Human metapneumovirus (HMPV) is a member of the subfamily Pneumovirinae in the family Paramyxoviridae and is a causal agent of respiratory diseases in all age groups. HMPV infection is clinically important, especially in the field of pediatric medicine, because HMPV, like human respiratory syncytial virus (HRSV) in the same subfamily, potentially causes severe lower respiratory tract infection in young children, such as infants with congenital heart disease and low-birth-weight babies. Pathogenesis of HMPV involves complicated processes that are affected by multiple factors, including viral evasion strategies for the host immune system. In general, silencing viral genes responsible for immune evasion resulted in attenuation of the virus. Therefore, a full understanding of viral immune evasion mechanisms contributes to not only elucidation of viral pathogenesis, but also development of effective vaccines and antiviral agents.
Interferons (IFNs) play a central role in innate immunity, constituting the first line of host defense. IFNs not only induce an antiviral state in cells via the JAK-STAT signaling cascade, but also contribute to activation of macrophages and T lymphocytes. A variety of cells produce IFNs, but plasmacytoid dendritic cells (pDCs) are unique in producing enormous amounts of alpha IFN (IFN-α). pDCs recognize pathogen-associated molecular patterns (PAMPs) derived from invading pathogens by using their endosomal Toll-like receptor 7 (TLR7) and TLR9 (TLR7/9). This recognition initiates signaling cascades, one of which leads to the ultimate activation of latent IFN regulatory factor 7 (IRF7). IRF7 is phosphorylated by the serine/threonine kinases, interleukin 1 (IL-1) receptor-associated kinase 1 (IRAK1) and/or IκB kinase alpha (IKKα), forms a homodimer, and translocates into the nucleus to activate the IFN-α genes (1–4
). The core of the TLR7/9-dependent signaling cascade consists of myeloid differentiation factor 88 (MyD88), IRAK4, tumor necrosis factor receptor-associated factor 6 (TRAF6), TRAF3, IRAK1, IKKα, viperin, and osteopontin (5–12
To maintain efficient virus growth in the body, viruses have independently developed strategies by which they minimize host IFN production. Indeed, it was found that members of the subfamily Paramyxovirinae
encode V and C proteins that have the ability to block the TLR7/9-dependent signaling cascade leading to IFN-α production in pDCs (13–16
). This activity is shared by all the C and V proteins tested in the subfamily Paramyxovirinae
, suggesting its importance for survival of members of the Paramyxovirinae
throughout evolution. We investigated their underlying molecular mechanisms and have presented evidence to indicate that the V protein of human parainfluenza virus type 2 (PIV2) targets TRAF6 for inhibition of K63-linked polyubiquitination and the C protein of Sendai virus (SeV) binds to IKKα to inhibit phosphorylation of IRF7 (13–15
). On the other hand, Pfaller and Conzelmann reported that the V protein of measles virus inhibited phosphorylation of IRF7 by serving as a decoy substrate for IKKα (16
). In contrast, little is known about the mechanisms by which members of the subfamily Pneumovirinae
inhibit IFN-α production by pDCs, although it has been suggested that HRSV and HMPV have strategies for suppressing IFN-α production by pDCs (17–19
). Guerrero-Plata et al. revealed that infection with HRSV or HMPV in vitro
inhibited IFN-α production by pDCs in response to a TLR9 ligand, synthetic CpG oligodeoxynucleotide (CpG-ODN), and that viral replication was necessary to elicit this effect (20
). They also found that lung pDCs isolated from mice infected with HRSV or HMPV in vivo
exhibited reduced ability to produce IFN-α in response to the synthetic CpG-ODN (19
Considering these circumstances, we designed this study to identify HMPV proteins responsible for the inhibition of TLR7/9-dependent signaling and to elucidate the underlying molecular mechanisms. Screening of the HMPV open reading frames (ORFs) for the ability to inhibit TLR7/9-dependent IFN-α induction revealed that the M2-2 protein was the most potent inhibitor. We show that the M2-2 protein directly binds to IRF7 and specifically prevents MyD88/TRAF6/IKKα-stimulated homodimerization of IRF7, possibly through inhibition of IRF7 phosphorylation.
