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Influenza A Virus Nucleoprotein Exploits Hsp40 to Inhibit PKR Activation

Introduction

Influenza A viruses (IAV) are negative sense segmented RNA genome viruses, which can rapidly develop resistance to the drugs available against them. These viruses pose a continuing threat of pandemics, thus it is imperative to develop novel strategies to prevent their infection and spread. Interactions between viral proteins and host factors are often crucial for successful replication of the virus in host cells. Many of these interactions are aimed at overcoming the early innate immune response of infected cells against the virus. Mammalian cells respond to viral infections through several innate immune mechanisms. One such crucial antiviral mechanism is activation of PKR (a dsRNA dependent protein kinase) which is phosphorylated upon encountering viral dsRNA. Activated PKR has several downstream substrates, one of which is the eukaryotic translation initiation factor 2 alpha subunit (eIF2α)–[11]. Phosphorylation of eIF2α by activated PKR renders it unable to participate in translation initiation leading to translation arrest and inhibition of protein synthesis from viral mRNAs,. Another effector function of PKR is activation of transcription factor IRF3, which leads to the expression of IFN β and inhibition of virus replication,. Being such a crucial component of the host innate immune system, PKR is tightly regulated by cellular inhibitors and very often targeted by viral proteins–[20]. For example, the non-structural protein 1 (NS1) of influenza virus directly binds to PKR and prevents its activation,. Apart from PKR inhibition, NS1 is also involved in the inhibition of other cellular signaling cascades, which lead to the activation of anti-viral interferon response,. PKR activity is also inhibited by a cellular 58 kDa protein, P58IPK, which promotes influenza viral replication,. In naïve cells, P58IPK exists in an inactive state in a complex with heat shock protein 40 (Hsp40), which becomes active upon release from this complex. Influenza virus infection leads to the dissociation of P58IPK from Hsp40 and suppression of the PKR response,. However, neither the viral component nor the mechanism responsible for this event is known to-date.

Segment 5 of the influenza virus genome encodes for 498 amino acids Nucleoprotein (NP) whose primary function is viral genome encapsidation. Apart from that, NP is also known to interact with several viral and host factors and play additional roles in the viral life cycle–[37]. We were interested in identifying new cellular interactors of NP from a highly virulent A/H5N1 bird-flu isolate {A/Hatay/2004(H5N1)}, which may facilitate viral replication. For this a H5N1 NP was used as bait to search for novel interactors in a yeast two-hybrid system based screen of human lung cDNA library. In the screen, we identified that IAV NP interacts with human chaperone heat shock protein 40 (Hsp40). Considering the known role of Hsp40 in regulation of PKR activity during influenza A virus infection, we explored the possibility of NP playing a regulatory role in this process. We observed that expression of IAV NP in mammalian cells lead to reduced phosphorylation of PKR and its substrate eIF2α. We thus hypothesize that influenza NP is the viral factor that facilitates the inhibition of PKR activation by releasing P58IPK from Hsp40-P58IPK complex. Consistent with this hypothesis, we observed that during IAV infection, the association of NP with Hsp40 coincided with the release of P58IPK from Hsp40. Also, RNAi-mediated inhibition of NP expression in IAV infected cells enhanced the phosphorylation of PKR and its downstream substrate eIF2α. NP inhibition also led to enhanced IRF3 phosphorylation and IFN β production which may be mediated by PKR activation. Collectively, these findings identify a novel role for influenza A virus NP in blocking the PKR-dependent antiviral response in influenza A virus infected cells.

