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Published ahead of print on January 24, 2008, doi:10.1165/rcmb.2007-0285OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 661-670, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0285OC

Human Metapneumovirus Inhibits IFN-{alpha} Signaling through Inhibition of STAT1 Phosphorylation

Darrell L. Dinwiddie1 and Kevin S. Harrod1

1 Infectious Diseases Program, Lovelace Respiratory Research Institute, Albuquerque, New Mexico

Correspondence and requests for reprints should be addressed to Kevin S. Harrod, MD, 2425 Ridgecrest Dr. SE, Albuquerque, NM 87108. E-mail: kharrod{at}lrri.org


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The recently discovered human metapneumovirus (hMPV) is a major cause of lower and upper respiratory tract infections worldwide. Acute viral infection initiates the interferon response that is critical in mediating viral clearance, viral host defense, and development of adaptive immunity. Mouse models of infection suggest that hMPV can cause persistent lung infections, yet the mechanisms of evading host viral clearance are unknown. Here we report that hMPV can subvert host type I interferon signaling by a mechanism distinct from other paramyxoviruses. Two lung epithelial cell lines and primary normal human bronchial epithelial cells (NHBE) were permissive for hMPV, consistent with its tropism for the respiratory tract. Treatment of hMPV-infected cells with exogenous IFN-{alpha} failed to reduce viral replication. Moreover, in lung epithelial cells, hMPV infection prevented IFN-{alpha}–mediated transactivation of the interferon-stimulated response element (ISRE) and up-regulation of interferon-stimulated genes (ISGs). Further examination of the IFN-{alpha} signaling cascade showed that hMPV infection prevented IFN-{alpha}–induced phosphorylation and nuclear translocation of STAT1. The inhibitory effects of hMPV on STAT1 phosphorylation and translocation were abolished by ultraviolet inactivation. Regulation of STAT1 by hMPV was specific, as phosphorylation of STAT2, Tyk2, and Jak1 by IFN-{alpha} and the surface expression of the IFN-{alpha} receptor were unaltered by hMPV infection. These findings demonstrate that hMPV can inhibit the type I interferon response through regulation of STAT1 phosphorylation, and provide important insight into the viral pathogenesis of hMPV infection in the respiratory tract.

Key Words: human metapneumovirus • interferon-{alpha} • type I interferon • innate immune response • STAT



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Our research with human metapneumovirus (hMPV) provides new insight into the viral mechanisms hMPV uses to inhibit the innate immune response. This will assist in the development of preventive vaccines and/or treatments of hMPV.

 
Human metapneumovirus (hMPV), first reported in 2001 (1), is a causative agent of acute respiratory tract infections of particular importance in pediatric, immunocompromised, and elderly populations (2–4). Closely related to respiratory syncytial virus (RSV), hMPV is a member of the Metapneumovirus genus within the Pneumovirinae subfamily of the Paramyxoviridae family. Both RSV and hMPV can cause severe infection resulting in bronchiolitis, bronchitis, or pneumonia (5, 6) and have been associated with exacerbations of chronic obstructive pulmonary disease and asthma (4, 7). Furthermore, neither RSV nor hMPV infection results in long-term protective immunity, and individuals can be infected repeatedly throughout life and possibly within the same season by the identical strain of virus (8, 9). Although hMPV and RSV infection result in similar clinical manifestations, they exhibit significant genetic variation at both the nucleotide and amino acid level (10). While RSV encodes 11 proteins, hMPV encodes only 9 proteins and lacks the nonstructural NS1 and NS2 proteins of RSV that have been shown to modulate the cellular innate immune response initiated by acute viral infection (11, 12). The lack of these immunomodulatory genes in hMPV suggests that the interaction between the virus and the innate immune response is significantly different from that of RSV. Further evidence of differences of the interactions between RSV and hMPV and the immune response is provided by mouse models of infection. In mice, hMPV can cause a persistent infection, demonstrating that hMPV possess mechanisms of evading host viral clearance (13, 14). However, to date, very little is known about the molecular mechanisms of hMPV pathogenesis and the immune response triggered by infection.

