c-jun phosphorylation at S73 (Fig. 8A). As determined before (Fig. 3A, data not shown), Rel A, IRF3 and c-jun phosphorylation at 4 and 8 hours post-infection was greater in human DCs infected with TX 1-99 than DCs infected with TX WT (Fig. 8A). Data shown in figure 8A indicates 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, a comparable amount of Rel A and IRF3 phosphorylation at 4 and 8 hours after TX 1-99 and deltans1 infections was seen. However, levels of c-jun phosphorylation in TX 1-99 infections were greater at 4 and 8 hours when compared with delta NS1 infections. Furthermore, it was observed, similar to previous studies (32), that the NS1 of influenza A/TX/91 allowed minimal activation of transcription factors, more so than PR8 infections. These results suggest faster kinetics of human DC activation in TX1-99 infections, and corroborate previous evidence that NS1 of TX WT does allow same activation of the type I IFN induction pathway as that of the NS1 from PR8 (32). These differences in transcription factor activation were also reflected in type I IFN production by DCs after infection with those influenza viruses (Fig. 8B). TX 1-99 infection initially resulted in the fastest secretion of IFNα at 4 hours (29.8 +/ pg/ml) and 8 hours (89.5 +/ pg/ml) post-infection, but these levels were taken over by IFNα secretion from human DCs infected with deltans1 after 8 hours post-infection with / pg/ml and TX 1-99 at 274 +/ pg/ml at 12 hours (Fig. 8B). Also corroborating previous data, type I IFN transcription in infected human DCs (Fig. 6), TX WT infections caused slightly more type I IFN secretion over PR8 infections with TX WT infection levels at / pg/ml at 4 hours, / pg/ml at 8 hours and / pg/ml at 12 hours and PR8 infection levels being below the detection limit at 4 and 8 hours, but at / pg/ml at 12 hours. 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 hpi, but also at 4 and 8 hpi (Fig. 8C), whereas STAT1 phosphorylation was only detected from 8 hours post-infection, again supporting the observation of faster kinetics of DC activation and establishment of an antiviral state in TX 1-99 infections. 14

15 Surprisingly, STAT1 phosphorylation in TX WT infections of human DCs was comparable to that seen in human DCs infected with TX 1-99, being markedly increased over the minimal STAT1 phosphorylation in PR8 infections of human DCs (Fig. 8C). Here, we not only show that type I IFN signaling in human DCs is highly sensitive, with immediate STAT1 phosphorylation from relatively low IFNα levels (as in TX WT infections, Fig. 8B, C and 6A, B), 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, IFNα 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 TX 1-99 infected DCs (Fig. 8C), the rate of MxA and IP10 induction was reduced, and induction even became blunted towards later time points (Fig. 8D). When MxA and IP10 induction 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 hours onward, Fig. 8D), PR8 expression levels of IFN inducible genes exceeded those from TXWT infections as previously illustrated (Fig. 6A, 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 anti-viral state is stronger than the antiviral induction capability of PR8 virus. Influenza/A/PR/8/34 virus infected DCs are better stimulators of naïve CD4 T cells than human influenza A/TX/91 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 DC infected with PR8 or TX WT at a MOI of 1 were co-cultured with naïve T cells for 2 and 4 days and assessed for IFNγ expression. Co-cultures with human DCs infected with PR8 virus had strikingly greater amounts of IFNγ secretion than cocultures with TX WT infected DCs (Fig. 9A). We then compared the PR8 background viruses to the TX background viruses in DC-mediated activation of naïve T-cells. As shown in Fig. 9B PR8 induces more IFNγ production in DC/naïve CD4 co-cultures than TX WT, while DeltaNS1 induces comparable levels of IFNγ in DCs as compared to TX 1-99 and TX We show here for the first time that a wildtype human isolate of influenza A is a better inhibitor of human DC stimulation of adaptive immunity than a wildtype strain of mouse-adapted influenza A virus. This data supports 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. 15

