Cell Type-Specific Recognition of Human Metapneumoviruses (HMPVs) by Retinoic Acid-Inducible I (RIG-I) and TLR7 and Viral Interference of RIG-I Ligand This information is current as Recognition by HMPV-B1 Phosphoprotein of October 2, 2021. Nadege Goutagny, Zhaozhao Jiang, Jane Tian, Peggy Parroche, Jeanne Schickli, Brian G. Monks, Nancy Ulbrandt, Hong Ji, Peter A. Kiener, Anthony J. Coyle and Katherine A. Fitzgerald Downloaded from J Immunol 2010; 184:1168-1179; Prepublished online 30 December 2009; doi: 10.4049/jimmunol.0902750 http://www.jimmunol.org/content/184/3/1168 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Cell Type-Specific Recognition of Human Metapneumoviruses (HMPVs) by Retinoic Acid-Inducible Gene I (RIG-I) and TLR7 and Viral Interference of RIG-I Ligand Recognition by HMPV-B1 Phosphoprotein

Nadege Goutagny,*,1 Zhaozhao Jiang,*,1 Jane Tian,† Peggy Parroche,* Jeanne Schickli,† Brian G. Monks,* Nancy Ulbrandt,† Hong Ji,† Peter A. Kiener,‡ Anthony J. Coyle,† and Katherine A. Fitzgerald*

Human metapneumoviruses (HMPVs) are recently identified Paramyxoviridae that contribute to respiratory tract in children. No effective treatments or vaccines are available. Successful defense against relies on early detection by germ line-encoded pattern recognition receptors and activation of cytokine and type I IFN . Recently, the RNA helicase Downloaded from retinoic acid-inducible gene I (RIG-I) has been shown to sense HMPV. In this study, we investigated the abilities of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I IFNs. Despite the abilities of both HMPV-A1 and HMPV-B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I–mediated sensing of HMPV viral 59 triphosphate RNA. In contrast to most cell types, http://www.jimmunol.org/ plasmacytoid dendritic cells displayed a unique ability to sense both HMPV-A1 and HMPV-B1 and in this case sensing was via TLR7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related , which operate in cell type‑specific manners. The Journal of Immunology, 2010, 184: 1168–1179.

