Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 Jul 1.
Published in final edited form as: Virology. 2016 Apr 27;494:248–256. doi: 10.1016/j.virol.2016.04.022

Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation

Andrew K Hastings 1, Katherine R Amato 2, Sherry C Wen 1, Laura S Peterson 1, John V Williams 3,‡
PMCID: PMC4930656  NIHMSID: NIHMS782499  PMID: 27131212

Abstract

Type I interferon (IFN) is a key mediator of antiviral immunity. Human metapneumovirus (HMPV) inhibits IFN signaling, but does not encode homologues of known IFN antagonists. We tested the hypothesis that a specific viral protein prevents type I IFN signaling by targeting signal transducer and activator of transcription-1 (STAT1). We found that human airway epithelial cells (capable of expressing IFNs) became impaired for STAT1 phosphorylation even without direct infection due to intrinsic negative feedback. HMPV-infected Vero cells (incapable of expressing IFN) displayed lower STAT1 expression and impaired STAT1 phosphorylation in response to type I IFN treatment compared to mock-infected cells. Transient overexpression of HMPV small hydrophobic (SH) protein significantly inhibited STAT1 phosphorylation and signaling, and recombinant virus lacking SH protein was unable to inhibit STAT1 phosphorylation. Our results indicate a role for the SH protein of HMPV in the downregulation of type I IFN signaling through the targeting of STAT1.

Keywords: metapneumovirus, paramyxovirus, interferon, small hydrophobic protein

Introduction

Human metapneumovirus (HMPV) is a negative sense, single-stranded RNA virus in the Paramyxoviridae family (van den Hoogen et al., 2001). HMPV is a leading cause of acute lower respiratory infection (LRI), and no treatment or vaccine is currently available (Boivin et al., 2003; Edwards et al., 2013; Esper et al., 2004; Papenburg et al., 2012; Widmer et al., 2012; Williams et al., 2004; Williams et al., 2006). By the age of five, nearly all individuals have been infected with HMPV (van den Hoogen et al., 2001) and produce neutralizing antibodies, but the very young, elderly, and immunocompromised are at high risk for developing severe complications from HMPV infection (Englund et al., 2006; Papenburg et al., 2012; Shahda et al., 2011; Walsh et al., 2008).

Type I IFN signaling begins with the recognition of viral nucleic acids by pattern recognition receptors (PRRs) in infected cells (Platanias, 2005), as well as in innate immune cells such as macrophages and dendritic cells (Gordon, 2002), which induce the production and release of type I IFNs IFNα and IFNβ. These molecules ligate the interferon α receptor (IFNAR), in both an autocrine and paracrine fashion, and lead to signaling events involving STAT1, STAT2, and IRF9, culminating in the expression of anti-viral effector molecules (Barber, 2001; Biron, 2001; Sadler and Williams, 2008). A key step in this pathway is phosphorylation of STAT1, which then heterodimerizes with STAT2 and traffics to the nucleus. In addition, this pathway is capable of modulating the adaptive immune response by contributing to both the presentation of antigen as well as the differentiation and maintenance of adaptive immune cells (Havenar-Daughton et al., 2006; Kolumam et al., 2005; Le Bon and Tough, 2008; Luft et al., 1998).

Related paramyxoviruses encode proteins responsible for targeting and inhibiting innate immune signaling. RSV encodes the NS1 and NS2 proteins, and the PIV genome contains an alternative reading frame of the P/C/V gene that encodes the V protein; both types of proteins specifically inhibit aspects of innate immunity (Andrejeva et al., 2004; Childs et al., 2012; Gainey et al., 2008; Lo et al., 2005). Previous data using human airway epithelial cell lines suggested that HMPV, like other members of the Paramyxoviridae family such as respiratory syncytial virus (RSV) and parainfluenza virus (PIV), is capable of blocking or modulating the innate immune response through targeting of molecules involved in signaling through the type I interferon (IFN) signaling pathway (Dinwiddie and Harrod, 2008; Ren et al., 2011). We sought to identify the viral protein responsible for this inhibition. MPV encodes 9 proteins: nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), M2 with two open reading frames (M2-1, M2-2), small hydrophobic (SH), glycoprotein (G), and polymerase (L). Most of the HMPV proteins are analogous to other paramyxovirus proteins, with analogous functions (Cai et al., 2015; de Graaf et al., 2008; Derdowski et al., 2008; Kitagawa et al., 2010; Leyrat et al., 2014a; Leyrat et al., 2013; Leyrat et al., 2014b; Miller et al., 2007; Sabo et al., 2011; van den Hoogen et al., 2002).

Interestingly, we found that the loss of the ability to phosphorylate STAT1 observed in human airway cells was not specific to infected cells, but instead reflected an intrinsic negative feedback loop related to prolonged exposure to IFNα produced by viral infection. We used Vero cells, which contain an intact type I IFN response pathway but are unable to produce IFNs under PRR stimulation, to avoid this negative feedback and show that HMPV is capable of specifically decreasing STAT1 expression and IFNα-induced phosphorylation. This led us to hypothesize that HMPV uses a specific viral protein to evade type I IFN signaling pathway by targeting STAT1.

MATERIALS AND METHODS

Cells and Virus

BEAS-2B (ATCC® CRL-9609™) cells were purchased from ATCC. Vero (ATCC® CCL-81™) cells were kindly provided by Dr. James Crowe. BEAS-2B cells were maintained in Opti-MEM (Life Technologies) with 2% FBS. Vero cells were maintained in DMEM (Life Technologies) with 10% FBS. HMPV (pathogenic clinical strain TN/94-49, genotype A2) was grown and titered in LLC-MK2 cells as previously described (Williams et al., 2005). Reverse engineered HMPV strain CAN/97-83 (A2 genotype) lacking the SH gene (kindly provided by Ursula Buchholz and Peter Collins) was generated as described (Biacchesi et al., 2004a; Biacchesi et al., 2004c) and grown in LLC-MK2 cells. For type I IFN treatment to measure STAT1 phosphorylation, cells were incubated with 1000U/mL recombinant human IFNα (Alpha-2a)(PBL) for 15 minutes. For long-term type I IFN treatment, cells were incubated with 1000U/mL IFNα for 24 hours. Poly(I:C) treatment was achieved by transfecting 5μg/well polyinosinic-polycytidylic acid sodium salt (Sigma) into a 24-well plate using Lipofectamine 2000 (Life Technologies) and incubating cells for 24 hours.

Plasmids

Mammalian optimized viral protein constructs were designed using the GeneOptimizer® software (GeneArt) by adjusting codon usage and optimizing GC content to be efficiently expressed in mammalian cells. Viral protein sequences were then cloned into a pcDNA3.1 plasmid, and into the pcDNA3.1-GFP plasmid to create viral protein-GFP fusion constructs. HMPV M, N and P proteins were tagged on their C-terminus, while the SH protein, a type II transmembrane protein, was tagged on the N-terminus. Transfection was performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. To confirm subcellular localization of GFP-tagged proteins, cells were transfected in 24-well plates on autoclaved glass cover slips, fixed and permeabilized in buffered formalin 48h after transfection, and protein expression and localization determined by epi-illumination fluorescence microscopy using a fluorescein filter set (Zeiss).

