RIG-I Signaling Is Essential for Influenza B Virus-Induced Rapid Interferon Gene Expression

ABSTRACT

Influenza B virus causes annual epidemics and, along with influenza A virus, accounts for substantial disease and economic burden throughout the world. Influenza B virus infects only humans and some marine mammals and is not responsible for pandemics, possibly due to a very low frequency of reassortment and a lower evolutionary rate than that of influenza A virus. Influenza B virus has been less studied than influenza A virus, and thus, a comparison of influenza A and B virus infection mechanisms may provide new insight into virus-host interactions. Here we analyzed the early events in influenza B virus infection and interferon (IFN) gene expression in human monocyte-derived macrophages and dendritic cells. We show that influenza B virus induces IFN regulatory factor 3 (IRF3) activation and IFN-λ1 gene expression with faster kinetics than does influenza A virus, without a requirement for viral protein synthesis or replication. Influenza B virus-induced activation of IRF3 required the fusion of viral and endosomal membranes, and nuclear accumulation of IRF3 and viral NP occurred concurrently. In comparison, immediate early IRF3 activation was not observed in influenza A virus-infected macrophages. Experiments with RIG-I-, MDA5-, and RIG-I/MDA5-deficient mouse fibroblasts showed that RIG-I is the critical pattern recognition receptor needed for the influenza B virus-induced activation of IRF3. Our results show that innate immune mechanisms are activated immediately after influenza B virus entry through the endocytic pathway, whereas influenza A virus avoids early IRF3 activation and IFN gene induction.

IMPORTANCE Recently, a great deal of interest has been paid to identifying the ligands for RIG-I under conditions of natural infection, as many previous studies have been based on transfection of cells with different types of viral or synthetic RNA structures. We shed light on this question by analyzing the earliest step in innate immune recognition of influenza B virus by human macrophages. We show that influenza B virus induces IRF3 activation, leading to IFN gene expression after viral RNPs (vRNPs) are released into the cytosol and are recognized by RIG-I receptor, meaning that the incoming influenza B virus is already able to activate IFN gene expression. In contrast, influenza A (H3N2) virus failed to activate IRF3 at very early times of infection, suggesting that there are differences in innate immune recognition between influenza A and B viruses.

INTRODUCTION

Influenza A and B viruses are important respiratory pathogens and cause seasonal epidemics with an estimated 250,000 to 500,000 deaths annually. Influenza A and B viruses are structurally similar: they are negative-sense RNA viruses with a single-stranded segmented genome. The genome is structured in eight viral ribonucleoprotein (vRNP) complexes where the single-stranded RNA (ssRNA) is associated with multiple nucleoprotein (NP) molecules and a polymerase complex consisting of the PB1, PB2, and PA proteins (1). The vRNP complexes are packaged in a matrix protein shell surrounded by a host-derived lipid envelope in which the viral glycoproteins hemagglutinin (HA) and neuraminidase (NA) are embedded. Influenza viruses bind to sialic acids on cell surface glycoproteins and enter the cells mainly via clathrin-mediated endocytosis but also by macropinocytosis and clathrin-independent entry pathways (2, 3). Influenza viruses take advantage of the host endocytic pathway; a reduction of pH during the maturation of endosomes induces a conformational change in viral HA molecules and triggers fusion between viral and endosomal membranes. Fusion is followed by the uncoating of the capsid by M1 dissociation due to acidification of the virion via the M2 ion channel protein. This results in the release of vRNPs into the cytosol. The influenza virus genome is then imported into the nucleus for transcription and replication of viral genes. Primary transcription of the viral genome is triggered by the virion-associated polymerase protein complex, which leads to the translation of early viral proteins in the cell cytoplasm. Newly synthesized polymerase, NP, and NS1 proteins are transported into the nucleus, where they initiate and regulate the replication and synthesis of cRNA and viral RNA (vRNA) molecules, followed by secondary rounds of transcription. At later stages of infection, new vRNP complexes are packaged in the nucleus, followed by M1- and nuclear export protein (NEP)-regulated export of vRNPs into the cytoplasm. Here they associate with viral envelope glycoproteins HA and NA on the plasma membrane, leading to budding of the newly formed viral particles (4).

Host cells respond to influenza virus infection by producing interferons (IFNs) and antiviral proteins, thus establishing an antiviral cellular state to restrict the spread of infection. The most important cellular sensors for RNA viruses are cytosolic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), RIG-I, and melanoma differentiation-associated protein 5 (MDA5), which recognize and bind virus-derived ssRNA and double-stranded RNA (dsRNA) structures (5–7). Endosomal Toll-like receptors (TLRs), such as TLR3 and TLR7/8, also recognize viral dsRNAs and ssRNAs, respectively (8–11). RLRs and TLRs regulate IFN and other proinflammatory cytokine responses during influenza virus infection in certain cell types. However, the point in the influenza virus entry and/or replication cycle at which viral RNA is sensed and IFN gene expression is induced remains unclear. One study suggested that influenza A virus (IAV) RNA synthesis and nuclear export but not viral replication trigger IFN gene expression (12). Other studies have proposed that RIG-I can recognize the incoming negative-sense RNA virus via its 5′-triphosphorylated genomic RNA, even though it is bound by NP proteins and the viral RNA polymerase complex (13–15). It has also been suggested that the incoming influenza A virus carries certain viral structures that inhibit RIG-I activation and IFN induction, even though the viral RNA is recognized by RIG-I at the time of entry into the cytosol (16).