uman metapneumovirus (HMPV) is a newly described may be attributed to HMPV infection (2). Retrospective studies virus responsible for lower respiratory tract infections in have revealed that HMPV is not a new virus but rather one that has H children (1). The virus was first isolated in The Neth- been circulating for ∼50 y (1). erlands in 2001. Compared to its closest human relative, re- There is currently no effective treatment or vaccine for HMPV. spiratory syncytial virus (RSV), HMPV has a worldwide Recent studies in mice have revealed an important role for T cells in by guest on October 2, 2021 prevalence and causes a broad spectrum of illness that ranges from antiviral immunity and pathogenesis (3); however, our un- asymptomatic infection to severe bronchiolitis. Serological studies derstanding of the innate immune response to HMPV is limited. have revealed that virtually every child has been exposed to HMPV is an enveloped virus containing a single-stranded negative- HMPV by the age of 5 y (1). Depending on the population ana- sense RNA genome encoding eight open reading frames. On the lyzed, 5‑15% of respiratory infections and 12‑55% of otitis media basis of its sequence homology to the avian pneumovirus, HMPV was assigned to the Metapneumovirus genus within the Para- myxoviridae family, which also contains RSV (4). Phylogenetic *Division of Infectious Diseases and Immunology, Department of Medicine, Univer- sity of Massachusetts Medical School, Worcester, MA 01605; †MedImmune, Gaithers- analysis has revealed two major genetic clusters, designated as burg, MD 20878; and ‡Zyngenia, Rockville, MD 20850 groups A and B, which have been further subdivided into four main 1N.G. and Z.J. contributed equally to this work. subtypes A1, A2, B1, and B2 (2, 5). Unlike most viruses that enter Received for publication August 20, 2009. Accepted for publication November 22, cells by receptor-mediated endocytosis, most paramyxoviruses 2009. deliver their genome into the cytoplasm directly by fusion with the This work was supported by National Institutes of Health Grant AI-067497 and by plasma membrane (6). The attachment (G, H, or HN) and fusion (F) a MedImmune Research Award (both to K.A.F). proteins are critical for mediating these events (reviewed in Ref. 7). Address correspondence and reprint requests to Dr. Katherine A. Fitzgerald, Division Most other viruses enter cells by receptor-mediated endocytosis and of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605. E-mail address: kate.fitzgerald are delivered to the endosomal compartment where the acidic en- @umassmed.edu vironment is critical for viral fusion and the release of viral genomes The online version of this article contains supplemental material. into the cytosol. Several classes of germ line-encoded pattern rec- Abbreviations used in this paper: A1R, human metapneumovirus-A1 recovered from ognition receptors have been identified that recognize different cDNA; APB, human metapneumovirus-A1 virus encoding the P protein from human components of viruses. In most cases, viruses are sensed via their metapneumovirus-B1; BDCA2, blood dendritic cell antigen 2; CIAP, calf intestinal genomes or their replicative or transcriptional activities (8). The alkaline phosphatase; HAU, hemagglutinating unit; HEK, human embryonic kidney; HMPV, human metapneumovirus; IRF, IFN regulatory factor; ISRE, IFN-stimulated recognition of RNA and DNA viruses has been shown to involve regulatory element; MAVS, mitochondrial antiviral signaling protein; MDA-5, mel- endosomally localized TLRs, including TLR7 and TLR9, which are anoma differentiation-associated gene 5; MOI, multiplicity of infection; NDV, New- castle disease virus; pDC, plasmacytoid dendritic cell; PRD, positive regulatory expressed on plasmacytoid dendritic cells (pDCs), the major pro- domain; RIG-I, retinoic acid-inducible gene I; RIG-IC, RIG-I consisting of the heli- ducers of IFN-a in vivo (9). Both influenza virus and vesicular case domain only; RNP, ribonucleoprotein; RSV, respiratory syncytial virus; TK, stomatitis virus (VSV) are sensed by TLR7 in pDCs (10), whereby thymidine kinase; VSV, vesicular stomatitis virus; WT, wild-type. recognition of the genomes of these ssRNA viruses is tightly linked Copyright 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 to viral fusion and uncoating (11). DNA viruses are sensed in pDCs www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902750 The Journal of Immunology 1169 via TLR9. Induction of IFN-a by the TLR7 and TLR9 pathway is a mixed C57/BL6 3 129 background were from Z. J. Chen (University of 2/2 2/2 mediated by the TLR adapter MyD88 and the transcription factor Texas Southwestern Medical Center, Dallas, TX). TLR7 and TLR9 IFN regulatory factor (IRF)-7 (12, 13) mice were from S. Akira (Osaka University, Osaka, Japan). Animal studies have been reviewed and approved by the University of Massachusetts Sensing of RNA and DNAviruses also occurs in the cytosol and in Medical School Institutional Animal Care and Use Committee. the case of RNA viruses is mediated by a second class of immune Reagents sensors, the RNA helicases, retinoic acid-induciblegene (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5). Genetic The trypsin-independent HMPV isolates A1 (NL\1\001) and B1 (NL\1\99) evidence has revealed that RIG-I and MDA-5 discriminate between were provided by MedImmune (Gaithersburg, MD) and were propagated as different classes of RNA viruses (9, 14). RIG-I is required for trig- described previously in IMDM containing 4% BSA and trypsin (24, 25). Influenza virus (strain A/PR/8/34) was from Charles River Laboratories gering antiviral responses against several Flaviviridae, Para- (Wilmington, MA). Newcastle diseasevirus (NDV; LaSota strain) was from P. myxoviridae, Orthomyxoviridae, and Rhabdoviridae, whereas Pitha (Johns Hopkins University, Baltimore, MD). HSV-1 (KOS strain) was MDA-5 is required for the response against picornaviruses such as from D. Knipe (Harvard Medical School, Boston, MA). HMPV and NDV encephalomyocarditis virus (9, 15). RIG-I senses the nascent 59 were inactivated by heating at 56˚C for 30 min or by UV cross-linking at a dose of 2 J/cm2. CpG-A (CpG 2216), CpG-2088 (26), and ISS661 (27) were triphosphate moiety of viral genomes or virus-derived transcripts of from Integrated DNA Technologies (Coralville, IA). Poly(dA-dT)•poly(dA- negative-sense ssRNAviruses, whereas MDA-5 is activated by long dT) was from GE Healthcare (Piscataway, NJ). Chloroquine and bafilomycin dsRNA, a typical intermediate of the replication of plus-sense A1 were from Sigma-Aldrich (St. Louis, MO). The monoclonal antibodies ssRNA viruses. RIG-I and MDA-5 induce type I IFN responses by specific for the HMPV F protein were generated and characterized by Med- recruiting a caspase-recruitment domain-containing adapter mole- Immune (24). Mouse mAb no. 338 was used as neutralizing Ab at a concen- tration of 10 mg/ml, and biotinylated hamster mAb no. 1017 was used at cule, mitochondrial antiviral signaling protein (MAVS) (16) [also a concentration of 1 mg/ml for staining of the F protein by immunoblottingand Downloaded from known as IPS-1, VISA or CARDif (17‑19)] and triggering IRF3 flow cytometry. Anti–b-actin was from Sigma-Aldrich. activation to regulate type I IFN gene transcription. Some DNA Plasmids viruses also are sensed by a pathway involving RIG-I; however, in this case viral DNA is transcribed by RNA polymerase III into The IFN-b luciferase, pGL3-positive regulatory domain (PRD) II, -PRDIII‑I, a RNA intermediate that is then recognized by RIG-I (20, 21). and -PRDIV luciferase reporter genes were from T. Maniatis (Harvard Uni- versity, Cambridge, MA). The IFN-a4 luciferase reporter gene was from S. A recent study has implicated the RIG-I pathway in sensing of the Akira. The ISG54 IFN-stimulated regulatory element (ISRE) was from HMPVstrainCAN97-83(HMPV-A2).HMPV-A2inducesIFN-b and Stratagene (La Jolla, CA). Human RIG-I flag and RIG-I consisting of the http://www.jimmunol.org/ chemokine gene expression in a RIG-I–dependent manner (22). In helicase domain only (RIG-IC) were from T. Fujita (Tyoto University, Tokyo, this study, we have examined in detail the role of TLRs and RNA Japan). A dominant-negative mutant form of MDA-5 (MDA-5-T789M) was from D. Conte (C. Mello Laboratory, University of Massachusetts Medical helicases in the detection of HMPVs by comparing two prototype School, Worcester, MA). pME18-NS3/4A-myc and pME18-NS3/4A-S139A- strains NL\1\00 (HMPV-A1) and NL\1\99 (HMPV-B1) in primary myc were from Z. J. Chen. pGL4-thymidine kinase (TK) Renilla luciferase cells and cell lines. Although closely related, only the HMPV-A1 and pGL3-control luciferase were from Promega (Madison, WI). All of the strain activated type I IFN gene transcription in most of the cell types viral proteins (with the exception of L) from HMPV-B1 were cloned in- examined. In both human cell lines as well as highly purified human dividually into pEF-Bos-Flag by PCR using full-length cDNA as a template (28). Clone sequences were verified by sequencing and protein expression in monocytes, HMPV-A1 but not HMPV-B1 induced type I IFN. In- 293T cells by Western blot analysis with anti-Flag M2 (Sigma-Aldrich). by guest on October 2, 2021 duction of IFN-a/b by HMPV-A1 was mediated by the recognition Mouse and human primary cell isolation of 59 triphosphate viral RNA through RIG-I and its downstream adaptor MAVS. The failure of HMPV-B1 to trigger type I IFN relates PBMCs were freshly isolated by density-gradient centrifugation using Ficoll to its ability to antagonize IFN production through the phospho- Hypaque (GE Healthcare). Monocytes and pDCs were purified from PBMCs using CD14 microbeads and the Diamond Plasmacytoid Dendritic Cell protein P. In the context of the virus, the HMPV-B1 phosphoprotein Isolation Kit, respectively, as recommended by the manufacturer (Miltenyi prevented RIG-I from sensing the viral genome during infection. In Biotec, Auburn, CA). Purity of these populations was assessed by staining for contrast to human cell lines and monocytes, pDCs produced type I CD14 and blood dendritic cell antigen 2 (BDCA2) expression by flow cy- IFN upon infection with both HMPV-A1 and HMPV-B1. In pDCs, tometry and was .95%. Mouse pDCs were purified from spleens of mice injected with BL6-FLT3L–producing cells. Briefly, mice were injected s.c. treatment of cells with lysosomotropic agents that prevented endo- with 5 3 106 cells in 300 ml PBS. Spleens were harvested within 2 wk after somal acidification blocked IFN-a induction by both viruses, which injection, and pDCs were isolated using the Plasmacytoid Dendritic Cell was mediated by TLR7. Taken together, these data emphasize the Isolation Kit II (Miltenyi Biotec). Purity was assessed by staining for CD11c, unique ability of pDCs to sense and induce type I IFN in response to Ly6C, and mPDCA1 expression by flow cytometry and was .85%. viruses that appeared “invisible” to most other cell types. These data Reporter assays also highlight the abilities of two closely related viruses to differ- Luciferase reporter assays were conducted as described previously (29). entially antagonize innate immune sensing mechanisms. Briefly, 293T or A549 cells (2 3 104 cells per well in 96-well plates) were transfected with the indicated luciferase reporter genes together with the TK Materials and Methods Renilla luciferase reporter gene and with the indicated expression plasmids Cells and mice using GeneJuice (Novagen, Madison, WI). Luciferase activity was measured as described previously (30). Huh7 and Huh7.5 cells (4 3 104 cells per well in Human embryonic kidney (HEK) 293, 293T, alveolar epithelial (A549), and 96-well plates) were transfected as above except that these cells were also Verocells were from the American Type Culture Collection (Manassas, VA). transfected with 40 ng of a pGL3-control reporter, which drives constitutive The human hepatocellular carcinoma cell lines Huh7 and Huh7.5 were from luciferase activity to normalize the data between the two cell lines. Data were C. Rice (Rockefeller University, New York, NY). B16-FLT3L–producing normalized according to the manufacturer’s recommendations (Promega). cells were from G. Dranoff (Harvard Medical School, Boston, MA) (23). Viral RNA Purification HEK293 cells stably expressing the P protein from HMPV-A1 and HMPV- B1 were generated by transfection with cDNAs encoding P proteins, which Viral RNA from HMPV-A1 and HMPV-B1 was extracted using the QIAamp were generated by PCR. All of the above cell lines were maintained in Viral RNA Mini kit (Qiagen, Valencia, CA). Viruses were concentrated by DMEM (Mediatech, Herndon, VA) supplemented with 5% FBS (Hyclone, ultracentrifugation for 2 h at 4˚C at a speed of 27,000 rpm using a SW28 Logan, UT) and 10 mg/ml ciprofloxacin (Mediatech). BSR-T7/5 cells were rotor (Beckman Coulter, Fullerton, CA). RNA (1.5 mg) was treated with or from K. Conzelmann (Ludwig Maximilian University of Munich, Munich, without 10 U of calf intestinal alkaline phosphatase (CIAP; Fermentas, Germany) and were cultured in DMEM containing 10% FCS and cipro- Glen Burnie, MD) for 3 h at 37˚C or RNase A (Promega) for 1 h at 37˚C. floxacin with G418 (0.5 mg/ml). C57/Bl6 and C57/BL6-129 F1 mice were All of the samples then were treated for 15 min at 85˚C to inactivate the from The Jackson Laboratory (Bar Harbor, ME). MAVS2/2 mice on enzymes. Viral RNA was transfected into 293T cells using Lipofectamine 1170 ROLE OF RNA HELICASES AND TLRs IN HMPV-DRIVEN IFN RESPONSES