Flow Cytometry

Cells were detached using 0.1% Trypsin-EDTA, washed, fixed with 3% buffered formalin for 10 minutes at room temperature, fixed and permeabilized with 100% methanol for 10 minutes at −20 °C, and washed with PBS/2% FBS. For labeling of HMPV-infected cells, samples were incubated with polyclonal guinea pig anti-HMPV sera and FITC-labeled anti-guinea pig IgG (Southern Biotech). Cells were also probed with a PE-labeled antibody to STAT1 (clone 1/STAT1, BD Phosflow) to determine overall expression levels, and with an AlexaFluor-647 labeled antibody to assess phosphorylation of STAT1 (pY701) (clone 4a, BD Phosflow) after treatment with IFNα.

Western Blot

Cells were mock- or HMPV-infected at ~80% confluency and infection was allowed to progress for 48 hours before IFNα treatment for 30 minutes. Cells were washed on ice with 1× PBS and then lysed using ice-cold RIPA buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, and 5 mM EDTA) supplemented with protease inhibitor (P8340, Sigma) and phosphatase inhibitor (Roche). Protein concentration was determined using a DC protein Assay (BioRad), and samples were diluted in a 5× sample buffer (250mM Tris HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, and 100mM DTT). Clarified lysates were boiled for ten minutes, subjected to SDS/PAGE, and transferred onto nitrocellulose membranes. Membranes were blocked for 1 hour in 5% nonfat dry milk in TBS-T buffer followed by incubation with antibodies against p-STAT1 (Clone A-2, Santa Cruz, sc-8394), STAT1 (Clone H-95, Santa Cruz, sc-98783), or β-tubulin (#T4026, Sigma). Blots were washed in TBS-T and subsequently incubated with HRP-conjugated secondary antibody (Promega). Signal was detected using the Clarity Western ECL substrate (Bio-Rad). Quantification of western blot analysis in Fig. 1B and C and Fig. 3B and C was performed using the Image J software to calculate band intensity. For STAT1 expression, intensity of band was normalized to the intensity of the band for the tubulin control. For STAT1 phosphorylation, intensity of the phosphorylated STAT1 band was calculated relative to STAT1 expression for each sample.

Fig. 1.

Fig. 1

Open in a new tab

HMPV inhibits STAT1 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were infected for 24 h with HMPV at an MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed with antibodies to phosphorylated STAT1, STAT1, and tubulin. (B) Quantification of western blot for STAT1 expression in HMPV-infected BEAS-2B cells. (C) Quantification of western blot for STAT1 phosphorylation in HMPV-infected BEAS-2B cells treated with type I IFN. (D) BEAS-2B cells were infected for 48 h with HMPV at MOI of 4 in 12-well plates, fixed and permeabilized, and fluorescent antibodies were used to probe for STAT1 expression using flow cytometry. (E) BEAS-2B cells were infected for 48 h with HMPV at MOI of 4, exposed to type I IFN for 15 m, fixed and permeabilized, and fluorescent antibodies used to probe for STAT1 phosphorylation by flow cytometry. N ≥ 3. Error bars represent SEM. **** P<0.0001, unpaired t test.

Fig. 3.

Fig. 3

Open in a new tab

HMPV inhibits STAT1 phosphorylation of Vero cells only in infected cells. (A) Vero cells were infected for 24 h with HMPV at MOI of 1 in 6-well plates. Cells were lysed with RIPA buffer, run on 4-15% bis-tris gel, transferred to nitrocellulose membrane, and probed with antibodies to STAT1, phosphorylated STAT1, and tubulin. (B, C) Quantification of western blot for STAT1 expression (B) and phosphorylation (C). (D) Vero cells were infected for 48 h with HMPV at MOI of 4 in 12-well plates, fixed and permeabilized, and STAT1 expression measured using flow cytometry. (E) Vero cells were infected for 48 h with HMPV at MOI of 4, exposed to type I IFN for 15 m, fixed and permeabilized, and STAT1 phosphorylation measured by flow cytometry. Groups were compared using unpaired t test or one-way ANOVA with Tukey's multiple comparisons test. N ≥ 3. Error bars represent SEM. * P<0.05.

Statistical Analyses

Data analysis was performed using Prism v4.0 (GraphPad Software). Groups were compared using unpaired t-test or one-way ANOVA with post-hoc Tukey test for multiple comparisons. P <0.05 was considered significant by convention.

RESULTS

HMPV infection increases STAT1 expression and inhibits type I IFN induced STAT1 phosphorylation in BEAS-2B cells

To characterize the effect of HMPV infection on STAT1 expression and phosphorylation, we first used the human bronchial epithelial cell line, BEAS-2B. After 24 hours of HMPV infection, cells were treated with IFNα for 15 minutes and STAT1 expression and phosphorylation levels were determined using western blot (Fig. 1A) and quantified using Image J software (Fig.s 1B, C, E). STAT1 expression did not change in cells infected with HMPV at an MOI of 1 (Fig. 1A-B). However, in infected BEAS-2B cells treated with IFNα, STAT1 phosphorylation was markedly lower than in IFN-treated uninfected cells (Fig. 1A and 1C). We next used flow cytometry to measure both expression and IFN-induced phosphorylation of STAT1 in bulk populations of BEAS-2B cells infected with HMPV. We observed an increase in total STAT1 expression in BEAS-2B cells over the course of 48 hours of HMPV infection (Fig. 1D), as well as a significant decrease in the ability of the infected BEAS-2B cells to phosphorylate STAT1 upon IFNα treatment as measured relative to expression (Fig. 1E). This suggested that HMPV is capable of inhibiting STAT1 phosphorylation in BEAS-2B cells. However, at an MOI of 1, only ~25% of cells are infected with HMPV (Tollefson et al., 2010), leading us to question whether this observation was specific to infected cells.

Type I IFNs produced by infected BEAS-2B cells induce high STAT1 levels, and upon long-term treatment lead to an inability to phosphorylate STAT1 in neighboring uninfected cells

In order to analyze the ability of HMPV to inhibit STAT1 phosphorylation specifically in the population of infected cells, we used fluorescent antibody staining to selectively label cells that were infected with HMPV and expressing viral proteins (Fig. 2A). Flow cytometry was then used to analyze STAT1 expression and IFN-induced phosphorylation in HMPV-infected and uninfected cells. STAT1 expression significantly increased by 48 hours in both infected and uninfected BEAS-2B cells in the same HMPV-exposed well compared to cells treated with uninfected LLC-MKC cell lysate (mock), but STAT1 expression in infected cells was lower than the uninfected cells in the same well (Fig. 2B). When cells infected with virus were treated with type I IFN at different time points during the course of infection, we observed that over 24 hours of HMPV infection, both infected cells and uninfected cells in the same well displayed a progressive loss of the ability to phosphorylate STAT1, relative to expression, in response to treatment with IFNα (Fig. 2C). Since uninfected cells in culture with HMPV-infected cells also lost the ability to phosphorylate STAT1, we tested the hypothesis that this inhibition of STAT1 phosphorylation was driven by host factors instead of a direct viral effect. We observed whether prolonged exposure to IFNα alone could induce this phenotype and found that after a 24-hour treatment of BEAS-2B cells with exogenous IFNα or endogenous type I IFN (the latter induced by poly(I:C)), these cells significantly upregulated STAT1 expression (Fig. 2D) and lost the ability to phosphorylate STAT1 when treated with IFNα (Fig. 2E). These findings led us to conclude that type I IFN produced and secreted by HMPV-infected cells was inducing a non-responsive state in surrounding uninfected cells in the well, thus hampering the ability to detect a specific viral effect within the infected cell population.