Identification of human Hsp40 as an interacting partner of H5N1 IAV NP

A human lung cDNA library was screened using IAV NP as bait, in GAL4 based Matchmaker yeast two-hybrid system (Clontech). Yeast cells (AH-109) were co-transformed with bait and prey plasmids, and selected for growth on selective L-T-H- plates supplemented with 50 mM aminotriazole. β-galactosidase positive colonies were further analyzed (Fig. S1A). Plasmids from positive colonies were isolated and subjected to DNA sequencing followed by BLAST analysis to identify their cDNA insert. The mammalian chaperone heat shock protein 40 (Hsp40/DNAJB11) was thus identified as an interacting partner of NP. The strength of the NP-Hsp40 interaction was determined using a quantitative β-galactosidase assay, and was found to be comparable to the positive control used in the assay (Fig. S1B, bars 6 and 7 respectively, p-value = 0.0668).

Transiently expressed IAV NP interacts with Hsp40 in mammalian cells

The NP-Hsp40 interaction in mammalian cells was ascertained using co-transfection of plasmids coding H5N1 NP and Hsp40 in the HEK293T cells. Transfected cells were metabolically labeled with S35, and co-immunoprecipitation was performed using the lysates with either NP- or Hsp40- specific antibodies. These results showed that NP co-precipitated with Hsp40 and vice-versa (Fig. 1A, lane 2 and 4). These results were confirmed using A549 lung epithelial cells, which were transfected with NP expressing plasmid, followed by immunoprecipitation. It was observed that ectopically expressed NP could immunoprecipitate endogenous cellular Hsp40 and vice-versa (Fig. 1B, panel 1 and 2). A direct interaction between NP-Hsp40 was further confirmed using a co-immunoprecipitation assay in which 35S labeled NP and Hsp40 proteins were expressed from plasmids using in-vitro coupled transcription-translation rabbit reticulocyte lysate system (TNT, Promega, Inc) (data not shown). Collectively, these results showed that IAV NP directly interacts with Hsp40.

IAV NP interacts with Hsp40 in human lung epithelial cells infected with different pandemic and seasonal influenza A viruses

To validate the interaction between IAV-NP and Hsp40 in virus-infected cells, we investigated the kinetics of expression of NP and Hsp40 in A549 cells. A549 cells were infected with a laboratory-adapted IAV, A/Puerto Rico/8/1934/A (H1N1) influenza virus (PR8) at a multiplicity of infection (MOI) of 1. Although NP expression was maximal at 24 h post-infection, Hsp40 expression levels remained unchanged throughout the course of infection (Fig. 2A). Therefore, in subsequent experiments, A549 cells were infected with PR8 at an MOI of 1 for 24 h and lysates were prepared. A co-immunoprecipitation assay was performed using infected and control cell extracts. PR8 NP was able to co-precipitate endogenous Hsp40 (Fig. 2B, panel 2). Conversely, Hsp40 was able to co-precipitate NP (Fig. 2B, panel 1). The NP-Hsp40 interaction was also observed when A549 cells were infected with influenza virus isolates belonging to various subtypes (Table 1). Co-immunoprecipitation of proteins from A549 cells infected with these select viral isolates showed that NP co-precipitated with Hsp40 in all cases without exception (Fig. 2C, panel 1). A phylogenetic analysis of influenza NP genes used in the experiment was constructed using the Neighbor-Joining method, nucleotide model Tamura-Nei, in MEGA version 4 (Fig. S2). The diversity of changes in NP amino-acid sequences of the strains used in this study are shown in Fig. S3. The NP genes of IAV used in the study had amino-acid sequence divergence in the range of 1% to 10% from Hatay/H5N1/2004 isolate. These results clearly indicated that the NP-Hsp40 interaction was conserved among seasonal human, avian H5N1 and the 2009 H1N1 pandemic influenza A viruses.