2000 at a ratio of 1:1 (w/v) along with the IFN-b luciferase and the TK with 80% acetone. Cells were incubated with HMPV-specific 1017 bio- Renilla reporter genes according to the manufacturer’s instructions (In- tinylated Ab (MedImmune) for 1 h at 37˚C, followed by incubation with HRP- vitrogen, Carlsbad, CA). Viral RNA from NDV and VSV were from T. labeled streptavidin (BD Biosciences, San Jose, CA). Plaques were quantified Morrison and S. Zhou, respectively (University of Massachusetts Medical after incubation with a freshly prepared solution of 3,39-diaminobenzidine School, Worcester, MA). (Vector Laboratories, Burlingame, CA) to determine viral titers. ELISA Statistical analysis For ELISA analysis, human PBMCs (2 3 105 cells per well), monocytes (1 3 Differences between groups were analyzed for statistical significance by 105 cells per well), and pDCs (4 3 103 cells per well) or mouse pDCs (5 3 104 using a t test in GraphPad Prism software (GraphPad, San Diego, CA). p , cells per well) were plated in 96-well plates in 100 ml and stimulated for 24 h. 0.05 was considered statistically significant. Three, two, and one asterisk Poly(dA-dT)•poly(dA-dT) was transfected as described previously (31) at represent p values of ,0.001, ,0.01, and ,0.05, respectively. a concentration of 5 mg/ml using Lipofectamine 2000 (Invitrogen) at a ratio of 2:1 (w/v). Human IFN-a was measured according to the manufacturer’s recommendations (Bender MedSystems, Vienna, Austria). A murine IFN-b Results sandwich ELISA was used as described previously (32). Differential induction of type I IFN by HMPV-A1 and HMPV-B1 RNA extraction and real time PCR In an effort to understand the underlying mechanisms regulating the innate immune response to HMPVs, we compared the abilities of The 293T cells (2 3 106 cells per 10-cm plate) were stimulated with HMPV-A1 or HMPV-B1 (3.2 3 105 PFU/ml) for 5‒48 h, and the RNA was two closely related strains HMPV-A1 (NL\1\00) and HMPV-B1 isolated using RNeasy (Qiagen, Valencia CA). cDNA was synthesized as (NL\1\99) to induce type I IFN by examining the IFN-b reporter described previously (29) using the SuperScript III enzyme (Invitrogen). gene activity in infected cells. 293T cells were transfected with Quantitative real-time PCR analysis was performed using SYBR green a reporter gene under the control of the IFN-b gene enhancer. reagent (Invitrogen) on a DNA engine Opticon 2 cycler (Bio-Rad, Her- b ∼ Downloaded from cules, CA) using the following primers: IFN-b F, CAGCAATTTT- HMPV-A1 induced the IFN- reporter ( 60-fold induction), CAGTGTCAGAAGC; IFN-b R, CATCCTGTCCTTGAGGCAGT; IFN-a whereas HMPV-B1 failed to drive this reporter (Fig. 1A). HMPV- F, GTGAGGAAATACTTCCAAAGAATCAC; IFN-a R, TCTCATGATT- A1 also induced an IFN-a4 reporter gene, whereas HMPV-B1 TTCTGCTCTGACAA; b-actin F, CCTGGCACCCAGCACAAT; b-actin R, failed to do so (Supplemental Fig. 1A). We observed similar re- GCCGATCCACACGGAGTA; L1 F, TTGCATGAGGTACCTTGGATTG; sults in the human hepatoma cell line Huh7 (Fig. 1B) and the L1 R, AGAGTGCATTATCACACATCA. The specificity of amplification was assessed for each sample by melting curve analysis, and the size of the human alveolar epithelial cell line A549 (Fig. 1C). Similar ob- amplicon was checked by electrophoresis. PCR efficiency was calculated servations were made when the endogenous IFN-b transcript http://www.jimmunol.org/ for both viral RNAs with 10-fold dilutions of the cDNA with the formula levels were measured. HMPV-A1 induced IFN-b gene transcrip- [21/slope] E =10 2 1 as described previously (33). Relative quantification was tion, whereas HMPV-B1 failed to do so (Fig. 1D). performed using standard curve analysis. All of the gene expression data were normalized with b-actin and are presented as a ratio of gene copy We also tested these responses in primary cells. Human PBMCs number per 100 copies of b-actin 6 SD. were purified from whole blood and infected with HMPVs, and the production of the type I IFN (IFN-a) was measured by ELISA. Western blot analyses Contrary to previous observations in human cell lines, both HMPV- Wholecellextractswerepreparedusinglysisbuffer(50mMTris[pH7.5],150 A1 and HMPV-B1 induced type I IFN in PBMCs (Fig. 1E). Both mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 10% glycerol), freshly IFN-a and IFN-b mRNA levels were induced as measured by by guest on October 2, 2021 supplemented with protease and phosphatase inhibitors. Cells were placed on quantitative PCR (Supplemental Fig. 1B). Monocytes and pDCs are ice for 15 min and centrifugedfor 15 min at 14,000 rpm. Thirty micrograms of protein was analyzed by 10% SDS-PAGE and subjected to immunoblot the major producers of type I IFN in PBMCs. We therefore purified analysis using anti-F and anti–b-actin Abs as indicated. The IRF3 di- monocytes and pDCs from total PBMCs. Monocytes responded to merization assay was performed as follows. A549 cells (10 3 106 cells per 10 HMPV-A1 but not HMPV-B1, whereas pDCs responded to both 5 ml in a 10-cm plate) were stimulated with HMPV-A1, HMPV-B1 (10 PFU/ viruses (Fig. 1F,1G). These results reveal the unique ability of ml), or NDV (40 hemagglutinating unit [HAU]) as indicated. Nuclear pro- teins were extracted using a Nuclear Extract Kit (Active Motif, Carlsbad, pDCs to induce type I IFN in response to HMPV-B1. CA). Twenty-five micrograms of protein was analyzed by 8.5% PAGE and To exclude the possibility that the failure of HMPV-B1 to induce subjected to immunoblot analysis using anti-IRF3. type I IFN induction was due to a failure to infect or replicate in these cells, we compared viral infection and replication in both Generation of chimeric viruses monocytes and pDCs. Upon infection with paramyxoviruses, the Chimeric viruses, where the P proteins of both HMPV strains were ex- viral F protein is first synthesized as an inactive precursor F0, which changed, were generated by PCR using the naturally occurring MluI and is subsequently converted into the fusogenic F1 form by cellular PacI enzymes and Esp31 enzyme to generate overlapping PCR fragments. PCR fragments were generated using full-length cDNA as described pre- proteases (34). A HMPV-specific anti-F Ab has been characterized viously (28). For each virus strain, three PCR products were generated and previously and shown to detect F0 and F1 forms of the F protein cloned in the TOPO cloning vector (Invitrogen): from the MluI site to the from both HMPV-A1 and HMPV-B1 (24). The level of F-protein ATG of the P protein, the P protein, and to the end of P up to the PacI site. expression was monitored in infected cells by flow cytometry. Ligations of MluI to P and P to PacI from each strain were ligated with the ∼ + P protein from the opposite virus strain into pCDNA3. Full-length HMPV- Similar percentages of purified monocytes ( 20% of CD14 cells) A1 and HMPV-B1 were generated by partial digestion using MluI and PacI and pDCs (∼45% of BDCA2 positive cells) expressing the F and used to clone the three pieces by PCR from pCDNA3. Colonies were protein were observed for both HMPV strains (Fig. 2A). This screened by digestion and sequenced by PCR. indicated that both strains can infect these cell types. Moreover, in Virus recovery and titration 293T cells, comparable levels of expression of the F protein (both F0 and F1 forms) were detected in both HMPV-A1– and HMPV- RecoveryofrecombinantHMPVwasperformedasdescribedpreviously(28). B1–infected 293T cells when examined by Western blot analyses Briefly, BSR-T7 cells were transfected with 5 mg full-length HMPV cDNA plasmid, 2 mg pCITE-N, 2 mg pCITE-P, 1 mg pCITE-L, and 1 mgpCITE- (Fig. 2B). Replication of HMPV-A1 and HMPV-B1 also was ex- M2.1 using GeneJuice (Novagen). Two days later, the BSR-T7 cells were amined by quantifying the levels of viral transcripts and measur- scraped and cocultured with Vero cells in IMDM with 4% BSA and 3.75 mg/ml ing viral titers in infected cells. To ensure similar PCR efficiencies trypsin(Invitrogen) for 7 d. Viruses were titered as followed. Twenty-four‑well for both strains, the primer-binding sites were designed in a region plates containing 95% confluent monolayers of Vero cells were inoculated with 200 ml 10-fold serial virus dilutions. After 2 h at 37˚C, 0.8 ml 0.5% of the gene encoding the L protein, which was identical in both methylcellulose/DMEM with 3% FCS was added. Cells were incubated for an strains. Equivalent levels of HMPV-A1 and HMPV-B1 transcripts additional 5 d. Methylcellulose overlays were removed, and cells were fixed were detected in infected cells throughout the course of infection The Journal of Immunology 1171