Upon binding of ssRNA or some dsRNA molecules, RIG-I oligomerizes and associates with the adaptor protein mitochondrial antiviral signaling protein (MAVS) on the mitochondrion-associated membrane (MAM) and the outer membrane of mitochondria (17, 18). This activates a signaling cascade, which leads to serine/threonine-protein kinase TBK1/IκB kinase ε (IKKε)-mediated phosphorylation and nuclear translocation of the transcription factor interferon regulatory factor 3 (IRF3) and induction of IFN genes (18, 19). RIG-I signaling can also induce the activation of the classical kinases IΚΚα and IΚΚβ, which promote signaling by the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways (19). To counteract virus-induced activation of IFN gene expression, influenza A and B viruses have evolved mechanisms to inhibit the activation of innate immunity. The best-known conserved virulence factor of influenza A and B viruses is the multifunctional NS1 protein, which can inhibit RIG-I signaling and interfere with the processing of host cell pre-mRNA molecules (20). Influenza A virus also encodes additional virulence factors (PB1-F2, PB1-N40, and PA-X proteins) that antagonize innate immune responses (21–23).

Most information on the influenza virus life cycle and immune restriction has been obtained from influenza A virus research, and it has been presumed that influenza B virus (IBV) behaves in a similar fashion. However, we suggest that there may be differences between the viruses regarding the mechanisms of activation of IFN gene expression. We have shown previously that influenza B virus induces a very rapid IFN response in human monocyte-derived dendritic cells (moDCs) that does not depend on the transcription or replication of the virus (24). Here we show that early influenza B virus-induced IFN gene expression is RIG-I dependent and takes place after the vRNPs are released into the cytosol. These conclusions are supported by detailed gene expression analyses, cell biological approaches, and the use of RIG-I and MDA5 knockout (KO) mouse cell lines.

MATERIALS AND METHODS

Ethics statement.

Adult human blood was obtained from anonymous healthy blood donors through the Finnish Red Cross Blood Transfusion Service (permission no. 29/2014, renewed annually). Animal immunizations related to this study were approved by the Ethical Committee of the National Institute for Health and Welfare (permission no. KTL 2008-02).

Cell cultures.

Monocytes were purified from freshly collected, leukocyte-rich buffy coats obtained from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland), as described previously (25). Human peripheral blood mononuclear cells were isolated by density gradient centrifugation over a Ficoll-Paque gradient (Amersham Biosciences). To obtain monocytes for macrophage differentiation, mononuclear cells were allowed to adhere to plates (Falcon; Becton Dickinson) for 1 h at +37°C in RPMI 1640 (Sigma-Aldrich) supplemented with 0.6 μg/ml penicillin, 60 μg/ml streptomycin, 2 mM l-glutamine, and 20 mM HEPES. Nonadherent cells were removed by washing with cold phosphate-buffered saline (PBS), and the remaining monocytes were cultured in macrophage/serum-free medium (Life Technologies) supplemented with antibiotics and human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) (10 ng/ml; Nordic Biosite). The cells were differentiated into macrophages for 7 days, with a change to fresh culture medium every 2 days. In each experiment, cells from 3 to 4 different donors were grown and used separately for infection experiments.

To obtain moDCs, Ficoll-Paque gradient centrifugation was followed by Percoll gradient (Amersham Biosciences) centrifugation. The top layer containing monocytes was collected, and the remaining T and B cells were depleted by using anti-CD3 and anti-CD19 magnetic beads (Dynal). Monocytes were allowed to adhere to plates for 1 h at +37°C in RPMI 1640 supplemented as described above. Adherent monocytes were washed with PBS, and immature moDCs were generated by cultivating cells in RPMI 1640 supplemented with 10% fetal calf serum (FCS) (Integro), 10 ng/ml human rGM-CSF, 20 ng/ml human recombinant interleukin-4 (rIL-4) (R&D Systems), and antibiotics. The cells were cultivated for 6 days, and fresh medium was added every 2 days.

Primary wild-type (wt) Irf3^−/− Irf7^−/− mouse embryonic fibroblasts (MEFs) and the Rela^−/− Rel^−/− Nfκb1^−/− (NF-κB KO) MEF cell line were kindly provided by A. Hoffmann, Signaling Systems Lab, Los Angeles, CA. Primary wild-type (Rig-I^+/+), Rig-I^−/−, Mda5^−/−, and Rig-I^−/− Mda5^−/− MEFs were obtained from mouse embryos, and they were immortalized by using a rigorous passaging protocol to obtain wt and KO cell lines. The MEF cell lines were cultured in DMEM supplemented with 0.6 μg/ml penicillin, 60 μg/ml streptomycin, 2 mM l-glutamine, 20 mM HEPES, and 10% FCS.

Viruses and infection experiments.

Human influenza A virus A/Beijing/353/89 (H3N2) (A/Beijing), influenza B virus B/Shangdong/7/97 (B/Shangdong), and Sendai virus (strain Cantell) were grown from a 10⁻⁵ dilution of stock virus in allantoic cavities of 11-day-old embryonated chicken eggs at +34°C for 3 days. Influenza stock virus titers were determined by a plaque assay on MDCK cells and were as follows: 6 × 10⁷ PFU/ml for A/Beijing and 18 × 10⁷ PFU/ml for B/Shangdong. The multiplicity of infection (MOI) was based on the titers determined in MDCK cells. The Sendai virus stock titer (6 × 10⁹ PFU/ml) in moDCs was previously determined by flow cytometry (26).