Fig. 2.

Fig. 2

Open in a new tab

Type I IFN secreted by infected BEAS-2B cells inhibits STAT1 phosphorylation in neighboring uninfected cells. (A) BEAS-2B cells were infected with HMPV for 24 h at an MOI of 1 in 6-well plates, then stained with polyclonal anti-HMPV antibody and gated into distinct uninfected (−) and infected (+) populations by flow cytometry. (B) Cells were stained with fluorescently labeled antibodies for STAT1 and expression was measured as mean fluorescence intensity (MFI) by flow cytometry. (C) BEAS-2B cells were infected with HMPV, treated with type I IFN at the indicated time points, fixed and stained to discriminate uninfected from infected as in A, and analyzed by flow cytometry for STAT1 phosphorylation. (D) BEAS-2B or Vero cells were treated with mock cell lysate, type I IFN, or poly(I:C) for 24 h, then analyzed by flow cytometry for STAT1 expression. (E) BEAS-2B or Vero cells were treated with mock cell lysate, type I IFN, or poly(I:C) for 24 h, then treated with type I IFN for 15 min and analyzed by flow cytometry for STAT1 phosphorylation. Groups were compared using one-way ANOVA with Tukey's multiple comparisons test. N = 2-3 duplicate experiments. Error bars represent SEM. * P<0.05, ** P<0.005, **** P<0.0001.

Vero cells are defective in the production of type I IFNs but can respond to exogenous type I IFN (Emeny and Morgan, 1979a, b) with STAT1 phosphorylation and provided a tractable alternative. We found no effect of poly(I:C) on either STAT1 expression (Fig. 2D) or IFNα-induced STAT1 phosphorylation (Fig. 2E) in Vero cells, confirming that these cells do not make IFN in response to poly(I:C). Treatment for 24 hours with IFNα did not effect STAT1 expression in Vero cells (Fig. 2D), as previously reported (Harle et al., 2001; Johnson et al., 2008), but did diminish the responsiveness of IFN-induced STAT1 phosphorylation (Fig. 2E), confirming that Vero cells can respond to exogenous IFNα. These data show that the inability of bulk populations of BEAS-2B cells, including uninfected cells, to phosphorylate STAT1 after HMPV infection is partly driven by paracrine type I IFN production by HMPV-infected cells, and that use of the Vero cell line can avoid this confounding effect.

HMPV infection of Vero cells reduces STAT1 expression and inhibits STAT1 phosphorylation specifically in infected cells

To determine the effect of HMPV infection on STAT1, Vero cells were infected at an MOI of 1, treated with IFNα 24 hours later, then analyzed for STAT1 expression and phosphorylation by western blot (Fig. 3A). No difference was detected in total STAT1 expression between infected and mock-treated bulk cell populations (Fig. 3A, B), but two isoforms of STAT1 appeared in Vero cells infected with HMPV (Fig. 3A)(Muller et al., 1993; Schindler et al., 1992). HMPV infection also modestly decreased STAT1 phosphorylation relative to expression in the bulk cell population (Fig. 3A, C). As in the previous experiments, we sought to look specifically at infected cells to determine a viral affect on STAT1. Flow cytometric analysis of Vero cells infected with an MOI of 4 was performed using an anti-HMPV antibody to differentiate between uninfected and infected cells as in Fig. 2. We found significantly lower STAT1 expression in HMPV-infected cells compared to uninfected cells in the same well (Fig. 3D). When these cells were treated with IFNα, there was significant defect in the ability of infected cells to phosphorylate STAT1 (normalized to expression) (Fig. 3E). These results show that HMPV is capable of decreasing STAT1 expression and inhibiting IFNα-induced STAT1 phosphorylation in Vero cells.

Vero cells transiently expressing the SH protein from HMPV display lower STAT1 expression and phosphorylation induced by both type I and II IFN

To identify which HMPV protein was responsible for the inhibition of IFNα-induced STAT1 phosphorylation, we generated GFP-tagged viral protein expression plasmids optimized for mammalian cell expression (Supplementary Fig. 1). We used flow cytometry to analyze Vero cells expressing viral proteins compared to non-transfected cells in the same well by gating on GFP (Fig. 4A and Supplementary Fig. 1A). We also performed western blot analysis of the GFP-fusion proteins, and showed that these proteins were expressed in transfected Vero cells (Supplementary Fig. 1B). The SH-GFP fusion protein was observed in multiple molecular weight bands, consistent with glycosylated and unglycosylated forms of SH as described previously (Masante et al., 2014), suggesting physiologic protein expression and processing (Supplementary Fig. 1B). The GFP-tagged viral proteins exhibited predominantly diffuse and/or punctate cytoplasmic distribution, with some nuclear localization of M protein (Supplementary Fig. 2). In cells expressing HMPV SH protein, there was significantly lower STAT1 expression compared to untransfected cells in the same well after 48 hours, but no difference in STAT1 expression was observed upon transfection of other viral proteins or GFP alone (Fig. 4B). When transfected cells were treated with IFNα and STAT1 phosphorylation relative to total expression was measured, the cells expressing SH were significantly inhibited in their ability to phosphorylate STAT1 (Fig. 4C). Similar inhibition of STAT1 phosphorylation relative to STAT1 expression was observed in SH-transfected Vero cells treated with IFNα (Fig. 4D). Cells expressing the HMPV N protein also showed a modest but significant defect in type II IFN-induced STAT1 phosphorylation (Fig. 4D). Thus, the HMPV SH protein is capable of preventing IFN-induced phosphorylation of STAT1.

Fig. 4.

Fig. 4

Open in a new tab

Transient expression of HMPV SH protein inhibits STAT1 phosphorylation. (A) Vero cells transfected with HMPV GFP-fusion proteins were analyzed compared to untransfected cells in the same culture using flow cytometry. (B) Transfected cells were analyzed by flow cytometry for STAT1 expression. (C, D) Transfected cells were treated with IFNα (C) or IFNγ (D) and STAT1 phosphorylation analyzed by flow cytometry. Transfected and non-transfected cells for each HMPV protein were compared using unpaired t test. N ≥ 3 duplicate experiments per group. Error bars represent SEM. * P<0.05, ** P<0.01, *** P<0.001.