FIGURE 1. HMPV-A1 and HMPV-B1 differ- entially induce IFN-b gene transcription (A‒C). The 293T, Huh7, and A549 cells were transfected with the full-length IFN-b promoter. Cells were infected with HMPV-A1 or HMPV-B1 (from 5 3 104 to 5 3 102 PFU/ml for 293T and 5 3 104 PFU/ ml for other lines) for an additional 24 h. Data are expressed as fold induction relative to the reporter- only control and presented as the mean 6 SD. D, The 293T cells were stimulated with HMPV-A1 or HMPV-B1 (5 3 104 PFU/ml) for the indicated time. Levels of human IFN-b and b-actin were quantified

in RNA samples by real-time PCR. Results are Downloaded from presented in arbitrary units as the ratio of IFN-b over 100 copies of b-actin. E‒G, Total PBMCs, monocytes, or pDCs were stimulated with HMPV- A1, HMPV-B1 (2 3 105 PFU/ml), CpG-A (3 mM), NDV (8 HAU/ml), or poly(dA-dT)•poly(dA-dT) (5 mg/ml) for 24 h. Protein levels were measured in the supernatant of the culture by ELISA and presented http://www.jimmunol.org/ as the mean 6 SD. by guest on October 2, 2021

(Fig. 2C). Viral titers were also equivalent for both strains (Fig. multimerized ISRE from the ISG54 promoter. Consistent with 2D). This was also true in 293T, A549, and Huh7 cells (Fig. 2E). a failure to induce the PRDIII‑I element from the IFN-B promoter, Indeed, the levels of HMPV-B1 exceeded those of HMPV-A1 at HMPV-B1 also failed to drive the ISG54 ISRE and IFN-a4re- later time points (data not shown). We conclude therefore from porters (data not shown and Supplemental Fig. 1A). these studies that both viruses infect and replicate in all of the We next monitored endogenous IRF3 activation by examining cells tested; however, pDCs were unique in their ability to induce the formation of IRF3 dimers in virus-infected cells. IRF3 is type I IFN in response to HMPV-B1. normally present in the cytoplasm of resting cells as a monomer. Virus infection triggers the phosphorylation and dimerization of HMPV-B1 fails to activate IRF3 IRF3 followed by its nuclear translocation. Infection of 293T cells The transcriptional enhancer of the IFN-b gene contains four PRDs with HMPV-A1 induced IRF3 dimerization in a manner similar to (PRDI–IV) that bind distinct transcriptional regulators that act co- that observed with NDV, our positive control (Fig. 3D). In contrast, operatively to activate IFN-b gene expression. The transcription cells infected with HMPV-B1 failed to lead to the dimerization of factors that bind to these elements include NF-kB, which binds to endogenous IRF3. These observations were confirmed using an PRDII; IRF3 and IRF7, which bind to adjacent PRDIII and PRDI in vitro assay for IRF3 and IRF7, which utilizes a hybrid protein sites, collectively referred to as PRDIII‑I; and the heterodimeric consisting of the yeast Gal4 DNA binding domain fused to IRF3 transcription factor activating transcription factor 2/c-Jun, which or IRF7 lacking its own DNA binding domain (37). Reporter gene binds to PRDIV. To define the effect of HMPV-B1 on each of these expression from the Gal4 upstream activation sequence in this pathways, we tested the effect of HMPV-B1 on transcription from assay requires IRF activation (37). HMPV-A1 but not HMPV-B1 individual IFN-b regulatory elements using multimerized reporter activated both of the Gal4-IRF3 and Gal4-IRF7 reporters (data not assays. Each of these promoter elements when present in multiple shown). Taken together, these data provide clear evidence that copies has been shown to be activated by Sendai virus (35, 36). In HMPV-B1 fails to trigger IRF3 and IRF7 activation and as a result agreement with the data presented above, only HMPV-A1 activated fails to trigger IFN-b production. the PRDIII‑I reporter, which responds to IRF3 and IRF7 (Fig. 3A). In contrast, HMPV-A1 and HMPV-B1 both induced the PRDII and HMPV-A1 triggers type I IFN gene transcription via RIG-I PRDIV reporter genes, which are activated by NF-kB and acti- Because RIG-I is a sensor of paramyxoviruses and HMPV has been vating transcription factor 2/c-Jun, respectively (Fig. 3B,3C). shown to trigger RIG-I signaling (9, 16, 22, 38), we monitored the Similar data were obtained when a multimerized NF-kB reporter contribution of the RIG-I pathway to IFN-b gene activation upon with canonical NF-kB binding sites was tested (data not shown). HMPV-A1 stimulation. We first examined the role of MAVS, which We also monitored the activation of a reporter gene containing relays signals from RIG-I to downstream kinases and transcription 1172 ROLE OF RNA HELICASES AND TLRs IN HMPV-DRIVEN IFN RESPONSES Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. HMPV-A1 and HMPV-B1 infect and replicate in primary cells and cell lines. A, Purified monocytes (6 3 105 cells per well) and pDCs (2 3 105 cells per well) were cultured for 24 h in the presence of HMPV-A1 and HMPV-B1 (3.2 3 105 PFU/ml). Cells were harvested and stained for specific cell surface Ag (CD14 or BDCA2) and F-protein expression using an anti-F biotinylated Ab and PE- or allophycocyanin-conjugated streptavidin. Results are presented as histogram overlays of anti-F Ab staining versus isotype control. Displayed percentages were subtracted from the isotype control. B, Infection of 293T cells by HMPV was assessed by immunoblot analysis. Cells (6 3 105 cells per well) were infected with HMPV-A1 or HMPV-B1 (5 3 104 PFU/ml) for 2‒48 h. A specific anti-F biotinylated Ab and HRP-conjugated streptavidin were used to detect F protein in cell lysates. F0 and F1 forms of the F protein are indicated by an arrow. Anti-F no. 338 neutralizing Ab was used to show the specificity. The lower panel shows b-actin levels in the samples. C, Replication of HMPV in 293T cells was quantified by real-time PCR. Cells were infected with HMPV-A1 or HMPV-B1 (5 3 104 PFU/ml) for the indicated time points. Levels of the HMPV L gene and human b-actin were quantified in RNA samples using specific primers. Results are presented in arbitrary units as the ratio of the HMPV L gene over 100 copies of b-actin. D and E, Virus titers were determined postinfection of several cell types (3 3 105 cells per well) with HMVP-A1 or HMPV-B1 (105 PFU/ml). Virus titers were measured for the indicated time points postinfection in Vero cells (D)or after 24 h postinfection in 293T, A549, and Huh7 cells (E) and presented as PFU per milliliter. factors. The involvement of MAVS was assessed using the NS3/4A induction of IFN-b by HMPV-A1 as well as that induced by Sendai protease from hepatitis C virus. NS3/4A cleaves and inactivates virus (Fig. 4A). Importantly, the protease inactive mutant (NS3/4A- MAVS, thereby disrupting RIG-I signaling (39). NS3/4A therefore S139A) had no effect. We also tested the involvement of RIG-I using can be used as a tool to implicate MAVS signaling in a particular the hepatoma cell line (Huh7) and the Huh7.5 subline that bears response. The effect of NS3/4A on the HMPV-A1–induced IFN a natural mutation in RIG-I (T55I) that renders it inactive (40). Huh7 response was tested in 293T cells. Increasing concentrations of and Huh7.5 cells were transfected with the PRDIII‑I reporter gene wild-type (WT) NS3/4A protease dose-dependently blocked the and then infected with HMPV-A1, HMPV-B1, or NDV (Fig. 4B). The Journal of Immunology 1173