In this study, we identified the M2-2 protein as a negative regulator of IFN-α production by pDCs. Recombinant ΔM2-2 virus, in which expression of M2-2 is abrogated, indeed induced a high level of IFN-α in human pDCs compared with wild-type HMPV (Fig. 1E
). This enhancement of IFN-α production seems to be causally related to two kinds of independent activities of M2-2 that appear to explain in part why the ΔM2-2 virus is attenuated (29
). One is the inhibitory activity for TLR7/9-dependent IFN-α induction. We demonstrated, by using TLR7/9-dependent signaling reconstituted in HEK293T cells, that M2-2 was the most potent inhibitor of all the proteins encoded by the HMPV genome (Fig. 1A
). The other is an inhibitory activity for viral RNA synthesis. ΔM2-2 virus produces high levels of viral mRNAs in Vero cells compared with wild-type virus (30
). The inhibitory effect of M2-2 on viral RNA synthesis was also confirmed by experiments with an HMPV minigenome construct carrying the luciferase reporter gene (31
). Possible enhancement of viral RNA synthesis in pDCs infected with ΔM2-2 virus may cause elevation of the PAMP level, which stimulates IFN-α production. However, it is difficult to determine the degree to which each activity contributes to suppression of IFN-α production.
How does the M2-2 protein block TLR7/9-dependent signaling? BRET and SRC assays revealed that the M2-2 protein inhibits MyD88/TRAF6/IKKα-induced homodimerization of IRF7 (Fig. 5
). This inhibition seems to be critical, since the homodimerization derepresses transactivation and allows specific DNA binding (23
). We initially assessed the IRF7-IRF7 interaction by immunoprecipitation with extracts from cells cotransfected with two kinds of tagged IRF7 in the absence of stimulation with upstream signaling components, according to the standard method described in other studies (22
). This experiment showed that expression of M2-2 had no appreciable effect on IRF7-IRF7 interaction (data not shown). This result was also confirmed by the SRC assay. Since in these experiments we could assess only the interaction between static unphosphorylated IRF7 molecules, we next attempted to examine interaction between phosphorylated IRF7 molecules after stimulation of the TLR7/9-dependent signaling pathway using extracts from cells cotransfected with MyD88, TRAF6, and IKKα. Unexpectedly, we found that phosphorylated IRF7 became insoluble in a lysis buffer containing Triton X-100 (even in radioimmunoprecipitation assay [RIPA] buffer) and was therefore removed from cell extracts by centrifugation before immunoprecipitation. It remains unclear why phosphorylated IRF7 becomes insoluble, although it may be related to TLR9 stimulation-dependent colocalization of IRF7 with viperin, which recruits IRAK1 and TRAF6 to lipid droplets after TLR9 stimulation (10
). We therefore used a BRET assay and an SRC assay in place of the immunoprecipitation method and succeeded in showing inhibition of the IRF7-IRF7 interaction by M2-2. To our knowledge, few reports on examining the IRF7-IRF7 interaction after TLR7/9 stimulation are available. BRET and SRC assays may be useful to study interaction of other viral proteins with TLR7/9 signaling components after TLR7/9 stimulation.
The M2-2 protein binds to the ID of IRF7 (Fig. 4
). Since the ID-ID interaction seems to be essential for IRF7 homodimerization (24
), we assumed that the IRF7-IRF7 interaction was interrupted by a steric effect of M2-2. However, it is unlikely, because the M2-2 protein inhibits neither IPS-1-induced homodimerization of IRF7 (Fig. 6B
) nor homodimerization of static unphosphorylated IRF7. IRF7 is subjected to two kinds of modification: K63-linked polyubiquitination by TRAF6 and subsequent serine phosphorylation by IKKα/IRAK1. We examined the effect of M2-2 on K63-linked polyubiquitination of IRF7 according to the procedure previously described (14
), but K63-linked polyubiquitination was not affected by M2-2 (data not shown). Instead, M2-2 was found to inhibit phosphorylation of IRF7 on Ser477, but not Ser471/Ser472 (Fig. 7A
). When an IRF7 mutant in which only Ser477 and Ser479 are replaced with the phosphomimetic Asp is transfected into cells, the mutant is homodimerized and activates the IFN-α promoter without transfection of upstream signaling components (22
). It is thus possible that inhibition of phosphorylation of IRF7 on Ser residues, including Ser477, is involved in inhibition of IRF7 homodimerization. To prove this hypothesis, it is necessary to examine the effect of M2-2 on phosphorylation of all the Ser residues in the regulatory domain and, further, to analyze the role of each phosphorylated Ser residue in IRF7 homodimerization.
It should be noted that inhibition of phosphorylation of IRF7 by M2-2 is partial. This indicates that M2-2 never inhibits the kinase activity per se
of the responsible kinases, IKKα and/or IRAK1, and does not target upstream components of the TLR7/9-dependent signaling pathway. This is in good agreement with the fact that M2-2 has no ability to inhibit MyD88-induced activation of NF-κB (Fig. 1D
). It is possible that the M2-2 protein masks Ser477, but not Ser471 and Ser472, by binding to the ID near the regulatory domain and thereby prevents IKKα and/or IRAK1 from gaining access to Ser residues near Ser477. The masking effect of the M2-2 protein might be insufficient to prevent IKKi/TBK1 from gaining access to Ser477.