16 DISCUSSION In generating NS1 C-terminal truncation mutants of a human influenza A strain, we were able to ablate the functions of NS1 in that strain, demonstrating its potent inhibition potential. Interestingly, using these human viruses, we were also able to compare the NS1 functions in a laboratory strain of influenza virus and have characterized key differences in their manner of inhibition of anti-viral responses. Importantly, we have demonstrated from our current study that the human influenza A NS1 protein has multiple capacities to inhibit the innate immune responses to influenza A infection, which gives it an advantage to human influenza. However, this strong virulence factor is rendered incompetent with C-terminal truncations, as these deletions leads to a gross destabilization of NS1 expression. This suggests that the C-terminus of human influenza A NS1 is important for stability of NS1 leading to viral pathogenesis, and mutants that contain this type of truncation would be virtual NS1 knockouts, and potential options for an efficacious vaccine. We have investigated, for the first time, an ex-vivo model of human influenza A infection using a human isolate of influenza A virus and human primary cells to better understand the role of the NS1 protein of influenza virus in its pathogenesis and to replicate efficiently and evade host immunity. Previous studies have demonstrated the importance of the C-terminal region of the influenza A virus NS1 protein during infection by showing that truncating the C-terminal domain of the NS1 results in decreased viral growth rate, and decreased viral NS1, HA and M protein expression (12, 54, 63, 66). However, our model is unique in its ability to study human NS1 mutant viruses using primary human cells. Using human TX WT virus, we found that this virus efficiently expressed viral proteins in a human lung epithelial cell line (A549), in primary human tracheo-bronchial epithelial (HTBE) cells and in DCs. As DCs support influenza virus infection but do not release infectious viral particles (shown in mice (35) and in humans, data not shown), viral replication was demonstrated in these cells by analyzing western blots for viral proteins in the present study. Our comparison of TX WT viral replication with the NS1 truncated TX 1-99 and TX viruses support a role for the NS1 protein in enhancing the expression of other viral proteins in relevant human cells. Although TX 1-99 and TX appeared to replicate well in the low-ifn environment of 7-day old eggs, they exhibited diminshed viral replication in A549 cells and DCs, as demonstrated by the poor expression of the HA, M1 and NS1 proteins. In HTBE cell infections, most of the infected cells were non ciliated, consistent with the known tropism of human influenza viruses (40). There were no significant differences on the number of infected cells during early TX WT or TX infections (9.5 hours post-infection). However, this was decreased at later times in HTBE infections with TX virus (25 hours post-infection). These results are consistent with the lack of differences in viral titers between those two viruses at 9 hours post-infection. However, at 25 hours, more than 50% of the cells on the culture were infected with TX WT and only approximately 30% 16

17 were infected with TX Poor viral replication can be explained by a possible impairment in the ability of the mutant NS1 protein to dimerize (70), resulting in lost augmentation of viral replication and aberrant IFN antagonistic functions. Nevertheless, the comparable viral titers at early time points of HTBE infections, and limited viral replication in HTBE, A549 and DCs suggest that NS1 C-terminal truncation mutants will replicate enough to provide antigen for immunity, but not replicate efficiently to become pathogenic, prerequisites for vaccine design. We previously showed that the NS1 protein of a mouse-adapted laboratory strain of influenza virus (PR8) has the ability to inhibit the production of IFN and the activation of human DCs, and therefore an influenza PR8 virus lacking the NS1 protein (deltans1) is an efficient stimulant of DC activation (15). We now analyzed human DC infections with PR8 influenza virus and human influenza virus strain TX WT. These two viruses displayed very different abilities to replicate, and stimulate innate and adaptive immune responses. TX WT viral infections demonstrated a marked increased production of NP and NS1 mrna and protein in human DCs when compared with PR8, suggesting