Acute viral infection initiates a type I interferon (IFN) response that is composed predominantly of interferon {alpha} and β (IFN-{alpha}/IFN-β) signaling through the IFN-{alpha} receptor. IFN-{alpha} receptor binding results in activation of the accessory kinase proteins Jak1 and Tyk2, which subsequently phosphorylate STAT2 and STAT1, leading to STAT2-STAT1 heterotrimerization with interferon regulatory factor (IRF) 9 and nuclear localization (reviewed in Ref. 15). In the nucleus these proteins serve to transactivate the interferon-stimulated response element (ISRE) found in the promoter of interferon-stimulated genes (ISGs). Viruses have evolved numerous unique mechanisms to inhibit the type I interferon response and have been reported to block every aspect of the signaling pathway (for recent reviews see Refs. 16, 17). Several members of the Paramyxovirus family have been shown to directly target STAT signaling through distinct mechanisms which include proteasomal degradation (18–21), sequestration in high-molecular-weight complexes (22, 23), and inhibition of nuclear localization of STAT proteins (24).

Despite the capacity of many of the paramyxoviruses to inhibit IFN signaling, the ability of hMPV to alter IFN signaling has not been addressed. The lack of homologous genes known to encode immunomodulatory proteins with other paramyxoviruses suggests that hMPV may encode unique mechanisms to subvert viral clearance. In this study, we evaluated the type I IFN response in hMPV-infected lung epithelial cells. Indeed, our results show that hMPV is capable of regulating IFN-mediated signaling in lung epithelial cells through regulation of STAT1 phosphorylation and nuclear translocation.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Culture and Reagents
Human lung epithelial A549 and H441 and monkey kidney Vero E6 and LLC-MK2 cells were maintained in MEM (Sigma, St. Louis, MO) supplemented with 2 mM L-glutamine, antibiotic/antimycotic, 1.5 g/L sodium bicarbonate, and10% fetal bovine serum (GIBCO, Invitrogen, Carlsbad, CA). Normal human bronchial epithelial cells (Cambrex, Walkersville, MD) were cultured on 100-mm collagen-coated BIOCOAT (Becton Dickinson, Bedford, MA) dishes in bronchial epithelial medium (BEGM) (Cambrex), supplemented with singlequots (Cambrex), retinoic acid (Sigma), and bovine serum albumin (Sigma). Recombinant human IFN-{alpha}A (Biosource, Camarillo, CA) was used at a concentration of 1,000 IU/ml, and recombinant IFN-{gamma} (Peprotech, Rocky Hill, NJ) was used at a concentration of 10 ng/ml, unless otherwise noted.

hMPV and Vesicular Stomatitis Virus Propagation
Human metapneumovirus strain CAN97-83, a generous gift of Ralph Tripp (University of Georgia, Athens, GA), was propagated in Vero E6 cells. Vero E6 cells at approximately 85 to 90% confluence were washed with PBS and infected with hMPV at a multiplicity of infection (MOI) of 0.1 diluted in serum-free MEM containing 25 mM HEPES, 2 mM L-glutamine, antibiotic/antimycotic, 1.5 g/L sodium bicarbonate, and 1 µg/ml TPCK-trypsin. Virus was allowed to adsorb for 1 hour at 37°C with rocking every 15 minutes to ensure even distribution of the viral inoculum, followed by the addition of serum-free MEM with 0.1 µg/ml TPCK-trypsin (Worthington Biochemical Corporation, Lakewood, NJ). Infected cells were incubated for 5 days at 37°C before harvesting. Virus was harvested after the addition of BSA to a final concentration of 0.5% by removal of cells from the surface of the flask using a cell scraper and subjecting cell containing media to two freeze–thaw cycles. Cell debris was removed by centrifugation at 4,000 x g for 20 minutes at 4°C and titer was determined by a focus forming assay. Vesicular stomatitis virus (VSV) Indiana strain (ATCC, Manassas, VA) was propagated and titrated in LLC-MK2 cells.