Recombinant HMPV lacking the SH protein does not affect STAT1 expression or phosphorylation

Recombinant HMPV lacking the SH protein (ΔSH-HMPV) displayed similar growth kinetics to WT virus in LLC-MKC cells but was inhibited for replication in a hamster model (Biacchesi et al., 2005b; Biacchesi et al., 2004b). We infected Vero cells with ΔSH-HMPV and measured STAT1 expression and IFN-induced STAT1 phosphorylation in uninfected and infected cells (Fig. 5A). We found that in contrast to WT virus, which results in significantly lower STAT1 expression and IFN-induced phosphorylation (Fig. 3), the ΔSH-HMPV infected cells showed no difference in STAT1 expression (Fig. 5B). We then infected Vero cells with WT HMPV or ΔSH-HMPV for 24 hours, treated with type I IFN for 15 minutes, and measured STAT1 phosphorylation normalized to expression. As expected, cells infected with WT HMPV inhibited IFN-induced STAT1 phosphorylation (Fig. 5C). However, cells infected with ΔSH-HMPV were able to phosphorylate STAT1 at the same level as uninfected cells in the same well (Fig. 5C). These results, taken with the data using transient transfection, further corroborate the capacity of the SH protein to inhibit STAT1 signaling.

Fig. 5.

Fig. 5

Open in a new tab

Recombinant HMPV lacking the SH protein does not affect STAT1 expression or phosphorylation. Vero cells were infected with recombinant HMPV lacking the SH protein, treated with type I IFN, stained for HMPV-infected and uninfected (A), and analyzed for STAT1 expression (B) and phosphorylation (C) via flow cytometry. N = ≥2 duplicate experiments per group. Groups were compared using unpaired t test or one-way ANOVA with Dunnett's multiple comparisons test. Error bars represent SEM. ** P<0.01.

Discussion

HMPV is an important human pathogen and is capable of causing significant morbidity and mortality, particularly in vulnerable populations (Edwards et al., 2013; Englund et al., 2006; Esper et al., 2004; Papenburg et al., 2012; Shahda et al., 2011; Walsh et al., 2008; Widmer et al., 2012; Williams et al., 2004; Williams et al., 2006). Clinical disease is similar between HMPV and other related viruses such as RSV and PIV (Fox, 2007; Freymuth et al., 1995; Freymuth et al., 2006; Garcia-Garcia et al., 2006), despite the absence of sequence homology in the HMPV genome for previously known innate immune antagonists (Piyaratna et al., 2011; van den Hoogen et al., 2002). Both type I and II IFN signaling rely on STAT1 phosphorylation as a crucial component in both pathways. After ligation of IFNα or β to the type I IFN receptor, Janus kinases (Tyk2/JAK1) associated with the receptor cytoplasmic tail phosphorylate STAT1 (Tyr701) and STAT2 (Tyr689), which together with IRF9 form a transcription factor, interferon stimulated gene factor 3 (ISGF3) capable of binding to promoter elements for genes associated with the anti-viral response (Platanias, 2005). For type II IFN signaling, IFNγ binds to the IFNGR inducing phosphorylation of STAT1 (Tyr701) by other Janus kinases (JAK1/JAK2) resulting in a homodimer of STAT1 that acts as a transcription factor for anti-viral proteins, cytokines, and chemokines (Platanias, 2005). Type I and II IFN signaling induce an overlapping set of anti-viral molecules and act in combination to produce a cellular anti-viral response (Decker et al., 2005; Pestka et al., 2004; Schroder et al., 2004).

In our initial studies, we used the physiologically relevant human bronchial epithelial cell line, BEAS-2B, to determine the affect of HMPV infection on IFN signaling. Experiments using total cell populations showed that viral infection upregulated STAT1 expression and diminished IFN-induced phosphorylation of STAT1. However, by using flow cytometry to discriminate uninfected and infected cells, we found that infection of BEAS-2B cells induced a significant increase in STAT1 expression in both infected and uninfected cells in the same well, along with greatly reducing the ability for IFNα to induce STAT1 phosphorylation in both populations. The effect on uninfected cells in the same well as HMPV-infected BEAS-2B cells led us to speculate that type I IFNs secreted by infected cells were working in a paracrine fashion on uninfected cells, thus confounding the HMPV-specific STAT1 inhibition in infected cells with type I IFN-induced STAT1 downregulation in nearby uninfected cells (Isaacs and Lindenmann, 1957). Treatment of BEAS-2B cells with exogenous IFNα or poly(I:C) confirmed that type I IFN induced a natural downregulation of STAT1 expression and phosphorylation in our assays, which has been reported by other groups (Larner et al., 1986). This phenomenon presented a challenge for analyzing virus-specific effects on STAT1 within the subset of infected cells within an entire cell population. Notably, many studies of related viruses use cell lines competent for type I IFN production, suggesting that paracrine effects of type I IFN may confound similar experiments (Ren et al., 2012).

Vero cells are defective for the production of type I IFN, but are capable of responding to exogenous type I IFN (Emeny and Morgan, 1979a; Hasler and Wigand, 1978). We confirmed that, unlike BEAS-2B cells, poly(I:C) treatment of these cells neither increased STAT1 expression nor inhibited IFNα-induced STAT1 phosphorylation. Analysis of Vero cells via western blotting analysis of bulk cell populations showed that HMPV infection had no effect on STAT1 expression, but induced an inability to phosphorylate STAT1 in response to IFNα. Further investigation using flow cytometry to discriminate uninfected from infected cells revealed that HMPV-infected cells displayed significantly lower levels of STAT1 expression and IFNα-induced phosphorylation of STAT1 compared to uninfected cells from the same well. Notably, the diminished STAT1 expression in HMPV-infected Vero cells cannot be due to an IFN-induced paracrine effect. This finding may reflect an effect of viral infection on protein synthesis that perturbs particular pathways. HMPV has been shown in A549 cells to decrease the expression of eukaryotic elongation factor-2 (EEF2), glycyl-tRNA synthase (GARS), and other factors involved in protein synthesis and apoptosis (van Diepen et al., 2010). In addition, Vero cells cannot manifest the increased STAT1 expression induced by paracrine type I IFN, as can BEAS-2B cells (Fig. 2B).

Paramyxoviruses evade innate immune signaling through a variety of inhibitory actions (Caignard et al., 2007; Takeuchi et al., 2003), including modulation of STAT1 expression and phosphorylation (Didcock et al., 1999a, b; Kubota et al., 2001; Ramaswamy et al., 2004; Rodriguez et al., 2004; Rodriguez et al., 2002; Takeuchi et al., 2003). The V protein from PIV5 and mumps virus in the Rubulavirus genus, and the NS1 protein from RSV have been shown to target STAT proteins for proteosomal degradation (Didcock et al., 1999a, b; Kubota et al., 2001; Ramaswamy et al., 2004). However, the HMPV genome contains no open reading frames analogous to identified innate immune antagonists found in related paramyxoviruses (Piyaratna et al., 2011; van den Hoogen et al., 2002). Measles virus, by contrast, does not affect STAT protein levels, but uses the V protein to prevent STAT1 and STAT2 phosphorylation and thereby block the association and nuclear translocation of the ISGF3 transcription factor (Takeuchi et al., 2003). The Nipah virus V protein binds to STAT1 and STAT2 proteins and induces the conglomeration of high-molecular-weight complexes, which blocks type I IFN signaling (Rodriguez et al., 2004; Rodriguez et al., 2002).