FIGURE 3. Differential activation of IRF3 by HMPV-A1 and HMPV-B1. A‒C, The 293T cells were transfected with the IFNB PRDIII-I, PRDII, or PRDIV reporter genes as detailed in the Materials and Methods. Cells were infected with HMPV-A1 or HMPV-B1 (range from 5 3 104 to 5 3 102 PFU/ ml) for an additional 24 h. Data are expressed as fold induction relative to the

reporter-only control and presented as the mean 6 SD. D, A549 cells were Downloaded from stimulated with viruses for the indicated period of time. Nuclear protein was extracted and analyzed by native PAGE. The monomer and dimeric forms of IRF3 are indicated by arrows.

Consistent with our data in 293T cells, only HMPV-A1 induced this reporter gene in Huh7 cells (Fig. 4B). Activation of the PRDIII‑I element was completely abrogated in the Huh7.5 cell line in re- http://www.jimmunol.org/ sponse to HMPV-A1. The IFN-b response following NDVinfection (which is known to be RIG-I–dependent) also was inhibited in the Huh7.5 cells, consistent with published results. Moreover, re- constitution of Huh7.5 cells with WT RIG-I fully restored the re- sponse to both HMPV-A1 and NDV.We also found that a dominant- FIGURE 4. HMPV-A1 induces IFN-b via the RIG-I/MAVS pathway. A, negative version of RIG-I, RIG-IC, dose-dependently inhibited the The 293T cells were transfected with the IFN-b reporter gene and in- b IFN- response in 293T cells elicited by HMPV-A1 (Fig. 5D; see creasing concentrations (5‒80 ng per well) of the WT or S139A inactive below). MDA-5 did not appear to be involved in the recognition of

NS3/4A protease from hepatitis C virus. Cells were stimulated for an by guest on October 2, 2021 HMPV-A1, because a dominant-negative mutant form of MDA-5 additional 24 h with HMPV-A1, HMPV-B1 (5 3 104 PFU/ml), or Sendai did not inhibit the induction of the IFN-b reporter by HMPV-A1 but virus (400 HAU/ml). Data are expressed as fold induction relative to the blocked the IFN response to WT MDA-5 (Fig. 4C,4D). In- reporter-only control and presented as the mean 6 SD. B, Parental human terestingly, PRDII (NF-kB) and PRDIV (MAPK) reporter gene hepatoma cell lines Huh7 and Huh7.5 were transfected with the IFN-b activation by HMPV-A1 and HMPV-B1 were not inhibited by RIG- reporter gene in the absence or presence of pEF-Bos-huRIG-I Flag (40 ng 3 4 IC overexpression in 293T or Huh7.5 cells (Supplemental Fig. 2A, per well). Cells were stimulated with HMPV-A1, HMPV-B1 (5 10 PFU/ml), or NDV (64 HAU/ml) for an additional 24 h. Results are nor- 2B). Collectively, these observations suggest that HMPVs can malized by Renilla luciferase and presented as arbitrary units after cor- trigger RIG-I to turn on type I IFN production and also trigger RIG- rection between the two cell lines using the pGL3-control. C, The ‑ k I independent signaling events leading to NF- B and AP-1 activa- 293T cells were transfected with the IFN-b reporter gene and (D)WTor tion. Thus, RIG-I as well as additional as yet unidentified pattern a dominant-negative mutant of MDA-5 (80 ng per well). Cells were recognition receptor-driven pathways contribute to the host re- stimulated with HMPV-A1 (5 3 104 PFU/ml) for additional 24 h. Data are sponse to this virus. expressed as fold induction relative to the reporter-only control and pre- We next examined the requirement for viral replication in the sented as the mean 6 SD. HMPV-A1–induced type I IFN response. We compared live virus to either heat- or UV-inactivated HMPV-A1 IFN-b reporter gene either CIAP or RNase A treatment. Moreover, RIG-IC also dose- activity in 293T cells. Heat and UV inactivation of HMPV-A1 and dependently inhibited the IFN-b response in 293T cells elicited by NDV completely blocked their abilities to induce IFN-b reporter either HMPV-A1 or HMPV-A1 or HMPV-B1 viral RNAs (Fig. gene activity (Fig. 5A). Similar results were observed in purified 5D). These data indicate that 59 triphosphate RNA is the ligand for human monocytes (Fig. 5B). The 59 triphosphate moiety of the RIG-I and also reveal that the naked viral genome of HMPV-B1 viral RNA is the major ligand recognized by RIG-I (41, 42). To can be sensed by RIG-I, if delivered to the cytosol. However, the further investigate the nature of the ligand that triggers RIG-I in inability of HMPV-B1 to be sensed suggests that in the context of HMPVs, viral RNA was purified from HMPV-A1 and transfected the virus the HMPV-B1 RNA is not detected. into the cytoplasm of 293T cells via lipofection. Transfection of the viral RNA induced the IFN-b reporter gene, and removal of HMPV-B1 P protein prevents sensing by RIG-I the phosphate groups by CIAP or digestion of the viral RNA by To test this hypothesis, we first tested whether preincubation with RNase A abrogated this response (Fig. 5C). Importantly, when we HMPV-B1 could interfere with signaling initiated by HMPV-A1 or isolated the viral RNA from HMPV-B1 and transfected this RNA other viruses that are sensed by RIG-I. Cells were transfected with into 293T cells, IFN-b was induced, and this response was also the IFN-b reporter gene and preincubated for 24 h with live or UV- sensitive to CIAP and RNase A treatment. IFN induction by the inactivated HMPV-B1 before infection with HMPV-A1 or NDV. dsDNA analogue, poly(dA-dT)•poly(dA-dT), was unaffected by Luciferase activity then was monitored 24 h postinfection. 1174 ROLE OF RNA HELICASES AND TLRs IN HMPV-DRIVEN IFN RESPONSES Downloaded from