its enhanced ability of replication in human DCs. There was some activation of the type I IFN induction pathway, secretion of type I IFN and stimulation of IFN signaling with minimal increases in IFN responsive gene expression, all evidence for the initiation of an anti-viral state in human DCs. However, during later stages of TX WT infections, the expression of IFN responsive genes was diminished, whereas PR8 infections of human DCs resulted in a gradual increase of IFN responsive gene expression which surpassed that in TX WT infections. And, this respective inability of the PR8 virus to completely block human DC activation resulted in increased naïve CD4 stimulation. A likely explanation is reflected in a recent study (32) which showed that the NS1 protein of TX WT is more potent at inducing a general host shut off of gene expression than that of PR8 virus. The amino acid residues F103 and M106 of NS1 mediated interaction with the cellular factor CPSF, resulting in inhibition of cellular mrna processing. Since most human influenza strains similarly to TX WT, bear F103 and M106 residues in their NS1 proteins (Influenza Virus Resource, NCBI), it is possible that NS1-CPSF interactions potentiate the ability of human influenza viruses to overcome the type I IFN system in humans, as demonstrated in this study with reduced IP10, MxA, STAT1 and IFI56K expression in human DCs infected with human influenza A compared to human DCs infected with PR8. Nevertheless, it remains to be determined how influenza viruses expressing NS1 proteins which do not interact with CPSF, such as the PR8 virus, are able to replicate to high levels and cause disease in their hosts. Nevertheless, our results underscore the need to study human influenza viruses in human systems to understand the impact during natural infection. RIG-I is the major sensor for detection of influenza virus infection by DCs and to the resultant IFN and cytokine expression in mouse systems (30). Furthermore, NS1 is known to bind to RIG-I containing complexes, 17

18 decreasing RIG-I mediated NFκB and IRF3 activation (48), which leads to decreased expression of type I IFN and IFN-related genes (22, 42). These lines of evidence taken together suggests that infections with TX WT would show greater inhibition of RIG-I mediated activation of downstream transcription factors such as NFκB, IRF3 and c-jun than infections with attenuated TX 1-99 and TX viruses (60). Activation of these proteins was determined by phosphorylation at specific serine residues. Indeed, an increase was observed in phosphorylation of NFκB/Rel A, c-jun and IRF3 at S396 in DCs infected with TX 1-99 and TX (data not shown) versus those infected with TX WT. There was also inhibition of Rel A, IRF3 and c-jun activation in PR8 infected human DCs. Nevertheless, there was gradual increase in the production of antiviral genes in those cells. Here we further demonstrate and support the hypothesis that the NS1 of TX WT can not only inhibit the establishment of an antiviral state by diminishing IFN responsive genes, but also by blocking RIG-I signaling in human DC infections, making TX WT more efficient at the evasion of innate and adaptive immunity. These results conflict with those obtained from experiments studying infections with the A/Udorn/72 laboratory influenza virus strain (43), underscoring again the need to conduct studies with human viruses in human cells to gain insights in mechanisms of regulation of host gene expression by influenza virus in humans. The augmented DC activation by NS1 mutant viruses was corroborated by the increased proinflammatory cytokine gene transcription and protein secretion including type I IFN following TX 1-99 and TX infections, which led to the activation of the IFN-responsive genes IP10, IFI56K, ISG54, as well as RIG-I and STAT1 (data not shown). Additionally, the NS1 protein of a human influenza virus isolate can efficiently block IFN related genes (antiviral state) in human DCs, contributing to the establishment of infection in humans. Moreover, although the NS1 of TX WT uses multiple mechanisms to inhibit establishment of an antiviral state (32), truncating the C- terminus of NS1 efficiently enhances the ability of the virus to activate DC and allows increased upregulation and secretion of IFN, IFN-related genes and proinflammatory cytokines (15, 17, 36, 37). TX 