The HMPV genome contains eight genes encoding nine known proteins (van den Hoogen et al., 2002). Other groups have shown possible roles for the phosphoprotein (P), M2-2, the glycoprotein (G), and the SH protein in the evasion of innate immune responses to HMPV. One study reported a differential ability for the A and B subgroups of HMPV to induce type I IFN, and suggested that the P protein of the B1 subgroup of HMPV was able to prevent RIG-I activation and the production of IFNs (Goutagny et al., 2010). It is unclear whether this inhibition resulted from direct interactions with host innate immune sensors, P protein interactions with viral RNA, or an effect on the translation of other viral proteins. The M2-2 protein is not required for viral growth, but might target the PRR mediator MAVS to prevent innate immune signaling and thus block type I IFN production (Ren et al., 2012). The G protein of HMPV has been suggested to play a role in the direct inhibition of RIG-I through interactions between the CARD domain of its N-terminus (Ren et al., 2012), leading to higher levels of inflammatory cytokine production in cells infected with HMPV lacking the G protein. We found that transient expression of the SH-GFP protein reduced the level of STAT1 expression and showed a block in IFNα- and IFNγ-induced STAT1 phosphorylation.

Recombinant HMPV lacking the SH protein showed no difference in viral kinetics or pathogenesis in a rodent model of HMPV (Biacchesi et al., 2004b), minimal attenuation in non-human primates (Biacchesi et al., 2005a) and gene expression analysis of A549 cells infected with the SH-deficient virus showed little discrepancy from cells infected with WT virus (de Graaf et al., 2013). In contrast, other paramyxoviruses reverse-engineered to delete known innate immune antagonists, such as RSV lacking NS1 and NS2 and PIV lacking V protein, display defects in replication in both cell culture and animal models (Spann et al., 2004). This difference in viral fitness might be due to cell type specific factors; alternatively, the inhibition of STAT1 by HMPV SH protein may be less important for replication and pathogenesis than the NS proteins of RSV.

The function of the SH protein of HMPV has not been fully characterized. The SH protein is predicted to be to be a type II transmembrane protein and is present across the Pneumovirinae subfamily, but HMPV encodes the longest SH protein at around 180 aa (van den Hoogen et al., 2002). Sequence homology between HMPV SH and related viruses is very low (Biacchesi et al., 2004a; Yunus et al., 2003) but maintains key characteristics such as similar hydrophilicity and a high percentage of threonine and serine residues (Piyaratna et al., 2011). Recent data indicate that, similar to the RSV SH protein, the HMPV SH protein is capable of forming a viroporin and increasing cell permeability (Masante et al., 2014). This is particularly intriguing in the context of our work, because dephosphorylated STAT1 has been shown to interact with the host nucleoporins, Nup153 and Nup214, in order to translocate out of the nucleus after signaling (Marg et al., 2004). The SH proteins of PIV5 and RSV are capable of inhibiting TNF-α signaling in vitro (Fuentes et al., 2007; Lin et al., 2003; Wilson et al., 2006), and a similar role for SH-HMPV has been proposed (Ren et al., 2012).

There are limitations in extrapolating animal and cell culture data to human disease, and as our BEAS-2B experiments showed, analysis of gene or protein expression in bulk cell populations may be confounded by paracrine artifacts. Nonetheless, our data show that HMPV SH protein is capable of inhibiting interferon signaling by blocking IFNα-induced phosphorylation of STAT1. Further studies are needed to elucidate the role of this inhibition in pathogenesis.

Supplementary Material

1
2

Research highlights.

  • Human metapneumovirus (HMPV) does not encode accessory proteins known to inhibit type I interferon (IFN).

  • Type I IFN produced by infected cells causes a paracrine effect on neighboring uninfected cells, suppressing STAT1 phosphorylation in uninfected cells and confounding interpretation of the effect of HMPV.

  • HMPV inhibits type I IFN-induced STAT1 phosphorylation specifically within infected cells.

  • HMPV short hydrophobic (SH) protein inhibits STAT1 phosphorylation.

Acknowledgements

We thank Ursula Buchholz and Peter Collins of the Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases for generously providing ΔSH-HMPV virus. We thank D. Flaherty, B. Matlock, and K. Weller at the Vanderbilt Flow Cytometry Shared Resource for providing technical support and assistance with development of flow cytometry reagents. We thank J. Crowe and N. Thornburg for providing the Vero cell line. Supported by NIH AI085062 (JVW) and GM007347 for the Vanderbilt Medical Scientist Training Program (SCW). The VMC Flow Cytometry Shared Resource is supported by the Vanderbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Digestive Disease Research Center (DK058404).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