MoDCs, macrophages, and MEFs were infected with influenza viruses for different times, as indicated in the figures. To obtain a synchronized infection for immunofluorescence assays, the viruses were adhered to cells on ice for 1 h, after which the medium was replaced with warm medium (37°C) to allow the viruses to enter the cells. For infectivity experiments in macrophages, the cells were washed, and medium was replaced 1 h after infection. If inhibitors were used, they were added to the cells 30 min before infection. Inhibitors were purchased from Sigma-Aldrich, and the pretitrated, functional concentrations used were 10 nM bafilomycin A1 (BafA1), 80 μM Dynasore (27, 28), and 10 μg/ml cycloheximide (CHX). For lipopolysaccharide (LPS) stimulation, 100 ng of Escherichia coli serotype O111:B4 LPS (Sigma-Aldrich)/well was used. Cells were harvested, and samples for quantitative reverse transcriptase PCR (qRT-PCR), immunoblotting, or immunofluorescence were prepared.

qRT-PCR.

Cells from different blood donors were harvested and pooled after virus infection, and total cellular RNA was isolated by using the RNeasy minikit (Qiagen), including DNase digestion (RNase-free DNase kit; Qiagen). One microgram of total cellular RNA was reverse transcribed into cDNA in TaqMan RT buffer with 5.5 mM MgCl₂, 500 μM deoxynucleoside triphosphates (dNTPs), 2.5 μM random hexamers, 0.4 U/μl RNase inhibitor, and 1.25 U/μl MultiScribe reverse transcriptase (Applied Biosystems). cDNA samples were then amplified in TaqMan universal PCR master mix buffer (Applied Biosystems) with Gene Expression system assay mix oligonucleotides (Applied Biosystems) to analyze mRNA levels for human IFN-β1 (identification number Hs00277188_s1), IFN-λ1 (Hs00601677_g1), RIG-I (Hs00225561_m1), and IFITM3 (Hs01922752_s1); mouse IFN-β1 (Mm00439552_s1), CXCL10 (Mm00445235_m1), and tumor necrosis factor alpha (TNF-α) (Mm00443260_g1); and IAV or IBV NP and NS1 (24). Each cDNA sample was amplified in duplicate or triplicate with Mx3500P (Agilent Technologies). The mRNA levels were normalized against human 18S rRNA or mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with TaqMan endogenous control kits (Applied Biosystems), and the amounts of cytokine mRNAs relative to those in unstimulated cells were calculated with the ΔΔC_T method, as instructed by the manufacturer.

Immunoblotting.

For protein analyses, cells from different blood donors were collected, washed with PBS, and pooled, and whole-cell lysates were prepared in passive lysis buffer from the Dual Luciferase assay kit (Promega) containing 1 mM Na₃VO₄ and Complete protease inhibitor (Roche). MEF cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate [pH 7.2]) containing 1 mM Na₃VO₄ and Complete protease inhibitor (Roche). Total cellular proteins were denatured in Laemmli sample buffer and boiled. Equal amounts of proteins were separated on SDS-PAGE gels and transferred onto Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were blocked for 1 h at room temperature (RT) with 5% milk in PBS (blocking buffer). Rabbit antibodies against human IRF3 and influenza A virus NS1 were prepared as described previously (26, 29). The antibody against influenza A virus NP was prepared in rabbits by immunizing the animals four times at 4-week intervals with an Escherichia coli-expressed glutathione-Sepharose-purified (Amersham Biosciences) glutathione S-transferase (GST)–NP fusion protein (100 μg/immunization/animal). The antibodies against influenza B virus NP and NS1 were prepared similarly except that 35 μg of baculovirus-expressed, histidine-tagged antigen purified with preparative SDS-PAGE and 20 μg of baculovirus-expressed preparative SDS-PAGE-purified antigen with Freund's adjuvant were used, respectively. Staining was performed in blocking buffer at room temperature for 1 h. Antibodies for human Ser396-phosphorylated IRF3 (p-IRF3) (catalog number 4947), IRF3 (catalog number 4302), IκBα (catalog number 9242), and GAPDH (catalog number 2118) were obtained from Cell Signaling Technology, and all these antibodies also recognize mouse proteins. Staining was performed with PBS containing 5% bovine serum albumin (BSA) at 4°C overnight. Antibody against IFITM3 (catalog number AP1153a) was obtained from Abgent, and antibody against actin (catalog number sc-10731) was obtained from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated antibodies (Dako) were used for secondary staining at RT for 1 h. Protein bands were visualized on HyperMax films by using an ECL Plus system (GE Healthcare).

Immunofluorescence microscopy.