IAV NP and Hsp40 co-localize primarily in the nucleus of mammalian cells

It is known that under stress conditions the expression level of Hsp40 is enhanced and its cellular localization changes from cytoplasmic to nuclear, however its distribution in influenza virus infected cells was not studied. Thus we investigated the cellular localization pattern of IAV NP in context to Hsp40 in mammalian cells. We transfected A549 cells with IAV NP expressing plasmid for 24 h, and an immunofluorescence staining was performed with specific antibodies. Results showed that NP and cellular Hsp40 colocalize primarily in the nucleus (Fig. 3A, Lower right panel). Similar results were obtained with A549 cells infected with PR8 virus. Confocal microscopy revealed that NP and Hsp40 were present primarily in the nucleus. However there was significant amount of NP present in the cytoplasm at 24 h post-infection (Fig. 3C, Lower right panel). We also observed that Hsp40 cellular levels were elevated after IAV infection, as compared to uninfected cells (Fig. 3 C and D, upper right panels).

NP expression and association with Hsp40 coincides with Hsp40-P58IPK dissociation and downregulation of PKR and eIF2α phosphorylation

Hsp40 is known to negatively regulate eIF2α phosphorylation through PKR (27). Therefore, we next assessed if changes in PKR and eIF2α phosphorylation occurred during the course of IAV infection of A549 cells. We found that the phosphorylation of both PKR and eIF2α increased initially between 1–2 h post-infection and subsequently declined between 4–8 h post-infection (Fig. 4A, panel 1,3). However the total PKR and eIF2α levels remained unchanged (Fig. 4A, panel 2,4). Interestingly, NP expression also was detected around 4 h post-infection and increased up to 8 h post-infection (Fig. 4A, panel 5) which coincided with a decline in p-PKR and p-eIF2α levels.

During IAV infection P58IPK activity was increased as it was released from Hsp40 binding. We hypothesized that NP might disrupt the P58IPK-Hsp40 complex too and liberate P58IPK. To investigate this possibility, we monitored changes in the cellular levels of P58IPK and NP associated with Hsp40 during IAV infection. A549 cells were infected with the PR8 virus, harvested at different time points after infection and immunoprecipitation was conducted using equal amounts of total protein and anti-Hsp40 antibody. Western blot analysis revealed that between 4 and 8 h post-infection, the levels of NP associated with Hsp40 continued to rise (Fig. 4B, panel 1) with a concomitant decline in P58IPK associated with Hsp40 (Fig. 4B, panel 2). During this period, total amounts of Hsp40 and P58IPK remained constant (Fig. 4B, panel 5, 6). These results were consistent with the hypothesis of replacement of P58IPK from Hsp40, by NP. These findings indicate that the dissociation of P58IPK-Hsp40 complex occurs around 4 to 8 h post-infection, and is associated with downregulation of eIF2α phosphorylation. Taken together, these results suggest that during IAV infection, NP induces the dissociation of the P58IPK-Hsp40 complex leading to an inhibition of PKR activation and downregulation of eIF2α phosphorylation.

IAV NP expression leads to an inhibition of PKR activity and downregulation of eIF2α phosphorylation

During IAV infection, the NS1 protein inhibits PKR activation by directly interacting with it, and thereby ensuring continued viral mRNA translation,. Results from our study indicated that NP may also play a role in inhibiting PKR activity by intercepting this pathway at the level of Hsp40. To examine this aspect, we performed a time course study in HEK293T cells transfected with the plasmid expressing IAV H5N1 NP by monitoring p-eIF2α and p-PKR levels. Western blot analysis of transfected cell lysate showed downregulation of both PKR and eIF2α phosphorylation, which began as early as 12 h and was most prominent at 36 h post-transfection (Fig. S4 A, B). The 36 h time-point was selected to confirm this action of NP, and Western blot analysis results from 3 independent experiments showed significant reduction in the levels of p-PKR (Fig. 5A, panel 2 and Fig. 5B) and p-eIF2α (Fig. 5C, panel 2 and Fig. 5D). To confirm the PKR inhibitory action of NP, we transfected HEK293T cells with NP-GFP expressing plasmid and inhibited NP expression using specific siRNA (Table 2) against it (Fig. S5A). Inhibition of NP expression led to an increase in p-PKR and p-eIF2α levels as compared to control NP-GFP transfected cells (Fig. S5B). These results suggest that the expression of NP leads to inhibition of PKR activity in a NS1 independent manner. This action of IAV NP is likely to be mediated through its interaction with Hsp40.