1-99 and TX NS1 C-terminal deletion mutants efficient activation of DCs leads to potent stimulation of T cells. This may explain the strong induction of B and T cell responses of TX virus in nonhuman primates (5). For an efficient induction of adaptive immune responses, DCs must upregulate MHC class I and II, as well as co-stimulatory molecules to the membrane surface (2, 3) for proper antigen presentation and stimulation of NK, T and B-cells. We observed increased MHC class II (data not shown) and CD86 expression in DCs infected with TX 1-99 and TX over that of TX WT infections. And, this increased CD86 surface expression was primarily in Flu NP positive cells suggesting that CD86 upregulation is mostly dependent on infection. Furthermore TX 1-99 and TX infected DCs stimulated increased IFNγ secretion from T cells compared to cdcs infected with TX WT. The increased IFNγ suggests a Th1 polarity, which is believed to be 18

19 beneficial for clearance of influenza virus infections (11, 19, 44, 45). These observations correlate well with the efficacy in protection achieved using NS1 C-terminal truncation mutants from other influenza virus strains in mice and pigs (55, 63, 66). In this study, we show that NS1 C-terminus deletions that disrupt NS1 functions in a human strain of influenza decrease the rate and efficiency of viral replication, which is concomitant with an impaired ability to prevent molecular and cellular antiviral mechanisms. This strongly supports the idea that such mutant viruses might be used as live attenuated vaccine viruses for human influenza. Live attenuated vaccine viruses must replicate sufficiently to produce viral antigen and stimulate immune response and memory, but not inflict serious pathology and disease (6, 45, 49, 50). Strong supporting evidence for demonstrations from our system that NS1 C-terminal deletion mutants are possible live-attenuated human influenza vaccines comes from a recent study using macaques (5). In this study, macaques were mock infected, vaccinated with inactivated TX WT virus or infected with live TX virus. Macaques infected with live TX acquired humoral and cellular immunity to influenza A virus that was more robust than that acquired with inactivated virus, as demonstrated by higher levels of specific HA antibody production, and an increase in the percentages of influenza specific CD4 and CD8 T cells. An added benefit to the use of these mutants as human influenza vaccines is that they are generated by reverse genetics techniques, which is predicted to curtail the length of production from the current time of months to weeks (28, 49). Availability of efficient live attenuated influenza virus vaccines would be advantageous in efforts to protect the public against the increasing resistance of influenza A virus to current antiviral treatments, as well as potential influenza A pandemics (26, 28, 67). 19

20 Acknowledgements We thank Richard Cadagan for excellent technical assistance, Alica Solorzano for helpful reagents, suggestions and discussions, Mike Pazos for technical assistance with flow cytometry and Sharon Czelusniak for her help in manuscript editing. This work was partly supported by NIAID funded Center for Investigating Viral Immunity and Antagonism (CIVIA, U19 AI62623) to TMM, AF-S and AG-S and Center for Research on Influenza Pathogenesis (CRIP, HHSN C) to AF-S and AG-S, by NIAID grant R01 AI46954 to AG-S and by a NCI fellowship to KH (1F31CA PI). REFERENCES 1. Aragon, T., S. de la Luna, I. Novoa, L. Carrasco, J. Ortin, and A. Nieto Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus. Mol Cell Biol 20: Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, B. Pulendran, and K. Palucka Immunobiology of dendritic cells. Annu Rev Immunol 18: Banchereau, J., B. Schuler-Thurner, A. K. Palucka, and G. Schuler Dendritic cells as vectors for therapy. Cell 106: Bandukwala, H. S., B. S. Clay, J. Tong, P. D. Mody, J. L. Cannon, R. A. Shilling, J. S. Verbeek, J. V. Weinstock, J. Solway, and A. I. Sperling Signaling through Fc gamma RIII is required for optimal T helper type (Th)2 responses and Th2-mediated airway inflammation. J Exp Med 204: Baskin, C. R., H. Bielefeldt-Ohmann, A. Garcia-Sastre, T. M. Tumpey, N. Van Hoeven, V. S. Carter, M. J. Thomas, S. Proll, A. Solorzano, R. Billharz, J. L. Fornek, S. Thomas, C. H. Chen, E. A. Clark, K. Murali-Krishna, and