Macrophages were differentiated on glass coverslips, infected with influenza viruses at a multiplicity of infection (MOI) of 30, and incubated on ice for 1 h, after which the virus inoculum was removed and replaced with warm medium to allow virus entry into the cells at 37°C with 5% CO₂. After the indicated times, the cells were fixed with 3% paraformaldehyde at room temperature for 20 min, washed with PBS, permeabilized with 0.1% Triton X-100 for 5 min, and washed and blocked with 0.5% BSA in PBS for 30 min. The cells were stained with guinea pig anti-NP antibodies (1:400 dilution) prepared similarly to the corresponding rabbit antibodies used for the immunoblotting method described above, rabbit anti-IRF3 (1:50 dilution), mouse anti-LAMP1 (catalog number sc-20011; Santa Cruz Biotechnology), or mouse anti-EEA1 (catalog number 610456; BD Biosciences) for 45 min in PBS containing 0.5% BSA at 37°C. Secondary antibodies were fluorescein isothiocyanate (FITC)-labeled goat anti-guinea pig, rhodamine Red-X-labeled goat anti-rabbit, and rhodamine-labeled goat anti-mouse diluted 1:100 (Jackson ImmunoResearch Laboratories). In infectivity experiments, NucBlue Fixed Cell ReadyProbes reagent (Life Technologies) was added to Alexa Fluor 488 (Life Technologies) secondary antibody to stain the nucleus. The coverslips were washed with PBS containing 0.05% Tween 20 and then washed with distilled water and mounted in 25% Mowiol (Polysciences) in a solution containing 25 mM Tris-HCl (pH 7.5), 50% glycerol, and 2.5% 1,4-diazabicyclo(2,2,2)octane. The cells were visualized under a Leica TCS SPE confocal microscope with a 63× 1.40-numerical-aperture (NA) oil objective with a pinhole of 1 airy unit, maintaining the same image acquisition settings for all acquired images. The cells in the infectivity experiments were analyzed with a Zeiss Stallion fluorescence microscope with a Hamamatsu ORCA-Flash 4.0 LT sCMOS camera and a 20× 0.4-NA objective by using Slidebook 6 software (Intelligent Imaging Innovations).

RESULTS

Influenza A and B viruses induce IFN gene expression with different kinetics in human macrophages.

We have shown previously that influenza B virus induces IFN gene expression more rapidly than does influenza A virus in human moDCs and A549 cells (24). To extend these studies further, we analyzed IFN responses in human monocyte-derived macrophages. These primary cells were infected with influenza A/Beijing/353/89 and B/Shangdong/7/97 viruses at an MOI of 5, based on the titers determined with a plaque assay in MDCK cells. The expression levels of IFN-λ1, IFN-β, viral NP and NS1, RIG-I, and IFITM3 mRNAs were analyzed by qRT-PCR at different time points. In these human macrophages, influenza B virus induced maximal IFN-λ1 and IFN-β mRNA expression within 2 to 4 h, whereas the maximum IFN mRNA levels after influenza A virus infection were seen at 16 to 24 h (Fig. 1A). No substantial differences were observed in viral NP gene expression, suggesting that both viruses replicated equivalently in macrophages. The kinetics of expression of the IFN-inducible genes RIG-I and IFITM3 after influenza A and B virus infection mirrored the kinetics of IFN mRNA expression. The effect of virus dose on the kinetics of IFN-λ1 and NP gene expression was studied with MOIs of 1, 5, 25, and 125 in macrophages (Fig. 1B). Even though increasing virus amounts accelerated early IFN-λ1 gene expression, influenza B virus was clearly more efficient in inducing IFN-λ1 at the 2-h time point. The MOI values are presented as titers determined in MDCK cells throughout the study to be able to better compare the virus amount used for different cell types. We also confirmed infectivity in macrophages from four different donors by an immunofluorescence assay (Fig. 1C). Based on qRT-PCR data, influenza A and B viruses appeared to replicate equally well in human macrophages (Fig. 1B), while influenza B virus showed apparently 5-fold better infectivity than influenza A virus, as judged by an immunofluorescence assay (Fig. 1C).

FIG 1.

Kinetics of influenza virus-induced IFN gene expression in human macrophages. (A) Human monocyte-derived macrophages were infected with influenza virus strains A/Beijing/353/89 (IAV) and B/Shangdong/7/97 (IBV) at an MOI of 5 for different times, as indicated. Cells from 3 donors were pooled; total cellular RNA was isolated; IFN, viral NP and NS1, RIG-I, and IFITM3 gene expression was measured by qRT-PCR; and fold induction over the mock-infected sample or 1-h-infected sample (NP and NS1) was calculated. (B) Macrophages from four donors were infected, as described above, at an MOI of 1, 5, 25, or 125 for different times, and IFN-λ1 and viral NP gene expression was analyzed by qRT-PCR. The results are presented as the means (+1 standard deviation of the means) of data from two to three technical replicates and are from a representative experiment out of three (A) or two (B). (C) Macrophages were grown on coverslips and infected with viruses as described above, at MOIs ranging from 25 to 0.04 for 16 h. The coverslips were prepared for immunofluorescence analysis and stained with guinea pig anti-influenza A or B virus NP and nuclear staining using Alexa Fluor 488-conjugated anti-guinea pig secondary antibody. The mean percentages of NP-positive cells from four donors and one standard deviation are presented, and altogether, cells from eight donors were analyzed.

Influenza virus-induced early IFN gene expression correlates with activation of IRF3.

IRF3 is a key transcription factor regulating early IFN gene expression, with NF-κB having a contributing role. To analyze whether there were differences in the kinetics and extent of activation of these transcription factors in influenza A and B virus-infected macrophages, we examined the phosphorylation status of IRF3 and the degradation of NF-κB inhibitor α (IκBα) by Western blotting (WB) at different time points of infection (Fig. 2). In addition, the protein expression of IFN-inducible MxA and viral NP and NS1 was studied. Also, the nuclear accumulation of IRF3 in macrophages was visualized by immunofluorescence microscopy (Fig. 3). The phosphorylation of IRF3 correlated with the kinetics of IFN-λ1 and IFN-β gene expression (Fig. 1A), as the phosphorylation of IRF3 was apparent at 1 h and peaked at the 2-h time point in influenza B virus-infected macrophages (Fig. 2). In comparison, influenza A virus-induced IRF3 phosphorylation was visible at 4 h and was further enhanced at the 8- and 24-h time points. The degradation of IκBα, which precedes the activation of NF-κB, was weakly detectable at the 2-h time point for influenza B virus-infected cells and at the 8- and 24-h time points in influenza A virus-infected cells.