siRNA mediated inhibition of NP expression in IAV infected cells, leads to the upregulation of PKR, eIF2α and IRF3 phosphorylation

We suppressed NP expression PR8 IAV infected A549 cells, using a pool of gene specific siRNAs (Table 2), and determined its effect on PKR activity. Expression of NS1 was also blocked using siRNA against NS1 alone and in combination with NP siRNA to establish their exclusive and combined contribution to PKR-inhibition. A549 cells were first transfected with the indicated siRNAs, and 6 hours later were infected with PR8 influenza virus. Infected cells were harvested 24 h post-infection and cell lysates were subjected to western blot analysis. We observed that inhibition of NP expression led to upregulation of PKR phosphorylation, as compared to the control (Fig. 6A, panel 1, Fig. 6B). Increased PKR activity resulted in enhanced phosphorylation of eIF2α and IRF3 (Fig. 6A, panel 2, 4, Fig. 6C, D). Inhibition of NS1 had a similar effect, whereas inhibition of both NP and NS1 had a cumulative effect on upregulation of PKR, eIF2α, and IRF3 phosphorylation (Fig. 6B, C, D).

Inhibition of NP expression during IAV infection leads to enhanced IFNβ production and reduced virus replication

PKR mediated activation of IRF3 should lead to an increased IFN response. Previous results confirmed the involvement of NP in the inhibition of PKR and IRF3. To check the further downstream effect of NP, we suppressed the expression of NP using siRNAs as mentioned earlier. 24 hours post-infection, the cells were harvested and RNA was isolated to determine IFNβ and viral RNA levels by real-time PCR using gene-specific primers. The inhibition of NP expression led to increased IFNβ production as compared to control (Fig. 7A, bar 2, 3). Furthermore, the inhibition of NS1 had greater impact on IFNβ production as compared to that of NP, and there was a synergistic effect when both NS1 and NP were inhibited (Fig. 7A, bar 4, 5). Increased IFNβ production should lead to reduced virus replication and reduced production of Influenza vRNA. To confirm this, influenza vRNA levels in the above mentioned samples were measured by real-time PCR. Inhibition of NP or NS1 led to reduced vRNA production as compared to control (Fig. 7A, bar 2, 3, 4), and inhibition of both NP and NS1 together had greater synergistic effect (Fig. 7B, bar 5).

Discussion

Heat shock proteins are stress response factors which also regulate several cellular processes. The Hsp40 family chaperones are known to play important roles in protein folding, translocation, cell signaling and apoptosis–[42]. Very often they are targeted by viral components for successful virus replication. For example, Hsp40 is known to interact with HIV type 2 Vpx protein and facilitate nuclear import of the pre-integration complex. HIV type 1 Nef protein interacts with Hsp40 to enhance viral gene expression. Hsp40 is also known to interact with the HBV core protein and affect viral turnover. Heat shock proteins are known to affect the viral replication of influenza viruses also. For example Hsp90 is known to interact with influenza virus polymerase components and aid in viral RNA synthesis. Hsp70 is also known to be involved in the nuclear export of the RNP complex and play a role in temperature dependence of IAV replication,. Likewise, Hsp40 is also known to regulate PKR signaling in influenza virus infected cells. Similarly, the IAV NP is also a multifunctional protein that interacts with a wide variety of viral and cellular macromolecules, including RNA, PB1 and PB2 subunits of the viral RNA-dependent RNA polymerase and the viral matrix protein–[33]. It also binds to several host factors which include CRM1, UAP56, Alpha-importin 1 and NF90–[37]. Through these interactions, IAV-NP is known to encapsidate the viral genome, regulate virus transcription and replication, contribute towards pathogenicity of virus, and help in interspecies transmission of the virus. However, so far IAV NP is not reported to play any role in modulating the host antiviral response.