REFERENCES

  1. Andrejeva J, Childs KS, Young DF, Carlos TS, Stock N, Goodbourn S, Randall RE. The V proteins of paramyxoviruses bind the IFN-inducible RNA helicase, mda-5, and inhibit its activation of the IFN-beta promoter. Proceedings of the National Academy of Sciences of the United States of America. 2004;101:17264–17269. doi: 10.1073/pnas.0407639101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barber GN. Host defense, viruses and apoptosis. Cell Death Differ. 2001;8:113–126. doi: 10.1038/sj.cdd.4400823. [DOI] [PubMed] [Google Scholar]
  3. Biacchesi S, Pham QN, Skiadopoulos MH, Murphy BR, Collins PL, Buchholz UJ. Infection of nonhuman primates with recombinant human metapneumovirus lacking the SH, G, or M2-2 protein categorizes each as a nonessential accessory protein and identifies vaccine candidates. J Virol. 2005a;79:12608–12613. doi: 10.1128/JVI.79.19.12608-12613.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Biacchesi S, Pham QN, Skiadopoulos MH, Murphy BR, Collins PL, Buchholz UJ. Infection of nonhuman primates with recombinant human metapneumovirus lacking the SH, G, or M2-2 protein categorizes each as a nonessential accessory protein and identifies vaccine candidates. Journal of virology. 2005b;79:12608–12613. doi: 10.1128/JVI.79.19.12608-12613.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Biacchesi S, Skiadopoulos MH, Tran KC, Murphy BR, Collins PL, Buchholz UJ. Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes. Virology. 2004a;321:247–259. doi: 10.1016/j.virol.2003.12.020. [DOI] [PubMed] [Google Scholar]
  6. Biacchesi S, Skiadopoulos MH, Yang L, Lamirande EW, Tran KC, Murphy BR, Collins PL, Buchholz UJ. Recombinant human Metapneumovirus lacking the small hydrophobic SH and/or attachment G glycoprotein: deletion of G yields a promising vaccine candidate. Journal of virology. 2004b;78:12877–12887. doi: 10.1128/JVI.78.23.12877-12887.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Biacchesi S, Skiadopoulos MH, Yang L, Lamirande EW, Tran KC, Murphy BR, Collins PL, Buchholz UJ. Recombinant human Metapneumovirus lacking the small hydrophobic SH and/or attachment G glycoprotein: deletion of G yields a promising vaccine candidate. Journal of virology. 2004c;78:12877–12887. doi: 10.1128/JVI.78.23.12877-12887.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Biron CA. Interferons alpha and beta as immune regulators--a new look. Immunity. 2001;14:661–664. doi: 10.1016/s1074-7613(01)00154-6. [DOI] [PubMed] [Google Scholar]
  9. Boivin G, De Serres G, Cote S, Gilca R, Abed Y, Rochette L, Bergeron MG, Dery P. Human metapneumovirus infections in hospitalized children. Emerg Infect Dis. 2003;9:634–640. doi: 10.3201/eid0906.030017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cai H, Zhang Y, Ma Y, Sun J, Liang X, Li J. Zinc binding activity of human metapneumovirus M2-1 protein is indispensable for viral replication and pathogenesis in vivo. Journal of virology. 2015;89:6391–6405. doi: 10.1128/JVI.03488-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Caignard G, Guerbois M, Labernardiere JL, Jacob Y, Jones LM, Infectious Mapping Project IM, Wild F, Tangy F, Vidalain PO. Measles virus V protein blocks Jak1-mediated phosphorylation of STAT1 to escape IFN-alpha/beta signaling. Virology. 2007;368:351–362. doi: 10.1016/j.virol.2007.06.037. [DOI] [PubMed] [Google Scholar]
  12. Childs K, Randall R, Goodbourn S. Paramyxovirus V proteins interact with the RNA Helicase LGP2 to inhibit RIG-I-dependent interferon induction. Journal of virology. 2012;86:3411–3421. doi: 10.1128/JVI.06405-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. de Graaf M, Herfst S, Aarbiou J, Burgers PC, Zaaraoui-Boutahar F, Bijl M, van Ijcken W, Schrauwen EJ, Osterhaus AD, Luider TM, Scholte BJ, Fouchier RA, Andeweg AC. Small hydrophobic protein of human metapneumovirus does not affect virus replication and host gene expression in vitro. PLoS One. 2013;8:e58572. doi: 10.1371/journal.pone.0058572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. de Graaf M, Herfst S, Schrauwen EJ, Choi Y, van den Hoogen BG, Osterhaus AD, Fouchier RA. Specificity and functional interaction of the polymerase complex proteins of human and avian metapneumoviruses. J Gen Virol. 2008;89:975–983. doi: 10.1099/vir.0.83537-0. [DOI] [PubMed] [Google Scholar]
  15. Decker T, Muller M, Stockinger S. The yin and yang of type I interferon activity in bacterial infection. Nature reviews. Immunology. 2005;5:675–687. doi: 10.1038/nri1684. [DOI] [PubMed] [Google Scholar]
  16. Derdowski A, Peters TR, Glover N, Qian R, Utley TJ, Burnett A, Williams JV, Spearman P, Crowe JE., Jr. Human metapneumovirus nucleoprotein and phosphoprotein interact and provide the minimal requirements for inclusion body formation. The Journal of general virology. 2008;89:2698–2708. doi: 10.1099/vir.0.2008/004051-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Didcock L, Young DF, Goodbourn S, Randall RE. Sendai virus and simian virus 5 block activation of interferon-responsive genes: importance for virus pathogenesis. Journal of virology. 1999a;73:3125–3133. doi: 10.1128/jvi.73.4.3125-3133.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Didcock L, Young DF, Goodbourn S, Randall RE. The V protein of simian virus 5 inhibits interferon signalling by targeting STAT1 for proteasome-mediated degradation. Journal of virology. 1999b;73:9928–9933. doi: 10.1128/jvi.73.12.9928-9933.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Dinwiddie DL, Harrod KS. Human metapneumovirus inhibits IFN-alpha signaling through inhibition of STAT1 phosphorylation. Am J Respir Cell Mol Biol. 2008;38:661–670. doi: 10.1165/rcmb.2007-0285OC. [DOI] [PubMed] [Google Scholar]
  20. Edwards KM, Zhu Y, Griffin MR, Weinberg GA, Hall CB, Szilagyi PG, Staat MA, Iwane M, Prill MM, Williams JV. Burden of human metapneumovirus infection in young children. The New England journal of medicine. 2013;368:633–643. doi: 10.1056/NEJMoa1204630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Emeny JM, Morgan MJ. Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production. J Gen Virol. 1979a;43:247–252. doi: 10.1099/0022-1317-43-1-247. [DOI] [PubMed] [Google Scholar]
  22. Emeny JM, Morgan MJ. Susceptibility of various cells treated with interferon to the toxic effect of poly(rI).poly(rC) treatment. J Gen Virol. 1979b;43:253–255. doi: 10.1099/0022-1317-43-1-253. [DOI] [PubMed] [Google Scholar]
  23. Englund JA, Boeckh M, Kuypers J, Nichols WG, Hackman RC, Morrow RA, Fredricks DN, Corey L. Brief communication: fatal human metapneumovirus infection in stem-cell transplant recipients. Ann Intern Med. 2006;144:344–349. doi: 10.7326/0003-4819-144-5-200603070-00010. [DOI] [PubMed] [Google Scholar]