FIG 2.

IRF3 activation correlates with an early influenza B virus-induced IFN response. Macrophages were infected with influenza A/Beijing/353/89 and B/Shangdong/7/97 viruses (MOI of 5), and cells from 3 donors were collected at different time points and pooled. Whole-cell lysates were prepared for SDS-PAGE, and immunoblot analysis with antibodies against p-IRF3, IκBα, IRF3, influenza A and B virus NP and NS1, and MxA was carried out. Actin and GAPDH protein levels were analyzed to control equal loading of the samples. The data are representative of results from two independent experiments.

FIG 3.

IRF3 and influenza B virus NP accumulate in the nucleus simultaneously in macrophages. Human macrophages were differentiated on coverslips, followed by infection with influenza B/Shangdong/7/97 (A) and influenza A/Beijing/353/89 (B) viruses at an MOI of 30 on ice for 1 h. Subsequently, virus entry proceeded after warming of the cells to 37°C, and cells were fixed with paraformaldehyde after the indicated incubation times. Guinea pig anti-NP antibodies were used to detect influenza A or B virus NP (green), rabbit anti-human IRF3 was used to stain for IRF3 (red), and secondary antibodies were FITC-labeled donkey anti-guinea pig and rhodamine Red-X-labeled donkey anti-rabbit immunoglobulins. The second rows of images show higher magnifications of insets of NP staining indicated by white rectangles. Data shown are representative of results from four independent experiments on cells from 11 donors.

In immunofluorescence microscopy experiments, macrophages were infected with influenza A and B viruses at a high MOI of 30 and placed on ice for 1 h to obtain a synchronized infection. The high MOI was used in order to detect the incoming virus with the NP antibody and to confirm that all the cells were infected. The infection and internalization of viruses were triggered by increasing the temperature to 37°C, and nuclear accumulation of viral NP and IRF3 in the nucleus was monitored over a 3-h time course. One hour after warming of cells, IRF3 was observed in the nucleus in influenza B virus-infected macrophages (Fig. 3A), whereas in influenza A virus-infected cells, IRF3 nuclear translocation was not detected during the early phase (Fig. 3B). By 3 h after synchronized infection, influenza A and B virus replication occurred, since viral NP accumulated in the nucleus.

IRF3 and IRF7 transcription factors are indispensable for influenza B virus-induced IFN-β and proinflammatory cytokine gene expression.

To define the signaling pathway(s) responsible for influenza B virus-induced IFN/cytokine gene expression, we took advantage of a cell line generated from MEF cells lacking functional IRF3 and IRF7 or NF-κB genes. wt and IRF3/7 double-KO cells were infected with influenza A and B viruses at an MOI of 15 for different times, and cytokine gene expression was analyzed by qRT-PCR. A high MOI of 15, compared to the MOI of 5 in macrophages, was used because the mouse cells appeared to be more resistant to human influenza viruses. In wt mouse fibroblasts, influenza B virus induced maximal IFN-β gene expression by 3 h, whereas in influenza A virus-infected cells, IFN-β gene expression did not peak until 24 h after infection (Fig. 4A). As expected, neither influenza B nor influenza A virus induced IFN-β gene expression in IRF3/7 KO cells, although both viruses infected wt and KO cells with almost equal kinetics and efficacies, as analyzed by monitoring viral NP and NS1 expression by WB and qRT-PCR (Fig. 4A and B). We also analyzed the expression of CXCL10 and TNF-α mRNAs and found that in influenza B virus-infected IRF3/7 KO cells, the expression of both of these genes was also absent. However, influenza A virus infection readily induced the expression of CXCL10 and TNF-α mRNAs in both wt and IRF3/7 KO cells. We also analyzed the phosphorylation of IRF3 in mouse cells by WB; although we found weaker IRF3 phosphorylation in general, there was a clear kinetic difference between influenza A and B viruses (Fig. 4B), as seen in human macrophages. In wt MEFs, IκBα degradation occurred at the 1-h time point in influenza A virus-infected cells, whereas no clear degradation of IκBα was observed in influenza B virus-infected cells. We also analyzed IFN-β gene expression in triple-KO MEFs lacking the NF-κB components p65, c-Rel, and p105. Early IFN-β gene expression was reduced to some extent (10- to 40-fold) in influenza B virus-infected NF-κB KO cells (Fig. 4C).

FIG 4.

IRF3 and IRF7 transcription factors are essential for influenza virus-induced IFN gene expression. (A and B) wt MEFs and MEFs lacking IRF3 and IRF7 transcription factors (IRF3/7) were infected with influenza A/Beijing/353/89 and B/Shangdong/7/97 viruses at an MOI of 15 for the indicated times. RNA samples were collected for analysis of gene expression of the IFN-β, viral NP and NS1, TNF-α, and CXCL10 genes by qRT-PCR (A), and protein samples were prepared for immunoblot analysis with antibodies against p-IRF3, IRF3, IκBα, and influenza A and B virus NP and NS1 (B). GAPDH protein levels were analyzed to control equal loading. (C) wt MEFs and MEFs lacking the NF-κB components p65, c-Rel, and p105 (NF-κB) were infected with influenza A/Beijing/353/89 and B/Shangdong/7/97 viruses at an MOI of 15 for the indicated times. RNA samples were collected for analysis of gene expression of IFN-β and viral NP genes by qRT-PCR. The qRT-PCR data are presented as relative gene expression levels for the viral NP gene in relation to that of the 30-min or 1-h sample and for cytokine genes in relation to those of the mock-infected samples. The results are presented as means from triplicate measurements with 1 standard deviation, and data from representative experiments out of two are shown.