In spite of the inhibition of IRF7 homodimerization, M2-2 did not inhibit nuclear translocation of IRF7. Similar observations have been reported for other viruses. LF2 of Epstein-Barr virus interacts with the ID of IRF7, and this interaction results in inhibition of IRF7 homodimerization (33
). However, nuclear translocation of IRF7 is not affected by the LF2 protein. The ML protein of Thogoto virus interacts with IRF7 and prevents homodimerization of IRF7 and interaction between IRF7 and TRAF6 (32
). However, it never inhibits nuclear translocation of IRF7. These findings, including ours, suggest that homodimerization is not a prerequisite for nuclear translocation of IRF7.
Although our present study defined the function of M2-2 in immune evasion, conflicting results have been reported (34
). Ren et al. showed that the M2-2 protein targeted MyD88 for inhibition of the activation of NF-κB and activation of the IFN-β promoter. They did not examine the effect of the M2-2 protein on IRF7 activation, but it is likely that activation of IRF7 is also inhibited, because MyD88 is an upstream component of the TLR7/9-dependent signaling pathway. However, we could confirm neither significant interaction between M2-2 and MyD88 (Fig. 3
) nor an inhibitory effect on MyD88-induced activation of NF-κB (Fig. 1D
). Also, not obtained were positive results supporting the previous finding reported by Ren et al. and Chen et al. regarding inhibition of MAVS (IPS-1)-dependent gene transcription by M2-2 (Fig. 6
). The reasons for these conflicting results are unclear. Compared with the CAN97/83 strain used in the other studies, the Jpn03-1 strain used in our study has a single amino acid difference at position 58 in M2-2 (I and L at this position in Jpn03-1 and CAN97-83, respectively). Position 58 is located in the C-terminal half of the M2-2 protein (72 amino acids [aa]). As the C-terminal half is responsible for binding to MyD88 (34
), it will be of interest to investigate whether the amino acid at position 58 determines the target of the M2-2 protein in immune evasion. In this regard, it should be noted that there is a study pointing out strain-dependent difference in HMPV immune evasion. Goutagny et al. reported that the P protein of strain B1 specifically impaired RIG-I-mediated sensing of viral 5′-triphosphate RNA derived from HMPV, but this activity was not seen in the P protein of strain A1 (37
In summary, our study reveals that HMPV employs M2-2 as an IFN antagonist that suppresses IFN-α production by pDCs and that the M2-2 protein blocks the TLR7/9-dependent signaling pathway by inhibiting IRF7 homodimerization, possibly through partial inhibition of IRF7 phosphorylation. Infection with RSV, a closely related virus, in vivo
or in vitro
, also results in reduction of the pDCs' ability to produce IFN-α in response to TLR7 or TLR9 stimulation (17
). Unlike HMPV, RSV carries additional viral genes, NS1 and NS2, and uses them as IFN antagonists that block the IFN-responsive JAK-STAT signaling pathway or inhibit activation of IRF3 (38–41
). What proteins does RSV use for regulation of IFN-α production by pDCs? An investigation of the underlying mechanism for RSV is now in progress.
MATERIALS AND METHODS
HEK293T, Vero, LLC-MK2, and HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 2 mM l
-glutamine, penicillin (100 IU/ml), streptomycin (100 μg/ml), and 10% fetal bovine serum (FBS). pDCs were isolated from human PBMCs by magnetically activated cell sorting with a Diamond plasmacytoid dendritic cell isolation kit II (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. The isolated pDCs were resuspended in RPMI medium (Nacalai Tesque) supplemented with 10% FBS, human IL-3 (10 ng/ml; Peprotech), 2 mM l
-glutamine, penicillin (100 IU/ml), and streptomycin (100 μg/ml) (14
Mammalian expression plasmids encoding viral or cellular protein were created by insertion of a cDNA fragment containing each ORF into the multicloning site downstream of the cytomegalovirus enhancer chicken β-actin hybrid promoter of pCA7. The cDNAs to be inserted were created by PCR or reverse transcription (RT)-PCR. Mutations, including deletion and protein fusion, were introduced by PCR-based overlap mutagenesis as described previously (13
). Newly created were cDNAs for N, P, M, F, M2-1, M2-2, SH, G, L (derived from HMPV strain Jpn03-1), SeV C (derived from strain Z), human MyD88, TRAF6, IKKα, IRF7 (isoform d; aa 1 to 516), the ID (aa 276 to 469) of human IRF7, human IRF7 lacking the ID (IRF7ΔID), TRAF6 C70A, IRF7-YFP, IRF7-Nluc, RlucN-IRF7 (IRF7 fused to the C terminus of RlucN [aa 1 to 229] with an intervening linker peptide [GGGGSG]), and IRF7-RlucC (IRF7 fused to the N terminus of RlucC [aa 230 to 331] with the same linker peptide) (25
). The pNL1.1 vector was purchased from Promega Corp. and was used as a template for Nluc in PCR. Construction of pCA7 encoding Fluc, PIV2 V (derived from strain Toshiba), HCV NS3/4A, or mIRF7 deletion mutants (with or without an N-terminal 3× FLAG, V5, or myc tag), and pEFneo-IPS-1 were described previously (13
). The sequence fidelity of all the plasmids was confirmed by sequence analysis.