A key component of mammalian antiviral response mechanism is dsRNA dependent protein kinase PKR, which is activated by viral dsRNA. Upon activation, PKR gets dimerized and autophosphorylated at multiple serine and threonine residues. Activated PKR phosphorylates eukaryotic translation initiation factor eIF2α, which in phosphorylated state cannot participate in mRNA translation. This is an important strategy of the host to arrest translation of viral mRNAs thereby limiting viral replication,. Another crucial host pathway which is activated by PKR is IRF3-mediated IFNβ production. Activation of PKR is known to enhance IRF3 phosphorylation and nuclear movement where it drives expression of Interferon β production and built up of antiviral host response. Similarly, PKR also has other substrates such as MAPK and iKKß which upon phosphorylation trigger various signaling pathways leading to apoptosis or interferon response,. Being such a crucial molecule, PKR is very often the target of viral factors–[18]. In case of influenza virus infection, viral NS1 protein is known to bind directly to PKR and inhibit its activation,. NS1 also inhibits the function of retinoic acid inducible gene-I (RIG-I), a cytosolic pathogen sensor involved in the antiviral response. Apart from that, PKR activity is controlled by another mechanism where P58IPK, the cellular inhibitor of PKR is activated in influenza virus infected cells,. Further, P58IPK itself is inhibited by Hsp40 and is present as P58IPK-Hsp40 complex under normal conditions. However upon influenza virus infection, it is released from the Hsp40 complex and inhibits PKR activation. In a recent report, it was shown that M2 protein of influenza A virus stabilizes the P58IPK-Hsp40 complex and activates PKR phosphorylation, probably during later stage of infection. However the mechanism of dissociation of Hsp40-P58IPK complex and concomitant PKR inhibition during influenza virus infection remain unknown.

Here, we report that IAV NP interacts with the human chaperone Hsp40 and employs this interaction to mitigate PKR-mediated antiviral response of the host. NP-Hsp40 interaction was identified through a yeast two-hybrid screen and confirmed in a cell-free translation system, in transfected cells and in influenza virus infected cells. The interaction was found to be conserved across different influenza A viruses, ranging from seasonal, avian H5N1 virus and the 2009 H1N1 pandemic virus despite substantial amino acid differences that range from 0–5% within a subtype/group and 6–10% between the subgroups in NP amino-acid sequence. Our findings demonstrate that IAV NP is the viral component that dissociates P58IPK from the P58IPK-Hsp40 complex during influenza virus infection in mammalian cells. It was observed that during the course of influenza virus infection in lung epithelial cells, a gradual increase in the association of NP with Hsp40 coincided with a concomitant decrease in P58IPK association with Hsp40. Increased activity of P58IPK, promoted by NP, should lead to the inhibition of PKR activation and subsequent downstream effects (Fig. 8). In accordance with the above hypothesis, we observed that ectopic expression of IAV NP in mammalian cells substantially reduced the phosphorylation levels of PKR and eIF2α. Furthermore, siRNA-mediated inhibition of NP expression during influenza virus infection led to increased phosphorylation of PKR and eIF2α, confirming the role of NP in the negative regulation of PKR. Although eIF2α is phosphorylated by other kinases also, namely, HRI, GCN2 and PERK which are activated during stress condition, only PKR is known to be targeted by viral inhibitors. In line with this, NP and NS1 had similar effects on PKR mediated eIF2α phosphorylation; however their synergistic effect was higher than their individual effects (Fig. 6 B).