IRF3 is activated after influenza B vRNPs are released into the cytosol from the endosomal pathway.

To correlate the stage in the viral life cycle with IRF3 activation by influenza B virus, we analyzed influenza B virus-infected macrophages at 15-min intervals and stained cells for viral NP protein (green) to visualize virus entry and IRF3 (red) to define its nuclear accumulation (Fig. 5). Viruses were allowed to attach to the cells on ice for 1 h, followed by a change of medium (37°C) and a chase for up to 3 h. In confocal images at the 0-min time point, NP was localized at the plasma membrane. Within 15 min of incubation at 37°C, we detected green granules inside the cells, and between 30 and 45 min, some NP was localized in the nucleus. At 1 h, viral NP accumulated in the nucleus, and at 3 h, newly synthesized NP was seen. We confirmed that NP at the 3-h time point was nascently generated, since enhanced accumulation of NP in the nucleus was not seen in CHX-treated cells (Fig. 5). The activation and nuclear import of IRF3 occurred within 45 to 60 min and correlated with the entry of viral vRNPs into the nucleus. This suggested that IRF3 is activated after influenza B vRNPs are released into the cytoplasm. To confirm this hypothesis, we used a specific inhibitor of vacuolar H⁺ ATPase, BafA1, which prevents the acidification of the endosomes required for influenza virus entry into the cytosol. In the presence of BafA1, nuclear accumulation of viral NP or IRF3 was not detected (Fig. 5, bottom).

FIG 5.

Endosomal acidification but not de novo protein synthesis is needed for influenza B virus-induced IRF3 activation. Human macrophages were differentiated on coverslips, followed by infection with influenza B/Shangdong/7/97 virus at an MOI of 30 on ice for 1 h. Subsequently, virus entry proceeded after warming of cells to 37°C, and cells were fixed with paraformaldehyde after the indicated incubation times. The inhibitors BafA1 and CHX or the dimethyl sulfoxide (DMSO) vehicle was added to the cells 30 min before infection. Guinea pig anti-NP antibodies were used to detect influenza B virus NP (green), rabbit anti-human IRF3 was used to stain IRF3 (red), and FITC-labeled donkey anti-guinea pig and rhodamine Red-X-labeled donkey anti-rabbit immunoglobulins were used as secondary antibodies. Below each frame, a higher-magnification image of the inset of one cell marked with a white rectangle is presented. Data shown are representative of results from two independent experiments on cells from 6 donors.

To link these findings to innate immune signaling phenotypes, we analyzed IFN-λ1 gene expression in BafA1-treated macrophages (Fig. 6A) and moDCs (Fig. 6B). BafA1 treatment reduced the early phase of influenza B virus-induced IFN-λ1 gene expression. The phosphorylation of IRF3 was also reduced in BafA1-treated cells at the 2-h and 3-h time points in moDCs (Fig. 6B, left), consistent with the observed inhibition of nuclear translocation of IRF3 in macrophages (Fig. 5). A dynamin inhibitor, Dynasore, which prevents clathrin-mediated endocytosis, also blocked the early phase of influenza B virus-induced IFN-λ1 gene expression and IRF3 phosphorylation in moDCs (Fig. 6B, left). As a control, we treated cells with LPS, which is recognized by TLR4 and does not need endosomal acidification for signaling. As expected, BafA1 did not inhibit LPS-induced IFN-λ1 gene expression in moDCs (Fig. 6B, right). However, TLR4 is sensitive to Dynasore, since it needs to be endocytosed before signaling via the Toll-interleukin-1 receptor domain-containing adapter protein inducing IFN-β (TRIF) adaptor leading to IFN gene expression (30). As another control, we used Sendai virus, which fuses directly at the plasma membrane. As expected, BafA1 did not inhibit IFN-λ1 gene expression in Sendai virus-infected macrophages (Fig. 6A, right).

FIG 6.

Inhibition of endosomal acidification decreases early influenza B virus-induced IFN gene expression in human monocyte-derived macrophages and dendritic cells. (A) Macrophages were treated with BafA1 or the vehicle control, followed by infection with influenza A/Beijing/353/89, B/Shangdong/7/97, or Sendai viruses at an MOI of 5 for the times indicated. Cells from 4 donors were pooled for isolation of total cellular RNA and qRT-PCR analysis of IFN-λ1 gene expression. The data are presented as fold induction over mock-infected samples and are the means of data from three technical replicates with 1 standard deviation. (B) MoDCs were treated with BafA1, Dynasore, or the vehicle control, followed by infection with influenza B/Shangdong/7/97 virus (MOI of 5) (left) or stimulation with LPS (right) for different times. Cells from 4 donors were pooled for qRT-PCR analysis of IFN-λ1 gene expression and for WB analysis of p-IRF3, IκBα, and influenza B virus NP expression. GAPDH protein levels were analyzed to control for equal protein loading. The data are representative of results from two to three independent experiments.