  24. Esper F, Martinello RA, Boucher D, Weibel C, Ferguson D, Landry ML, Kahn JS. A 1-year experience with human metapneumovirus in children aged <5 years. J Infect Dis. 2004;189:1388–1396. doi: 10.1086/382482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Fox JD. Respiratory virus surveillance and outbreak investigation. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology. 2007;40(Suppl 1):S24–30. doi: 10.1016/S1386-6532(07)70006-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Freymuth F, Eugene G, Vabret A, Petitjean J, Gennetay E, Brouard J, Duhamel JF, Guillois B. Detection of respiratory syncytial virus by reverse transcription-PCR and hybridization with a DNA enzyme immunoassay. Journal of clinical microbiology. 1995;33:3352–3355. doi: 10.1128/jcm.33.12.3352-3355.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Freymuth F, Vabret A, Cuvillon-Nimal D, Simon S, Dina J, Legrand L, Gouarin S, Petitjean J, Eckart P, Brouard J. Comparison of multiplex PCR assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness. Journal of medical virology. 2006;78:1498–1504. doi: 10.1002/jmv.20725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Fuentes S, Tran KC, Luthra P, Teng MN, He B. Function of the respiratory syncytial virus small hydrophobic protein. Journal of virology. 2007;81:8361–8366. doi: 10.1128/JVI.02717-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Gainey MD, Dillon PJ, Clark KM, Manuse MJ, Parks GD. Paramyxovirus-induced shutoff of host and viral protein synthesis: role of the P and V proteins in limiting PKR activation. Journal of virology. 2008;82:828–839. doi: 10.1128/JVI.02023-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Garcia-Garcia ML, Calvo C, Perez-Brena P, De Cea JM, Acosta B, Casas I. Prevalence and clinical characteristics of human metapneumovirus infections in hospitalized infants in Spain. Pediatric pulmonology. 2006;41:863–871. doi: 10.1002/ppul.20456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Gordon S. Pattern recognition receptors: doubling up for the innate immune response. Cell. 2002;111:927–930. doi: 10.1016/s0092-8674(02)01201-1. [DOI] [PubMed] [Google Scholar]
  32. Goutagny N, Jiang Z, Tian J, Parroche P, Schickli J, Monks BG, Ulbrandt N, Ji H, Kiener PA, Coyle AJ, Fitzgerald KA. 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. J Immunol. 2010;184:1168–1179. doi: 10.4049/jimmunol.0902750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Harle P, Lauret E, Pitha PM, De Maeyer E, Carr DJ. Expression of human and macaque type I IFN transgenes interferes with HSV-1 replication at the transcriptional and translational levels: IFN-beta is more potent than IFN-alpha 2. Virology. 2001;290:237–248. doi: 10.1006/viro.2001.1178. [DOI] [PubMed] [Google Scholar]
  34. Hasler P, Wigand R. The susceptibility of Vero cell cultures for human adenoviruses. Medical microbiology and immunology. 1978;164:267–275. doi: 10.1007/BF02125495. [DOI] [PubMed] [Google Scholar]
  35. Havenar-Daughton C, Kolumam GA, Murali-Krishna K. Cutting Edge: The direct action of type I IFN on CD4 T cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection. J Immunol. 2006;176:3315–3319. doi: 10.4049/jimmunol.176.6.3315. [DOI] [PubMed] [Google Scholar]
  36. Isaacs A, Lindenmann J. Virus interference. I. The interferon. Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society. 1957;147:258–267. doi: 10.1098/rspb.1957.0048. [DOI] [PubMed] [Google Scholar]
  37. Johnson KE, Song B, Knipe DM. Role for herpes simplex virus 1 ICP27 in the inhibition of type I interferon signaling. Virology. 2008;374:487–494. doi: 10.1016/j.virol.2008.01.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kitagawa Y, Zhou M, Yamaguchi M, Komatsu T, Takeuchi K, Itoh M, Gotoh B. Human metapneumovirus M2-2 protein inhibits viral transcription and replication. Microbes Infect. 2010;12:135–145. doi: 10.1016/j.micinf.2009.11.002. [DOI] [PubMed] [Google Scholar]
  39. Kolumam GA, Thomas S, Thompson LJ, Sprent J, Murali-Krishna K. Type I interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection. J Exp Med. 2005;202:637–650. doi: 10.1084/jem.20050821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kubota T, Yokosawa N, Yokota S, Fujii N. C terminal CYS-RICH region of mumps virus structural V protein correlates with block of interferon alpha and gamma signal transduction pathway through decrease of STAT 1-alpha. Biochem Biophys Res Commun. 2001;283:255–259. doi: 10.1006/bbrc.2001.4764. [DOI] [PubMed] [Google Scholar]
  41. Larner AC, Chaudhuri A, Darnell JE., Jr. Transcriptional induction by interferon. New protein(s) determine the extent and length of the induction. The Journal of biological chemistry. 1986;261:453–459. [PubMed] [Google Scholar]
  42. Le Bon A, Tough DF. Type I interferon as a stimulus for cross-priming. Cytokine Growth Factor Rev. 2008;19:33–40. doi: 10.1016/j.cytogfr.2007.10.007. [DOI] [PubMed] [Google Scholar]
  43. Leyrat C, Paesen GC, Charleston J, Renner M, Grimes JM. Structural insights into the human metapneumovirus Glycoprotein ectodomain. Journal of virology. 2014a doi: 10.1128/JVI.01726-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Leyrat C, Renner M, Harlos K, Grimes JM. Solution and crystallographic structures of the central region of the phosphoprotein from human metapneumovirus. PLoS One. 2013;8:e80371. doi: 10.1371/journal.pone.0080371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Leyrat C, Renner M, Harlos K, Huiskonen JT, Grimes JM. Structure and self-assembly of the calcium binding matrix protein of human metapneumovirus. Structure. 2014b;22:136–148. doi: 10.1016/j.str.2013.10.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Lin Y, Bright AC, Rothermel TA, He B. Induction of apoptosis by paramyxovirus simian virus 5 lacking a small hydrophobic gene. Journal of virology. 2003;77:3371–3383. doi: 10.1128/JVI.77.6.3371-3383.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Lo MS, Brazas RM, Holtzman MJ. Respiratory syncytial virus nonstructural proteins NS1 and NS2 mediate inhibition of Stat2 expression and alpha/beta interferon responsiveness. Journal of virology. 2005;79:9315–9319. doi: 10.1128/JVI.79.14.9315-9319.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Luft T, Pang KC, Thomas E, Hertzog P, Hart DN, Trapani J, Cebon J. Type I IFNs enhance the terminal differentiation of dendritic cells. J Immunol. 1998;161:1947–1953. [PubMed] [Google Scholar]
  49. Marg A, Shan Y, Meyer T, Meissner T, Brandenburg M, Vinkemeier U. Nucleocytoplasmic shuttling by nucleoporins Nup153 and Nup214 and CRM1-dependent nuclear export control the subcellular distribution of latent Stat1. The Journal of cell biology. 2004;165:823–833. doi: 10.1083/jcb.200403057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Masante C, El Najjar F, Chang A, Jones A, Moncman CL, Dutch RE. The human metapneumovirus small hydrophobic protein has properties consistent with those of a viroporin and can modulate viral fusogenic activity. Journal of virology. 2014;88:6423–6433. doi: 10.1128/JVI.02848-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Miller SA, Tollefson S, Crowe JE, Jr., Williams JV, Wright DW. Examination of a fusogenic hexameric core from human metapneumovirus and identification of a potent synthetic peptide inhibitor from the heptad repeat 1 region. Journal of virology. 2007;81:141–149. doi: 10.1128/JVI.01243-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Muller M, Laxton C, Briscoe J, Schindler C, Improta T, Darnell JE, Jr., Stark GR, Kerr IM. Complementation of a mutant cell line: central role of the 91 kDa polypeptide of ISGF3 in the interferon-alpha and -gamma signal transduction pathways. EMBO J. 1993;12:4221–4228. doi: 10.1002/j.1460-2075.1993.tb06106.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Papenburg J, Hamelin ME, Ouhoummane N, Carbonneau J, Ouakki M, Raymond F, Robitaille L, Corbeil J, Caouette G, Frenette L, De Serres G, Boivin G. Comparison of Risk Factors for Human Metapneumovirus and Respiratory Syncytial Virus Disease Severity in Young Children. J Infect Dis. 2012 doi: 10.1093/infdis/jis333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Pestka S, Krause CD, Walter MR. Interferons, interferon-like cytokines, and their receptors. Immunological reviews. 2004;202:8–32. doi: 10.1111/j.0105-2896.2004.00204.x. [DOI] [PubMed] [Google Scholar]