M. G. Katze Functional genomic and serological analysis of the protective immune response resulting from vaccination of macaques with an NS1-truncated influenza virus. J Virol 81: Belshe, R. B., and W. C. Gruber Safety, efficacy and effectiveness of cold-adapted, live, attenuated, trivalent, intranasal influenza vaccine in adults and children. Philos Trans R Soc Lond B Biol Sci 356: Bergmann, M., A. Garcia-Sastre, E. Carnero, H. Pehamberger, K. Wolff, P. Palese, and T. Muster Influenza virus NS1 protein counteracts PKR-mediated inhibition of replication. J Virol 74: Brown, D. M., E. Roman, and S. L. Swain CD4 T cell responses to influenza infection. Semin Immunol 16: Chen, Z., Y. Li, and R. M. Krug Influenza A virus NS1 protein targets poly(a)-binding protein II of the cellular 3'-end processing machinery. Embo J 18:

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25 FIGURE LEGENDS Figure 1. Replication of wild-type and NS1 mutant human influenza viruses in human respiratory cells. (A) 1.0 x 10 6 A549 cells were mock infected (NI) or infected with TX WT, TX 1-99 and TX viruses at a MOI of 2 for 10hrs. Total cell lysates were made and western blot was performed for viral HA, NP, M1 and NS1 proteins. Actin is shown as 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 viruses at a MOI of 2 at the shown times, cells were fixed and stained with anti-influenza NP antibodies and anti-b-tubulin antibodies conjugated with FITC (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 hpi as detected by western blot. Triplicate lanes represent the same sample loaded in triplicate as a loading control. (D) Virus release over time from HTBE cells infected with TX WT and TX viruses at MOI of 2 or (E) an MOI of Error bars are the standard deviations derived from triplicate samples of one experiment. (F) Release of IFNα/β from HTBE cells infected with TX WT and TX viruses at MOI of 2. Antiviral activity was detected using a Vero cell bioassay.using NDV-GFP virus. The GFP fluorescence was measured. The level 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. Figure 2. Viral protein expression in influenza virus-infected human DCs. (A) Western blot of protein lysates from infected DCs. 1.0 x 10 6 human DCs were infected with TX WT, TX 1-99 and TX viruses at a MOI of 0.5 or 1 for 18hrs. Western blot are shown for viral NP and NS1 proteins. (B) Kinetics of viral protein expression in infected DCs. 2.0 x 10 6 human DCs were infected with TX WT, TX 1-99 and TX viruses at an MOI of 2 for 4, 8 and 16hrs. Total cell lysates were made and western blot was performed for viral HA, NP, M1 and NS1 proteins. Levels of cellular actin are shown as loading control. Results are representative of two different donors. Figure 3. Increased type I IFN and IFN-responsive gene expression in influenza virus-infected human DCs correlate with increased IRF3 activation. (A) Human DCs were mock infected (NI) or infected with TX WT, or with NS1 C-terminal deletion mutant viruses TX 1-99 or TX at a MOI of 2 for 4 and 8hrs. Total cell lysates were made and western blot 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 viruses TX 1-99 or TX at a MOI of 2 for 2, 4, 6, 8 and 16hrs. Total RNA was isolated from 5x10 5 cells and qrt-pcr was performed for IFNα and IFNβ mrna. (C) 2 x 10 6 human DCs were mock infected (NI) or infected with TX WT, or with NS1 C- terminal deletion mutant viruses TX 1-99 or TX at an MOI of 0.5 or 2 for 16hrs. Supernatants were then analyzed by ELISA for IFNβ. (D) Human DCs were mock infected (NI) or infected with TX WT, or with NS1 C- terminal deletion mutant viruses TX 1-99 or TX at a MOI of 2 for 2, 4, 6, 8 and 16hrs and qrt-pcr was performed for IFN responsive genes IP10, IFI56K and ISG54 as previously described. 25