Although the kinetics of the different phases of influenza A virus entry were described previously (31, 32), such information for influenza B virus is limited. In our experiments, the entry of influenza B virus seemed to be rapid, as the viral NP entered the nucleus in <1 h. To define this further, we performed immunofluorescence microscopy experiments to determine whether influenza B virus also uses early and late endosomes for its entry. We costained cells with EEA1 for the early endosome or LAMP1 for the late endosome/lysosome and influenza B virus NP. Colocalization of EEA1 with influenza B virus NP was detected within 15 to 30 min, and some colocalization with LAMP1 and NP was seen at 30 min (Fig. 7, bottom, insets). In BafA1-treated macrophages, influenza B virus NP did not enter the nucleus, and the colocalization with the endosomal markers was sustained for 1 to 2 h with EEA1 and even up to 4 h with LAMP1. We also analyzed IRF3 activation in the same experiment and found that it occurred at the 1-h time point, after colocalization of viral NP with the endosomal markers (Fig. 7, top).

FIG 7.

Influenza B virus NP colocalizes with classical markers of the endosomal pathway. Human macrophages were differentiated on glass coverslips and infected with influenza B/Shangdong/7/97 virus at an MOI of 30 on ice for 1 h. Virus entry proceeded after warming of the cells to 37°C, and cells were fixed with paraformaldehyde after the indicated incubation times. BafA1 or dimethyl sulfoxide (DMSO) was added to the cells 30 min before infection. Primary antibodies used were guinea pig anti-NP IBV, rabbit anti-human IRF3, mouse anti-EEA1, and mouse anti-LAMP1. Secondary antibodies were FITC-labeled donkey anti-guinea pig, rhodamine Red-X-labeled donkey anti-rabbit, and rhodamine-labeled goat anti-mouse immunoglobulins. Insets marked with white rectangles in the top panels for the 15-, 30-, and 60-min time points are shown in the bottom panels at a higher magnification. Data from one representative experiment out of two are shown.

RIG-I is essential for influenza B virus-induced early IRF3 activation.

To elucidate which RLR was responsible for the early innate immune recognition of influenza B virus infection, we used MEFs lacking RIG-I, MDA5, or both proteins (7, 33). We infected wt and single- and double-receptor-KO MEFs with influenza B and influenza A viruses at an MOI of 30 and analyzed the expression of IFN-β and viral NS1 RNA by qRT-PCR (Fig. 8A) and the phosphorylation of IRF3 and the expression of NP and NS1 by WB (Fig. 8B). As the phosphorylation of IRF3 was weak in mouse cells (Fig. 4B), a large amount of virus was used to better visualize the activation of IRF3. In RIG-I^−/− cells, influenza B virus-induced IFN-β gene expression was completely absent, whereas in influenza A virus-infected cells, primarily early IFN gene expression (2 and 4 h) was missing or reduced (Fig. 8A). The early activation of IRF3 was RIG-I dependent, since influenza B virus-induced IRF3 phosphorylation did not take place in RIG-I^−/− MEFs (Fig. 8B). In contrast, in MDA5 KO cells, IRF3 phosphorylation was induced normally by influenza B virus infection. The experiment also revealed differences between influenza A and B viruses. Unlike influenza B virus, influenza A virus also showed some residual IFN gene expression activity in RIG-I KO cells, and IRF3 phosphorylation was reduced in both RIG-I and MDA5 single-KO cells.

FIG 8.

RIG-I receptor is responsible for early influenza B virus-induced IRF3 phosphorylation and activation. (A) wt (RIG-I^+/+) MEFs and MEFs lacking RIG-I, MDA5, or both receptors were infected with influenza A/Beijing/353/89 and B/Shangdong/7/97 viruses at an MOI of 30 for the indicated times. Cells were collected, and total cellular RNA was isolated for qRT-PCR analysis of IFN-β and viral NS1 gene expression. The qRT-PCR data are presented as fold induction over mock-infected samples, and the means and standard deviations from triplicate measurements are shown. (B) Protein samples were isolated for analysis of p-IRF3, viral NP and NS1, and GAPDH by WB. Data from a representative experiment out of three are shown.

DISCUSSION

In the present study, we analyzed the mechanisms of activation of innate immune responses in human monocyte-derived macrophages and dendritic cells after influenza A and B virus infections. We show that in human macrophages, influenza B virus activates IRF3 phosphorylation and induces its nuclear accumulation soon after infection. This is followed by the activation of IFN-λ1 and IFN-β gene expression and the induction of IFN-stimulated genes (ISGs), including RIG-I and IFITM3. In influenza A virus-infected cells, these genes were induced with slower kinetics. IRF3 activation after influenza B virus infection occurred in the presence of the protein synthesis inhibitor CHX, indicating that its activation and IFN gene expression are triggered directly by the incoming virus. Blocking of influenza B vRNP release from endosomes with pharmacological inhibitors prevented the early activation of the innate immune response. Finally, by using KO MEFs, we demonstrate that IRF3 and RIG-I play a crucial role in influenza B virus-induced early IFN gene expression.

The present study was carried out mainly with human macrophages, in contrast to moDCs used previously (24). Tissue-resident alveolar macrophages are the first-line innate immune cells that fight influenza infection and also are possible targets of infection in the lungs (34). Analogous to studies with moDCs and A549 cells (24), influenza B virus induced IRF3 phosphorylation and IFN and ISG gene expression in macrophages at early times after infection. Influenza B virus does not readily infect mouse cells, and this species specificity depends partially on the viral NS1 protein, which binds and inhibits the antiviral action of human but not mouse ISG15 (35). In MEFs, influenza B virus was previously shown to induce IFIT2 and ISG15 in a RIG-I-dependent manner (7), and in agreement with these data, we also detected RIG-I- and IRF3/7-dependent IFN-β gene expression and IRF3 activation in MEFs.