Activation of PKR signaling during virus infections is known to result in IRF3 phosphorylation and concomitant IFNβ production. However IRF3 is not a direct substrate of PKR and it can get activated by the RIG I pathway, NFκB pathway and other unknown mechanisms,. Influenza NS1 protein is known to inhibit PKR, RIG I and NFκB pathways, thus it is expected to have greater impact on IRF3 phosphorylation as compared to NP, which may inhibit only PKR mediated IRF3 phosphorylation,. In line with this, we observed that NP inhibition during IAV infection led to enhanced IRF3 phosphorylation, IFNβ production and reduced viral replication; however inhibition of NS1 had greater impact on these events. As expected the synergistic effect of NP and NS1 inhibition on IRF3 activity was higher than their individual effects. The effect of NP on IFNβ production is also reflected on virus replication as siRNA-mediated inhibition of NP led to reduced vRNA production. This effect may also be attributed to the essential requirement of NP for proper functioning of influenza virus polymerase. However the inhibitory action of NP on PKR-mediated host response may also contribute to the reduced virus replication in case of siRNA-mediated inhibition of NP.

Based on our findings, we proposed a model for PKR inhibition by influenza virus nucleoprotein as shown in Fig. 7. According to this model, IAV NP interacts with Hsp40 and facilitates the release of P58IPK from it, which in turn inhibits PKR activation (Fig. 8). Reduced PKR activity, on one hand leads to reduced eIF2α phosphorylation and ensures continued translation from viral mRNAs and on the other hand leads to reduced IRF3 mediated IFNβ production. Therefore, apart from the NS1 protein which is already known to inhibit PKR activation and IRF3 phosphorylation,, NP also participates in this process, but through a different mechanism involving Hsp40. With structure information of both NP and Hsp40 being available,, it would be interesting to see which domains and key amino-acid residues are involved in this interaction. Since the NP-Hsp40 interaction is conserved across influenza viruses of various subtypes including the 2009 pandemic H1N1 virus, it serves as an important target for developing anti-viral strategies.

Yeast Two-Hybrid Screening

GAL4 based Matchmaker (Clontech) yeast two-hybrid system was used for screening human lung cDNA library, as described previously. H5N1 NP (A/Hatay/2004) gene cloned in pGBK vector (Clontech) was used as bait and a mammalian cDNA library cloned in pGAD (Clontech) vector was used as prey. The AH109 strain of yeast was used for co-transformation of bait and prey plasmids. The full-length Hsp40 gene was cloned into pGAD vector and used in yeast two-hybrid assays. Colonies which grew on L-T-H- plates (Leucine, Tyrosine and Histidine dropout standard dextrose media) supplemented with 50 mM Aminotriazole were considered positive. ß-Gal assays (liquid and filter) were performed as per manufacturer's protocols.

Cell culture and Plasmids

HEK 293T (human embryonic kidney) and A549 (adenocarcinomic human alveolar basal epithelial) cells were used for transfection and infection experiments respectively. All cells were grown in DMEM medium (Hyclone) supplemented with 10% FCS (Hyclone), 100 units/ml Penicillin Streptomycin solution (Invitrogen). NP gene of A/ Hatay/ 2004 (H5N1) influenza virus was cloned in pCDNA3.1 myc his vector (Invitrogen), pEGFPN1 vector (Clontech) and pGBKT7 vectors (Clontech) to generate myc-tagged, GFP-tagged and GAL4 DNA BD fused NP, respectively. Full-length human Hsp40 gene cloned in the pCDNA3.1 myc his vector was kindly provided by M. D. Amaral (Centri de Genetica Humana Instituto Nacional de Saude Dr. Ricardo Jorge, Lisboa, Portugal).

Transfection and virus infection assays

All DNA transfections were done using Lipofectamine 2000 (Invitrogen) and cells were maintained in DMEM medium devoid of serum and antibiotics. Six hours post-transfection, culture medium was supplemented with 5% FCS and 24 h post-transfection the medium was replaced with fresh culture medium. All virus infections were done at multiplicity of infection (MOI) of 1 for 1 h in DMEM medium supplemented with 2% BSA (GIBCO). After 1 h incubation, the cells were washed with DMEM once and then grown with DMEM supplemented with 0.2% BSA and 1 µg/ml N-p-tosyl-1-phenyl alanine chloromethyl ketone (TPCK) (Sigma Aldrich). The virus strains used in infection experiments are listed in Table 1.