Recovery of rHMPV-GFPΔM2-2.
The genome construction of rHMPV-GFP derived from strain Jpn03-1 was described previously (44
). To create rHMPV-GFPΔM2-2, in which the M2-2 ORF is silenced, two putative start codons of M2-2 at positions 5236 and 5248 were mutagenized from AUG to ACG. In addition, UUA at position 5272 was mutagenized to UAA to introduce a stop codon, and the major part of the M2-2 ORF (from positions 5288 to 5445), the sequence downstream of the stop codon of the M2-1 ORF, was deleted according to the method of Buchholz et al. (30
). These nucleotide changes do not affect the amino acid sequence of the M2-1 protein. rHMPV-GFPΔM2-2 was recovered by transfecting the M2-2-silenced antigenome plasmid into BSR T7/5 cells (a gift from K. K. Conzelmann), which constitutively express T7 polymerase, as described previously (44
). Sequence fidelity was confirmed by sequence analysis of the genome of the recovered virus. The recovered rHMPV was propagated in LLC-MK2 cells in the presence of 4 μg/ml of N
-acetyl trypsin. Trypsin was added to culture medium at a final concentration of 4 μg/ml every 3 days to promote multistep replication.
An IFN-α6 promoter-driven Fluc reporter (13
) (80 ng/well), an NF-κB Fluc reporter (Clontech) (100 ng/well), or an ISRE-driven Fluc reporter (Clontech) (80 ng/well) plasmid was transfected into HEK293T (∼1.0 × 105
) or Vero (∼2.0 × 104
) cells cultured in a 24-well plate in triplicate, together with pRL-TK (10 ng/well; Promega Corp.) and various combinations of plasmids that express MyD88 (25 ng/well), TRAF6 (25 ng/well), IKKα (25 ng/well), IPS-1 (50 ng/well), IRF7 (15 ng/well), IRF7ΔID (15 ng/well), N (100 ng/well), P (100 ng/well), M (100 ng/well), F (100 ng/well), M2-1 (100 ng/well), M2-2 (100 ng/well), SH (100 ng/well), G (100 ng/well), L (100 ng/well), PIV2 V (100 ng/well), TRAF6 C70A (100 ng/well), SeV C (100 ng/well), or HCV NS3/4A (100 ng/well), using polyethyleneimine (PEI) (Polysciences) (13
). The total mass of transfected DNA was held constant in all the experiments by adding an appropriate amount of pCA7 empty plasmid. The cells were lysed at 24 h posttransfection, and the relative luciferase activity was determined with the dual-luciferase reporter assay system (Promega). In certain experiments, transfected cells were treated with recombinant human IFN-α2b (1,000 IU/ml; Schering-Plough) for 6 h at 24 h posttransfection.
Measurement of IFN-α.
Levels of human IFN-α in culture media were measured by enzyme-linked immunosorbent assay (ELISA) with a human IFN alpha ELISA kit (PBL Interferon Source, Piscataway, NJ) according to the manufacturer's instructions.
HEK293T cells (∼5.0 × 105/well) in a 6-well plate were transfected with various combinations of plasmids (500 ng/well each), using PEI. At 24 h posttransfection, the cells were lysed in 400 μl of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail). Then, the cell lysates were incubated with anti-V5 mouse monoclonal antibody (MAb) (SV5-Pk1; Invitrogen), anti-FLAG mouse MAb (1E6; Wako), or anti-myc mouse MAb (9B11; Cell Signaling Technology), together with SureBeads protein G (Bio-Rad) at 4°C for 2 h. In certain experiments, cell-free protein synthesis was performed using the TNT SP6 high-yield wheat germ protein expression system (Promega). Mixtures of the products synthesized in vitro were incubated in place of cell lysates. After washing the beads five times with the lysis buffer, proteins were eluted from the beads by boiling with Laemmli sample buffer (50 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, 10% glycerol, and 5% 2-mercaptoethanol), and then the samples were subjected to immunoblot analysis.