Previously, we showed that UV-treated, inactivated influenza B virus still induced IFN gene expression in human moDCs, whereas UV-treated influenza A virus did not (24). UV irradiation of the virus is thought to block primary transcription, possibly due to the uracil dimers generated by radiation. A recent study analyzed influenza A virus-induced IRF3 activation in the presence of chemical inhibitors, including actinomycin D, which blocks primary transcription of the virus (12). That study showed that primary transcription but not replication of influenza A virus was needed to activate IRF3. Another recent study carried out with several negative-sense RNA viruses suggested that the incoming viral nucleocapsid containing a 5′-triphosphate genome can activate RIG-I signaling without transcription of viral genes (13). A more recent study showed that RIG-I is activated by the incoming influenza A virus 5′-triphosphate dsRNA panhandle (14). Our data support the latter model well, at least for influenza B virus infection, which triggers IRF3 activation at the earliest phase of infection when NP starts to accumulate in the nucleus and before any viral protein synthesis or replication takes place. However, we did not observe IRF3 activation after the entry of influenza A virus, which is consistent with our previously reported data showing that UV-treated influenza A virus is incapable of inducing IFN gene expression (24). In a study by Killip and coworkers, innate immune responses and IRF3 activation were analyzed at a relatively late time point (8 h) (12). This time point may reflect the detection of newly synthesized viral RNAs or replication intermediates. However, we clearly show that influenza B virus activates IRF3 at 1 h postinfection in a RIG-I-dependent manner, which means that RIG-I must sense the incoming vRNA structures. We hypothesize that even if the incoming influenza A virus nucleocapsids were recognized by RIG-I, the virus could have some RIG-I and/or MAVS signaling inhibitors that prevent IRF3 activation. Supporting this theory, Liedmann and coworkers described motifs in the polymerase proteins PB1 and PA of influenza A that seemed to inhibit IRF3 activation. When these sites were mutated, the mutant but not the wild-type virus activated IRF3 at the 8-h time point (16). To further complicate things, Weber and coworkers (14) showed that RIG-I itself could function as an antiviral protein by binding and inhibiting the incoming influenza A virus nucleocapsids, independently of IFN induction, and that mammal-adapted influenza A viruses with PB2-627K are more resistant to the direct antiviral action of RIG-I than are avian influenza A viruses with PB2-627E sequences (14). Whether influenza B virus has evolved similar RIG-I-counteracting strategies remains to be investigated. Regardless, our results indicate that in human macrophages and moDCs, IFN signaling is activated by the incoming influenza B virus. Perhaps, the influenza B virus polymerase complex is not as tightly associated with the viral genome in the vRNP complex as human-adapted influenza A viruses, and thus, the influenza B virus panhandle structures are more accessible for RIG-I recognition.

Banerjee and coworkers recently developed an image-based assay to study the precise kinetics of influenza virus entry (31). The steps from endocytosis to HA acidification and from acidification to membrane fusion were rapid, but after membrane fusion, a 45-min lag period between membrane fusion and vRNP uncoating was estimated to occur. We already observed influenza B virus NP in the nucleus at 1 h postinfection and speculate that in the case of influenza B virus, viral and endosomal membrane fusion and uncoating may occur more rapidly than for influenza A virus, resulting in faster entry into the nucleus. For this phenomenon, there may be a couple of explanations: (i) the influenza B virus HA fusion peptide is more exposed, which may lead to membrane fusion at a higher pH and accelerated fusion kinetics compared to those of influenza A virus (32), and (ii) the uncoating of influenza A virus takes place when the virion becomes acidified via the viral M2 ion channel, leading to the dissociation of the M1 protein. As influenza B virus has an additional ion channel, NB, the virion may become acidified more efficiently, and thus, the vRNPs may be released into the cytoplasm earlier. However, the NB ion channel is likely not the only contributing factor, as mutation of this ion channel leads to reduced IRF3 activation compared to that in the wt virus, but still, the activation of IRF3 is more efficient than with influenza A virus (data not shown). As vRNPs are released earlier from the endosome and uncoated, they may be recognized sooner as foreign RNA structures via cytosolic pattern recognition receptors (PRRs).

Innate immune receptors known to recognize viral RNA structures are RIG-I, MDA5, TLR3, TLR7, and TLR8. Macrophages and dendritic cells are equipped with a range of innate immune receptors to recognize foreign microbial structures. As influenza viruses enter the cells via an endosomal pathway, where TLR3, -7, and -8 are localized, viral entry could be recognized in the endosomes. This occurs in plasmacytoid DCs (pDCs), which express low levels of RLRs (36) but high levels of endosomal TLR7 and IRF7 and produce large amounts of IFN-α during influenza A virus infection (11). It is possible that in pDCs, some viruses cannot escape the endosomal pathway before degradation, and thus, viral RNA structures are released from the virion to be recognized by endosomal TLRs. Results of our experiments with BafA1 that resulted in the retention of influenza viruses in the endosomal pathway suggest that the incoming influenza B virus RNA is not recognized by TLRs in macrophages or moDCs. However, BafA1 treatment may affect the maturation and signaling of TLR receptors (37), so we cannot fully exclude their role. Nevertheless, experiments with knockout MEFs suggested that early influenza B virus-induced innate immune responses are mediated by RIG-I in the cytosol. In summary, we have demonstrated significant differences between influenza A and B viruses in their ability to activate early innate immune responses in primary human immune cells. A better understanding of early entry and immune recognition events may help to explain the pathogenesis of influenza A and B virus infections and provide us the means to develop efficient intracellular viral inhibitors.