FIGURE 5. Induction of IFN-b by HMPV-A1 requires viral replication. A, The 293T cells were transfected with the full-length IFN-b promoter. Cells were stimulated 24 h later with live or heat- or UV-inactivated HMPV-A1, HMPV-B1 (2 3 105 PFU/ml), or NDV (8 HAU/ml) for an additional 24 h. Data are expressed as fold induction relative to the reporter-only control and presented as the mean 6 SD. B, Freshly isolated monocytes were stimulated for 24 h with live or heat- or UV-inactivated HMPV-A1,HMPV-B1(2 3 105 PFU/ml), or NDV (8 HAU/ml). Human IFN-a protein levels were measured in the supernatant of http://www.jimmunol.org/ culture by ELISA and presented as the mean 6 SD. C, The 293T cells were transfected with viral RNA purified from HMPV-A1 or HMPV-B1 (80 ng) or poly (dA-dT)•poly(dA-dT) (20 ng) along with the IFN-b and TK Renilla reporter genes. Viral RNAs and poly(dA-dT)•poly(dA-dT) were treated with CIAP or RNase A prior to stimulation as indicated. D, The 293T cells were transfected with increasing amounts of RIG-IC (0, 2, or 20 ng per well) along with the IFNB reporter and viral RNAs (80 ng) or stimulated with HMPV-A1 (5 3 104 PFU/ml). In all of the cases, luciferase activity was measured 24 h posttransfection, and data are expressed as fold induction relative to the reporter-only control and presented as the mean 6 SD.

Preincubation with live HMPV-B1 reduced IFN-b induction upon consistent with the possibility that the HMVP-B1 P protein HMPV-A1 infection (Fig. 6A). In contrast, preincubation with UV- functions in a specific manner as an inhibitor, blocking IFN pro- by guest on October 2, 2021 inactivated virus did not block the induction of IFN-b. The acti- duction in response to HMPV RNA. vation of the IFN-b reporter gene in response to NDV was not Tofurther examine the inhibitoryeffect ofthe HMPV-B1P protein affected (Fig. 6A). Moreover, IFN-b reporter gene induction in- in a more physiologically relevant manner and in the context of the duced by overexpression of RIG-I, the downstream adapter virus, we generated chimeric viruses in which the P protein from MAVS, or the IRF3 kinase TANK-binding kinase 1 also was un- HMPV-A1 was replaced with that of HMPV-B1. Both WT and affected (data not shown). These data suggest that HMPV-B1 in- chimericHMPV-A1wererecoveredfromcDNA,andtheirabilitiesto terferes with the induction of the IFN-b reporter by HMPV-A1 induce type I IFN were examined by reporter assays. Consistent with and that the inhibitory effect was not due to antagonism down- our data with the HMPV-A1 clinical isolate, WT HMPV-A1 re- stream of RIG-I itself. Therefore, HMPV-B1 likely blocks sensing covered from cDNA (A1R) induced IFN-b reporter in HEK293 of HMPV RNA and not RIG-I function per se. cells; however, when A1R expressing the P protein from HMPV-B1 Each individual cDNA from HMPV-B1 (with the exception of (APB) was examined, no reporter gene activity was detected (Fig. the polymerase L) was cloned into mammalian expression vectors, 6D). Similar data were obtained when A549 cells were examined and their effects on type I IFN induction upon infection with (Fig. 6D). Because both HMPV-A1 and HMPV-B1 could induce HMPV-A1 were examined by reporter assay. Plasmids encoding PRDII and PRDIV luciferase reporters, we also compared the ef- HMPV-B1 cDNAs were transfected into 293T cells together with fects of A1R and APB on induction of these reporters in both the PRDIII‑I reporter gene, whose activity was monitored post- HEK293 and A549 cells. In contrast to the IFN-b reporter, both A1R infection of cells with HMPV-A1. Table I shows the percentage and APB induced both of these reporter genes (Fig. 6E). Moreover, identity between HMPV-A1 and HMPV-B1 proteins. Although the analysis of F-protein levels in virus-infected cells revealed similar M, SH, M2-1, N, M2-2, F, and G proteins had little or no effect, levels in cells infected with all of the viral strains (Fig. 6F). These the P protein blocked the induction of the reporter (Fig. 6B). results provide compelling evidence that the HMPV-B1 P protein Importantly, Western blot analysis revealed equivalent expression prevents RIG-I‑mediated sensing and signaling, resulting in IRF3- levels of all of these proteins in transfected cells (data not shown). dependent type I IFN gene transcription. We next generated HEK293 cell lines stably expressing the HMPV-B1 P protein to test whether the HMPV-B1 protein could Detection of HMPVs in pDCs does not involve RIG-I but prevent the induction of IFN-b upon the delivery of viral RNA into occurs via an endosomal sensing pathway the cytoplasm. Viral RNA from both HMPV-A1 and HMPV-B1 As shown in Fig. 1, both HMPV-A1 and HMPV-B1 induce type I triggered a strong IFN-b response in parental HEK293 cells, but IFN production in pDCs. The studies outlined above indicate that the response was largely impaired in HEK293 cells expressing it is unlikely that RIG-I mediated these events. Quantitative PCR HMPV-B1 P protein. Importantly, IFN-b induced by viral RNA analysis revealed that pDCs express RIG-I and MAVS, albeit at from NDV or VSV was unaffected (Fig. 6C). These data are much lower levels than those found in monocytes (43). To The Journal of Immunology 1175 Downloaded from http://www.jimmunol.org/