  55. Piyaratna R, Tollefson SJ, Williams JV. Genomic analysis of four human metapneumovirus prototypes. Virus Res. 2011;160:200–205. doi: 10.1016/j.virusres.2011.06.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Platanias LC. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nature reviews. Immunology. 2005;5:375–386. doi: 10.1038/nri1604. [DOI] [PubMed] [Google Scholar]
  57. Ramaswamy M, Shi L, Monick MM, Hunninghake GW, Look DC. Specific inhibition of type I interferon signal transduction by respiratory syncytial virus. Am J Respir Cell Mol Biol. 2004;30:893–900. doi: 10.1165/rcmb.2003-0410OC. [DOI] [PubMed] [Google Scholar]
  58. Ren J, Kolli D, Liu T, Xu R, Garofalo RP, Casola A, Bao X. Human metapneumovirus inhibits IFN-beta signaling by downregulating Jak1 and Tyk2 cellular levels. PLoS One. 2011;6:e24496. doi: 10.1371/journal.pone.0024496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Ren J, Wang Q, Kolli D, Prusak DJ, Tseng CT, Chen ZJ, Li K, Wood TG, Bao X. Human metapneumovirus M2-2 protein inhibits innate cellular signaling by targeting MAVS. Journal of virology. 2012;86:13049–13061. doi: 10.1128/JVI.01248-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Rodriguez JJ, Cruz CD, Horvath CM. Identification of the nuclear export signal and STAT-binding domains of the Nipah virus V protein reveals mechanisms underlying interferon evasion. Journal of virology. 2004;78:5358–5367. doi: 10.1128/JVI.78.10.5358-5367.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Rodriguez JJ, Parisien JP, Horvath CM. Nipah virus V protein evades alpha and gamma interferons by preventing STAT1 and STAT2 activation and nuclear accumulation. Journal of virology. 2002;76:11476–11483. doi: 10.1128/JVI.76.22.11476-11483.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Sabo Y, Ehrlich M, Bacharach E. The conserved YAGL motif in human metapneumovirus is required for higher-order cellular assemblies of the matrix protein and for virion production. Journal of virology. 2011;85:6594–6609. doi: 10.1128/JVI.02694-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Sadler AJ, Williams BR. Interferon-inducible antiviral effectors. Nature reviews. Immunology. 2008;8:559–568. doi: 10.1038/nri2314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Schindler C, Fu XY, Improta T, Aebersold R, Darnell JE., Jr. Proteins of transcription factor ISGF-3: one gene encodes the 91-and 84-kDa ISGF-3 proteins that are activated by interferon alpha. Proceedings of the National Academy of Sciences of the United States of America. 1992;89:7836–7839. doi: 10.1073/pnas.89.16.7836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-gamma: an overview of signals, mechanisms and functions. Journal of leukocyte biology. 2004;75:163–189. doi: 10.1189/jlb.0603252. [DOI] [PubMed] [Google Scholar]
  66. Shahda S, Carlos WG, Kiel PJ, Khan BA, Hage CA. The human metapneumovirus: a case series and review of the literature. Transpl Infect Dis. 2011;13:324–328. doi: 10.1111/j.1399-3062.2010.00575.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Spann KM, Tran KC, Chi B, Rabin RL, Collins PL. Suppression of the induction of alpha, beta, and lambda interferons by the NS1 and NS2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages [corrected]. Journal of virology. 2004;78:4363–4369. doi: 10.1128/JVI.78.8.4363-4369.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Takeuchi K, Kadota SI, Takeda M, Miyajima N, Nagata K. Measles virus V protein blocks interferon (IFN)-alpha/beta but not IFN-gamma signaling by inhibiting STAT1 and STAT2 phosphorylation. FEBS letters. 2003;545:177–182. doi: 10.1016/s0014-5793(03)00528-3. [DOI] [PubMed] [Google Scholar]
  69. Tollefson SJ, Cox RG, Williams JV. Studies of culture conditions and environmental stability of human metapneumovirus. Virus Research. 2010;151:54–59. doi: 10.1016/j.virusres.2010.03.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. van den Hoogen BG, Bestebroer TM, Osterhaus AD, Fouchier RA. Analysis of the genomic sequence of a human metapneumovirus. Virology. 2002;295:119–132. doi: 10.1006/viro.2001.1355. [DOI] [PubMed] [Google Scholar]
  71. van den Hoogen BG, de Jong JC, Groen J, Kuiken T, de Groot R, Fouchier RA, Osterhaus AD. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med. 2001;7:719–724. doi: 10.1038/89098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. van Diepen A, Brand HK, Sama I, Lambooy LH, van den Heuvel LP, van der Well L, Huynen M, Osterhaus AD, Andeweg AC, Hermans PW. Quantitative proteome profiling of respiratory virus-infected lung epithelial cells. J Proteomics. 2010;73:1680–1693. doi: 10.1016/j.jprot.2010.04.008. [DOI] [PubMed] [Google Scholar]
  73. Walsh EE, Peterson DR, Falsey AR. Human metapneumovirus infections in adults: another piece of the puzzle. Arch Intern Med. 2008;168:2489–2496. doi: 10.1001/archinte.168.22.2489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Widmer K, Zhu Y, Williams JV, Griffin MR, Edwards KM, Talbot HK. Rates of Hospitalizations for Respiratory Syncytial Virus, Human Metapneumovirus, and Influenza Virus in Older Adults. J Infect Dis. 2012 doi: 10.1093/infdis/jis309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Williams JV, Harris PA, Tollefson SJ, Halburnt-Rush LL, Pingsterhaus JM, Edwards KM, Wright PF, Crowe JE., Jr. Human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children. N Engl J Med. 2004;350:443–450. doi: 10.1056/NEJMoa025472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Williams JV, Tollefson SJ, Johnson JE, Crowe JE., Jr. The cotton rat (Sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity. Journal of virology. 2005;79:10944–10951. doi: 10.1128/JVI.79.17.10944-10951.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Williams JV, Wang CK, Yang CF, Tollefson SJ, House FS, Heck JM, Chu M, Brown JB, Lintao LD, Quinto JD, Chu D, Spaete RR, Edwards KM, Wright PF, Crowe JE., Jr. The role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience. J Infect Dis. 2006;193:387–395. doi: 10.1086/499274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Wilson RL, Fuentes SM, Wang P, Taddeo EC, Klatt A, Henderson AJ, He B. Function of small hydrophobic proteins of paramyxovirus. Journal of virology. 2006;80:1700–1709. doi: 10.1128/JVI.80.4.1700-1709.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Yunus AS, Govindarajan D, Huang Z, Samal SK. Deduced amino acid sequence of the small hydrophobic protein of US avian pneumovirus has greater identity with that of human metapneumovirus than those of non-US avian pneumoviruses. Virus Res. 2003;93:91–97. doi: 10.1016/s0168-1702(03)00074-1. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS782499-supplement-1.pdf (1.3MB, pdf)
2
NIHMS782499-supplement-2.pdf (2MB, pdf)

RESOURCES