26 Figure 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 viruses TX 1-99 or TX at a MOI of 2 for 2, 4, 6, 8 and 16hrs. Total RNA was isolated from 5x10 5 cells and qrt-pcr was performed for TNFα, IL6, IL12p35 and MIP1β mrnas. (B) Type I IFN and proinflammatory cytokine protein secretion in influenza virus-infected human DCs. 2 x 10 6 human DCs were mock infected (NI) or infected with TX WT, or with NS1 C-terminal deletion mutant viruses TX 1-99 or TX at an MOI of 0.5 or 2 for 16hrs. Supernatants were then analyzed by ELISA for TNFα and IL6. Error bars are the standard deviations derived from triplicate samples. This experiment is representative of three different donors. Figure 5. Levels of activation and T cell priming by influenza virus infected human DCs. (A) 5 x 10 6 human DCs were mock infected (NI) or infected with TX WT, or with NS1 C-terminal deletion mutant viruses TX 1-99 or TX at an MOI of 0.5 for 16hrs. Cells were then stained with anti-cd86-fitc for assessing CD86 expression by FACS analysis. (B) 1 x 10 6 human DCs were mock infected (NI) or infected with TX WT, TX 1-99 or TX at an MOI of 1 for 16hrs. Cells were then stained with rabbit anti-flu NP (and then goat anti-rabbit alexa 488) as well as anti-cd86-pe for assessing CD86 upregulation on infected DCs by FACS analysis. (C) Stimulation of T cells by influenza virus-infected human DCs. 2.0 x 10 4 human DCs mock infected (NI) or infected with TX WT, or with NS1 C-terminal deletion mutant viruses TX 1-99 or TX at an MOI of 0.5 were incubated with 1.0 x 10 5 cells naïve CD4 T cells for 3 days. Supernatants from the co-cultures were then analyzed by ELISA for IFNγ. Results are representative of more than three indepenedent donors. Figure 6. Human isolates of influenza A virus are potent inhibitors of type I IFN responses in human DCs. Samples of 1 x 10 6 cells were mock infected (NI) or infected with Influenza A/WYM, A/NY, TX WT, and PR8 influenza A virus at a MOI of 0.5 for 16hrs. Total RNA was isolated from samples and analyzed by qrt-pcr for the expression of the indicated genes. (A) IFNα, IP10, MxA and IFI56K (B) IFNβ, STAT1, RIG-I. Error bars are the standard deviation of triplicate samples. Results are representative of at least three different donors. Figure 7. Replication of Influenza A/TX/91 viruses and A/PR8/34 viruses in human DCs. Human DCs were mock infected (NI) or infected with PR8, deltans1, TX WT and TX 1-99 at a MOI of 1 for 4 and 8hrs. (A) Total RNA was isolated from 1x10 5 cells and qrt-pcr was performed for viral NP and NS1 mrna. (B) Total cell lysates were made and western blot was performed for viral NP and NS1 proteins. Levels of cellular actin are shown as loading control. Results are representative of two different donors. Figure 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 a MOI of 1 for 4 and 8hrs. (A) Total cell lysates were made and western blot was performed for phosphorylated Rel A, IRF3 and c-jun as well as total Rel A, IRF3 and c-jun. (B) Supernatants were analyzed by ELISA for IFNα at 4, 8 and 12hrs post-infection. (C) Human DCs infected at a MOI of 1 for 1, 4 and 8hrs and harvested for total lysate for western blot 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. Figure 9. Difference in PR8 and TX WT-mediated T cell activation. (A) 2.0 x 10 4 human DCs mock infected (NI) or infected with PR8 or TX WT at an MOI of 1 were incubated with 1.0 x 10 5 cells naïve CD4 T cells for 2 and 4 26

27 days. Supernatants from the co-cultures were then analyzed by ELISA for IFNγ. Error bars are the standard deviation 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 at an MOI of 0.5 and incubated with naïve CD4 T-cells for 2, 4 and 6 days. Supernatants from the co-cultures were then analyzed by ELISA for IFNγ. Error bars are the standard deviation of triplicate samples. Results are representative of similar experiments with three different donors. Supplemental Figure 1. (A) Antiviral gene induction. Samples of 5 x 10 5 cells were mock infected (NI), infected with TX WT or TX for 6 or 18hrs. Total RNA was isolated from samples and analyzed by qrt-pcr for the expression of IFNβ, RIG-I, IFI56K and TNFα. Error bars are the standard deviation of single samples from two different donors. (B) Samples of 1 x 10 6 cells were mock infected (NI) or infected with TX WT, TX 1-99 or TX at a MOI of 2 for 16hrs. Total RNA was isolated from samples and analyzed by qrt-pcr for the expression of the indicated genes. (A) IFNα, MIP1β, IP10, RANTES, IFI56K and TNFα. Error bars are the standard deviation of triplicate samples. Results are representative of at least three different donors. Supplemental Figure 2. 1 x 10 6 human DCs were mock infected (NI) or infected with TX WT, TX 1-99 or TX at an MOI of 1 for 16hrs. Cells were stained with (A) anti-cd86-pe for assessing CD86 upregulation and (B) Flu NP for infectivity. Downloaded from on January 12, 2019 by guest 27

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