Samples were resolved by SDS-10 to 15% polyacrylamide gel electrophoresis and then electroblotted onto a membrane filter (Immobilon-P; Millipore). The membrane was blocked in phosphate-buffered saline (PBS) containing 5% skim milk and 0.05% Tween 20, and was incubated at 4°C overnight with anti-FLAG mouse MAb (1E6), anti-V5 mouse MAb (SV5-Pk1), anti-myc mouse MAb (9E10; Wako), anti-phospho-IRF7 (anti-pIRF7) (S471/472) rabbit polyclonal antibody (Cell Signaling Technology), anti-pIRF7 (S477) rabbit MAb (D7E1W; Cell Signaling Technology), anti-IRF7 mouse MAb (F-1; Santa Cruz), anti-actin mouse MAb (AC-74; Sigma), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) rabbit MAb (D16H11; Cell Signaling Technology), or anti-histone H1 mouse MAb (AE-4; Santa Cruz). The membrane was then incubated at room temperature for 2 h with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody (GE Healthcare Bio-Science). Immunoreactive bands were visualized by using the ECL select substrate (GE Healthcare Bio-Science).
HEK293T cells (∼1.0 × 105
) in a 24-well plate were transfected with various combinations of plasmids that express MyD88 (25 ng), TRAF6 (25 ng), IKKα (25 ng), YFP (85 ng), IRF7-YFP (85 ng), Nluc (15 ng), IRF7-Nluc (15 ng), or viral proteins (100 ng). At 24 h posttransfection, the cells were suspended in Dulbecco's PBS and transferred to a 96-well microplate. After the addition of furimazine, a substrate for Nluc from the Nano-Glo luciferase assay system (Promega), luminescence and fluorescence signals were immediately detected using an Infinite F500 microplate reader (Tecan). The BRET ratio is defined as the light signal emitted by YFP (530 to 570 nm) relative to the light signal emitted by Nluc (370 to 450 nm) (45
). The actual BRET ratio was calculated by subtracting a background BRET ratio, which was obtained for cells expressing Nluc-IRF7 alone, from the directly measured BRET ratio of each sample.
HEK293T cells (∼1.0 × 105) in a 24-well plate were transfected with various combinations of plasmids that expressed MyD88 (25 ng), TRAF6 (25 ng), IKKα (25 ng), IPS-1 (50 ng), RlucN-IRF7 (15 ng), IRF7-RlucC (15 ng), or viral proteins (100 ng), along with an internal control, pCA7-Fluc (10 ng). The cells were lysed at 36 h posttransfection. Rluc and Fluc activities were measured with an Rluc assay system (Promega) and an Fluc assay system (Promega), respectively. The relative activity was defined as the ratio of Rluc activity to Fluc activity.
HeLa cells (∼1.0 × 104/well) cultured in an 8-well glass chamber slide (Matsunami Glass) were transfected with various combinations of plasmids that expressed V5-MyD88 (25 ng), V5-TRAF6 (25 ng), V5-IKKα (25 ng), myc-IRF7 (15 ng), FLAG-M2-1 (100 ng), FLAG-M2-2 (100 ng), or FLAG-PIV2 V (100 ng). At 24 h posttransfection, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.2% Triton X-100 for 5 min, followed by three washes with PBS. The cells were incubated with anti-myc mouse MAb (9B11) or anti-FLAG rabbit polyclonal antibody (Cell Signaling Technology) as the primary antibody, followed by anti-mouse IgG(H+L) antibody conjugated to Alexa Fluor 488 or anti-rabbit IgG(H+L) antibody conjugated to Alexa Fluor 647 as the secondary antibody. After each incubation step, the cells were washed with PBS three times. The stained cells were mounted with ProLong Gold reagent with DAPI (4′,6-diamidino-2-phenylindole) (Thermo Fisher Scientific) and then visualized under a BX-61 fluorescence microscope (Olympus).
Preparation of cytoplasmic and nuclear fractions.
HEK293T cells (∼5.0 × 105/well) in a 6-well plate were transfected with various combinations of plasmids that expressed MyD88 (125 ng), TRAF6 (125 ng), IKKα (125 ng), IRF7 (150 ng), or viral proteins (500 ng). At 24 h posttransfection, nuclear and cytoplasmic protein extracts were prepared by using nuclear and cytoplasmic extraction reagents (Thermo Fisher) according to the manufacturer's instructions.