FIGURE 6. HMPV-B1 P protein prevents RIG-I from sensing HMPV viral RNA. A, The 293T cells were transfected with the IFN-b reporter gene 1 d prior to stimulation. Cells were preincubated with live or UV-inactivated (2 J/cm2) HMPV-B1 (105 PFU/ml) for 24 h. Cells were then stimulated with HMPV-A1 (5 3 104 PFU/ml) or NDV (8 HAU) for an additional 24 h. Results are normalized by Renilla luciferase and presented as fold induction relative to the reporter- only control and presented as the mean 6 SD. B, The 293T cells were transfected with the IFN-b PRDIII‑I reporter gene and 40 ng of plasmids encoding by guest on October 2, 2021 HMPV-B1 proteins as indicated. Cells were stimulated for an additional 24 h with HMPV-A1 (5 3 104 PFU/ml). Data were normalized by Renilla luciferase and are expressed as a percentage of activation relative to conditions with the virus in the absence of exogenous proteins and presented as the mean 6 SD. C, HEK293 cells and HEK293 cells stably expressing the P protein from HMPV-B1 were transfected with the full-length IFN-b reporter gene along with the viral RNA (80 ng) purified from HMPV-A1, HMPV-B1, NDV, or VSVor with total RNA (80 ng) purified from Vero cells as a control. Results are normalized by Renilla and pGL3-control luciferase and presented as the mean 6 SD. D‒F, The 293Tand A549 cells were transfected with the full-length IFNB or the PRDIV reporter along with the TK Renilla reporter gene 1 d prior to stimulation. Cells were infected for 24 h with the following viruses: HMPV-A1, HMPV-B1, A1R, or APB (5 3 104 PFU/ml). Results are normalized by Renilla luciferase and are presented as fold induction relative to the reporter-only control and presented as the mean 6 SD. A Western blot of F-protein levels is shown (F). examine the contribution of the RIG-I pathway in pDC responses were treated with chloroquine or bafilomycin A1, two lysoso- to HMPVs, we monitored pDCs from mice lacking MAVS. pDCs motropic agents that act by raising the intraendosomal pH or by from MAVS-deficient mice responded normally to both HMPVs specific inhibition of the vacuolar adenosine triphosphatase, re- (as well as to NDV, HSV, and influenza viruses) (Fig. 7A). These spectively (11, 44). Induction of IFN-a by HMPV-A1 and HMPV- latter three viruses have been shown to signal in pDCs via TLRs B1 in pDCs was totally abrogated when cells were pretreated with (see below). R848 and CpG DNA, ligands for TLR7 and TLR9 either chloroquine or bafilomycin A1 (Fig. 7C). Responses to respectively, also induced IFN-b normally in MAVS-deficient CpG-A were also completely blocked by chloroquine and bafilo- pDCs. Of note, MAVS-deficient myeloid dendritic cells or mac- mycin A1, consistent with published results (44). In contrast to rophages are severely compromised in NDV- or synthetic tri- phosphate RNA-induced IFN responses (data not shown). Table I. Percentage of identity between HMPV-A1 and HMPV-B1 To define the mechanisms sensing these viruses in human pDCs, proteins we investigated the requirement for viral replication in mediating these responses. In contrast to our observations in 293T cells and Protein Identity (%) monocytes (Fig. 5), heat and UV inactivation of HMPV-A1, P 86.1 HMPV-B1, and NDV only partially affected their abilities to in- N 94.9 duce IFN-a induction (Fig. 7B). Altogether, these studies con- M 97.3 firmed that the sensing of HMPVs in pDCs is not mediated via the F 93.7 M2-2 88.9 RIG-I cytosolic pathway. M2-1 8.6 Because pDCs express high levels of TLR7 and TLR9 and sense SH 8.9 viruses via endosomally localized TLRs, we next examined the role G 11.3 of this system in the detection of HMPV-A1 and HMPV-B1. pDCs L 91.7 1176 ROLE OF RNA HELICASES AND TLRs IN HMPV-DRIVEN IFN RESPONSES

FIGURE 7. Induction of type I IFN in pDCs in re- sponse to both HMPV-A1 and HMPV-B1 is TLR7- mediated. A, pDCs from C57Bl6/129 and MAVS2/2 mice were stimulated for 24 h with CpG-A (2 mM), heat-inactivated influenza (multiplicity of infection [MOI] = 0.1), HSV (MOI = 100), HMPV-A1, and HMPV-B1 (2 3 105 PFU/ml). Mouse IFN-b protein levels were measured in the supernatant of culture by ELISA and presented as the mean 6 SD. B, Freshly isolated pDCs were stimulated for 24 h with live or heat- or UV-inactivated HMPV-A1, HMPV-B1 (2 3 105 PFU/ml), or NDV (8 HAU/ml). Human IFN-a protein levels were measured in the supernatant by ELISA and presented as the mean 6 SD. C and D, Freshly isolated pDCs and monocytes were stimulated with HMPV-A1, HMPV-B1 (2 3 105 PFU/ml), NDV (8 HAU/ml), heat- inactivated influenza (MOI = 0.1), or CpG-A (2 mM) for Downloaded from 24 h. Cells were preincubated with chloroquine (1 mM) or bafilomycin A1 (4 mM for pDCs and 10 mM for monocytes) for 1 h were indicated. Human IFN-a protein levels were measured in the supernatant by ELISA and presented as the mean 6 SD. E, Mouse pDCs from C57Bl6, TLR72/2, and TLR92/2 mice were stimulated for 24 h with CpG-A (2 mM), heat- http://www.jimmunol.org/ inactivated influenza (MOI = 0.1), HSV (MOI = 100) HMPV-A1, and HMPV-B1 (2 3 105 PFU/ml). Mouse IFN-b protein levels were measured by ELISA and presented as the mean 6 SD. F, Freshly isolated pDCs were stimulated with HMPV-A1, HMPV-B1 (2 3 105 PFU/ml), R848 (10 mM), or CpG-A (2 mM) for 24 h. Cells were preincubated with CpG-2088 (0.1 mM) or ISS661 (2 mM) for 1 h were indicated. Human IFN-a

protein levels were measured in the supernatant by by guest on October 2, 2021 ELISA and presented as the mean 6 SD.