Western blotting and antibodies

Cells were lysed using a buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100) supplemented with protease-inhibitors (Roche Diagnostics) and the lysates were subject to SDS PAGE. Anti-NP antibodies were obtained from Abcam and the Immunology and Pathogenesis Branch, Influenza Division, Centres for Disease Control and Prevention, Atlanta, GA, USA. Antibodies against PKR, p-PKR, eIF2α, p-eIF2α, P58IPK and Hsp40 were obtained from Cell Signaling. Anti-ß-actin antibody was purchased from Sigma-Aldrich. Anti-myc tag and anti-NS1 antibodies were purchased from Santa Cruz.

Immunoprecipitation assays

Cellular lysates were incubated with primary antibody overnight followed by incubation with protein A Dyna beads (Invitrogen) for 2 hours. Beads were washed thrice and the IP products were subjected to Western blotting. NP was immunoprecipitated using anti-NP monoclonal antibody (Immunology and Pathogenesis Branch/IPB, CDC, Atlanta) in case of infection or anti-myc tag antibody in case of transfection. Hsp40 was immunoprecipitated using anti-Hsp40 monoclonal antibody (Cell Signaling).

Immunofluorescence microscopy

After infection or transfection for 24 h, A549 cells were fixed with 2% paraformaldehyde for 30 min at room temperature. They were permeabilized with 0.5% Triton X-100 for 5 min at room temperature and blocked with PBS containing 2% bovine albumin. Immunostaining was performed using rabbit anti-Hsp40 (Cell Signaling) and mouse anti-NP (IPB, CDC, Atlanta) antibodies. Unbound antibodies were washed away with PBS and cells were incubated with Alexa488 tagged Goat anti-rabbit antibodies and Alexa594 tagged Goat anti-mouse. Nuclei were stained with DAPI. Photomicrographs were captured at 100× magnification using a Leica DM6000B confocal microscope. Images were processed using NIS Elements AR 3.0 software (Nikon).

RNAi

Control (non-targeting) and NP- and NS1- specific siRNAs of PR8 were purchased from Dharmacon and the cells were transfected using the Dharmafect 1 transfection reagent (Dharmacon). In each case, a pool of three specific siRNAs capable of targeting different regions of NP or NS1 were used (Table 2). A549 cells at a density of 106/well of a 6-well plate were transfected with 90 nM of the indicated siRNA for 6 h prior to infection with A/PR/8/34 at a MOI of 1. Lysates were prepared 24 h post-infection and analyzed for the expression of NP, NS1 and other cellular proteins by Western blotting.

RNA isolation, cDNA preparation and Real time PCR

Total RNA was isolated from cells using the RNAeasy kit (Qiagen, Valencia, CA, USA) and real-time RT-PCR was conducted using a Stratagene Q3005 PCR machine for expression of IFNβ, β-actin mRNA and NP vRNA. For each sample, 2 µg of RNA was reverse transcribed using Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's directions. Oligo dT primers were used for IFNβ and β-Actin cDNA synthesis. For NP vRNA, cDNA was synthesized as described by Ge et al

[52]. (Parallel reactions without reverse transcriptase were included as negative controls. Reverse transcription reactions (1/50th of each reaction) were analyzed in using syber green Q-PCR reagents (Stratagene, La Jolla, CA, USA). PCR condition was kept as 94°C for 15 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s for a total of 45 cycles. The threshold cycle number for cDNA was normalized to that of β-actin mRNA, and the resulting value was converted to a linear scale. Data from three independent experiments were taken account for analysis. All data points fell into a normal distribution and there were no outliers. Primer sets used for these studies are provided in Table 3.