pDC responses, induction of IFN-a by HMPV-A1 in monocytes TLR9 signaling (27, 45), had no effect. These results reveal that was less affected (Fig. 7D). IFN-a induction in response to in- the unique ability of pDCs to induce type I IFN in response to fluenza is presumable in chloroquine- or bafilomycin A1-treated HMPV-B1 correlates with the sensing of HMPVs through TLR7 conditions because of a failure of the virus to infect. and not the cytosolic RNA helicase pathway. For viruses that enter cells by receptor-mediated endocytosis, viral fusion and uncoating events are tightly coupled to recognition Discussion of viral ligands by endosomally localized TLRs. Therefore, in- HMPVs and RSVs are major contributors to respiratory tract hibition by chloroquine and bafilomycin A1 could implicate TLRs infections in infants and young children. In most infants, these in the sensing of these viruses. To examine the precise role of viruses cause symptoms resembling those of the common cold. endosomal TLRs in the recognition of HMPV-A1 and HMPV-B1, However, in infants born prematurely, children with chronic lung pDCs were isolated from WT and TLR7- and TLR9-deficient mice, disease, or children with congenital heart disease, these viruses can and IFN-b secretion was examined postinfection. As expected, the result in a severe or even life-threatening disease. As many as induction of IFN-b by CpG-A and HSV-1 was entirely dependent 125,000 hospitalizations occur annually in children ,1 y old due on TLR9 (Fig. 7E). In contrast, IFN-a induced in response to to lower respiratory infection or bronchiolitis (46). Developing influenza was unaffected in TLR9-deficient pDCs but was com- new therapeutics to prevent and treat these infections is therefore pletely defective in TLR7-deficient pDCs. HMPV-A1– and of considerable importance. HMPV-B1–induced IFN-b production in pDCs was induced nor- Limitingvirusinfectionrequiresrapidlymounteddefenses,which mally in TLR9-deficient pDCs and was fully impaired in TLR7- include in large part the release of type I IFN (IFN-a/b). IFN limits deficient pDCs. We also confirmed the TLR7 dependency in the viral replication directly and enhances viral clearance by activating human system using ISS661, a previously characterized oligonu- adaptive immunity. Understanding how viruses are sensed and how cleotide-based inhibitor for TLR7 signaling (26). Pretreatment type I IFN is regulated may facilitate the rational design of novel with ISS661 blocked the induction of IFN-a by HMPV-A1 and antiviral therapeutics or better vaccine candidates useful in the HMPV-B1 (Fig. 7F). Pretreatment with CpG-2088, an inhibitor of prevention or treatment of lower respiratory tract infections in The Journal of Immunology 1177 children. In this study, we demonstrate that type I IFN production because of higher levels of expression of the P protein in HMPV-B1 during infection with HMPVs involves differential sensing mecha- rather than what is found in HMPV-A1. Another possibility is that nisms that work in a cell type‑specific manner. Sensing of HMPV-A1 the HMPV-B1 P protein could have a higher affinity for the RNA or occurs via the cytosolic RNA helicase RIG-I in most cell types, with for other components of the RNP complex (HMPV RNA or N and the exception of pDCs, where TLR7 mediates these responses. A L), which would preclude the RNA from being sensed. The two P recent study by Casola and colleagues (22) also implicated RIG-I in proteins share 86% identity (Table I), and therefore unique resi- the sensing of HMPV in airway epithelial cells. We have confirmed dues in the HMPV-B1 P protein could account for these effects. these observations using mice with targeted deletions in the RIG-I Usurping IFN induction pathways is a common tactic employed pathway and have extended these studies to include an analysis of by viruses to enable their replication within host cells. Viral IFN additional cell types and a comparison of two closely related clinical antagonists strike at just about every level of the IFN regulatory viral isolates. We have identified 59 triphosphate RNA as the HMPV network, but by far the best-studied strategies relate to the ability of ligand triggering the RIG-I IFN-b response. Importantly, we also viral proteins to counteract RNA sensing and signaling events. identified a RIG-I–independent pathway for sensing HMPV-A1 and Examples include influenza virus nonstructural (NS) 1, which HMPV-B1 in epithelial cell lines. HMPV-A1 and HMPV-B1 acti- inactivates RIG-I (42); hepatitis C virus, NS3/4A, which cleaves vated NF-kB– and AP-1–dependent reporter genes in a RIG- and inactivates MAVS (18); and the phosphoprotein of Borna I–independent manner. These data suggest an additional mechanism disease virus and rabies virus, which target the IRF3 kinase, of HMPV sensing. The Nod-like receptor family member nucleo- TANK-binding kinase 1 (48, 49). In the case of RSV, the genome tide-binding oligomerization domain containing 2 was recently encodes two NS1 and NS2 proteins known to inactivate the IFN shown to act as a cytosolic sensor for RSV infection (47). Further response (50). A recent study from Casola and colleagues (51) studies should delineate whether nucleotide-binding oligomeriza- implicated the G protein from another strain of HMPV in evasion Downloaded from tion domain containing 2 also senses HMPV to regulate NF-kB and of the RIG-I signaling pathway. Recombinant HMPV lacking the AP-1 signaling described herein. G protein (rHMPV-DG) was developed as a potential vaccine A major focus of this study was a comparison of the innate candidate and shown to be attenuated in the respiratory tract of response to two closely related strains. Although both strains in- a rodent model of infection. Casola and colleagues found that duced type I IFN responses in pDCs, HMPV-B1 failed to elicit rHMPV-DG–infected airway epithelial cells produced higher a type I IFN response in monocytes and cell lines, despite its ability levels of chemokines and type I IFN compared with those of cells http://www.jimmunol.org/ to infect and replicate as efficiently as HMPV-A1 and despite the infected with rHMPV-WT. They showed that RIG-I was the target ability of naked viral RNA to trigger RIG-I if delivered by lip- of G protein inhibitory activity. Indeed, the G protein associated ofection to the cytoplasm. The fact that HMPV-B1 could prevent with RIG-I and inhibited RIG-I–dependent gene transcription. Our type I IFN induction by HMPV-A1 but not that induced by NDV data with HMPV-B1, however, do not support a role for the indicates that RIG-I signaling per se is not blocked by HMPV-B1. HMPV-B1 G protein but rather indicate that the HMPV-B1 Like HMPV-A1, purified HMPV-B1 RNA could trigger IFN, and phosphoprotein can prevent RIG-I from sensing the viral RNA. pretreatment of the HMPV-B1 RNA with RNase or removal of The inhibitory effect of the HMPV-B1 P protein was restricted to phosphate groups with alkaline phosphatase ablated sensing of the the RIG-I pathway, because HMPV-B1 did not prevent the in- by guest on October 2, 2021 viral RNA by RIG-I. These studies suggest that RNAs of both duction of IFN-a/b in pDCs. IFN production in pDCs was sen- strains are ligands for RIG-I; however, in the context of the virus, sitive to chloroquine and bafilomycin A1 and was dependent on the B1 viral RNA is prevented from being sensed by RIG-I. During TLR7. The current model of antiviral sensing in pDCs suggests viral infection, RNA viruses like HMPV fuse with the cell that TLR-mediated recognition of viruses occurs without direct membrane and deliver their ribonucleoprotein (RNP) complex into infection and that the presence of viral genomic nucleic acids the cells. The RNP complex consists of the viral RNA associated within the endosomal/lysosomal compartment is sufficient to with the viral polymerase L, the nucleoprotein N, and the phos- trigger TLRs. Iwasaki and colleagues (52) demonstrated recently phoprotein P. Upon fusion, the viral RNA is protected from free that RNA viruses, such as VSV, which do not enter cells via the cellular RNases by this protein complex. Proteins within the RNP endosomal compartment, replicate in the cytosolic compartment therefore could prevent the recognition of the viral RNA by the where cytosolic viral replication intermediates are then delivered RIG-I pathway. In fact, our studies using overexpressed HMPV-B1 into the lysosomal compartment by the process of autophagy to proteins indicated that the HMPV-B1 P protein but not other HMPV trigger TLRs. proteins could block IFN production by live HMPV-A1 or viral Generally cell entry of paramyxoviruses requires two glyco- RNA. Although this approach indicated that the HMPV-B1 P proteins: the attachment (G, H, or HN) and fusion (F) proteins. In protein was the most likely candidate, we found that if the HMPV- the case of HMPVs, however, analysis of recombinant viruses A1 P protein were overexpressed that an inhibitory effect also could lacking the G protein has suggested that attachment and fusion are be observed. To determine whether the HMPV-B1 P protein was mainly dependent on the F protein (53). The F protein is a type I responsible for the inhibitory effect in the context of the virus, we glycoprotein, synthesized as an inactive precursor F0, and sub- generated recombinant viruses where we replaced the P protein in sequently converted into its biologically active form, the hetero- the HMPV-A1 with that from HMPV-B1. This is a more physio- dimer F1/F2. The majority of Paramyxoviridae F proteins are logically relevant system to assess its contribution where associ- cleaved intracellularly by host cellular proteases, most notably ations of the HMPV-B1 P protein with other viral proteins also furin. Cleavage of the F protein from HMPV, however, requires could be accounted for. Recombinant HMPV-A1 encoding the P secretory proteases, which restrict HMPVs to the lumen of the protein from HMPV-B1 was generated, and its ability to induce respiratory and enteric tract for replication in vivo. In vitro the RIG-I signaling was examined. Unlike WT HMPV-A1 recovered addition of trypsin to process the F0 protein into its mature form from cDNA, the recombinant HMPV-A1 containing the HMPV-B1 allows efficient propagation of the virus (25). In contrast to most P protein had a substantially reduced ability to induce IFN, sug- other Paramyxoviridae F proteins that require neutral pH for gesting that in the context of the entire virus the HMPV-B1 P membrane fusion, cleavage of the HMPV F protein therefore protein may indeed prevent RIG-I from sensing HMPV. Specific might require low-pH conditions (54). These findings might in- inhibition of RIG-I sensing by the HMPV-B1 P protein could be dicate a requirement for low-pH compartments, such as the 1178 ROLE OF RNA HELICASES AND TLRs IN HMPV-DRIVEN IFN RESPONSES endosome, for the entry of HMPVs in pDC. Receptor-mediated 19. Xu, L. G., Y. Y. Wang, K. J. Han, L. Y. Li, Z. Zhai, and H. B. Shu. 2005. 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