hMPV Titration
Titration of hMPV was performed in Vero E6 cells using an immunostaining focus-forming assay. hMPV was diluted by serial 10-fold dilutions in serum-free MEM (GIBCO, Invitrogen) and added to approximately 95% confluent Vero E6 cells cultured in serum-free MEM (GIBCO, Invitrogen). After 2 hours of incubation at 37°C, viral inoculum was aspirated off and replaced with agarose overlay media. Agarose overlay media is composed of a 1:1 ratio of 2x MEM without phenol red (GIBCO, Invitrogen) 2 mM L-glutamine, 25 mM HEPES, antibiotic/antimycotic, and 0.1 µg/ml TPCK-trypsin to 0.6% sea plaque agarose in water (Cambrex). At 96 hours after infection, the cells were fixed in 10% formalin and the agarose overlay was carefully removed. The endogenous peroxidase activity of the cells was quenched by the addition of 3% hydrogen peroxide in methanol for 20 minutes at room temperature and washed five times in PBS with 0.5% Tween 20 and blocked for 2 hours with PBS containing 10% normal horse serum. Cells were immunostained using a purified mouse anti-human metapneumovirus monoclonal antibody (Chemicon, Temecula, CA) diluted 1:1,000 in 1% horse serum PBS-0.5% Tween 20 for 2 hours at 37°C. After washing five times with PBS-0.5% Tween 20 cells were stained with a horse anti-mouse biotinylated secondary antibody diluted 1:500 in 1% horse serum PBS-0.5% Tween 20 for 30 minutes at 37°C and labeled using the Vector ABC kit per the manufacturer's instructions. Foci were enumerated using 3',3'-diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA).

Detection of hMPV by Immunofluorescence Microscopy
For detection of hMPV by immunofluorescence, cells were grown and infected on glass chamber slides (Nalgene Nunc International, Naperville, IL). Fixation of cells was performed by the addition of 200 µl of 3% paraformaldehyde and 3% sucrose in PBS for 30 minutes, followed by washing three times with PBS and permeablization using 0.2% triton X-100 in PBS for 30 minutes. After three washes with PBS, cells were blocked with PBS containing 10% normal goat serum (Vector) for 1 hour and then incubated with a primary antibody specific to hMPV. For hMPV detection, a mouse anti-hMPV (Chemicon) antibody that detects the matrix protein at a dilution of 1:400 was used. The nuclei of all cells were stained using a 1:400 dilution of Hoechst (Molecular Probes, Invitrogen, Carlsbad, CA), while virus was visualized using either a goat anti-mouse IgG Cy3- or Cy5-conjugated antibody (Vector). Fluorescence microscopy was performed on a Zeiss Axioplan 2 (Zeiss, Thornwood, NY) with an Intelligent Image Innovations CCD Camera using Slidebook software version 4.1.0.7 (both from Intelligent Image Innovations, Denver, CO).

IFN-{alpha} Sensitivity of hMPV and VSV
A549 cells were incubated with media or pretreated with media containing IFN-{alpha} (1,000 U/ml) for 20 hours before infection with VSV or hMPV. After 1 hour of absorption, viral inoculum was removed and cells were washed three times with PBS and replaced with serum-free Dulbecco's modified Eagle's medium. For hMPV infections, trypsin (1 µg/ml) was present in the media during infection and after removal of virus inoculum. Supernatant was collected at 6, 24, 48, and 72 hours after infection and viral titration was performed on LLC-MK2 cells.

Cellular Localization of STAT1
Cells were grown and infected as described in detection of hMPV by immunofluorescence microscopy section. After 48 hours of infection with hMPV, media was removed and replaced with new media containing 1,000 IU/ml of IFN-{alpha} for 30 minutes. Cells were fixed, permeabilized, and blocked as before and co-stained for STAT1, hMPV, and nuclei. STAT1 was detected using a rabbit anti-STAT1 (Cell Signaling, Danvers, MA) at a dilution of 1:100 added to the primary antibody step for hMPV detection. Secondary antibody incubation consisted of a 1:400 dilution of Hoechst (Molecular Probes), a 1:200 dilution of a goat anti-mouse IgG Cy5-conjugated antibody to detect hMPV, and a 1:200 dilution of a goat anti-rabbit IgG Cy3 (Vector)-conjugated antibody in 3% goat serum PBS for 1 hour at room temperature.

Quantitative RT-PCR Analysis of ISG Induction
RNA from A549 cells infected with hMPV for 48 hours and treated with IFN-{alpha} for 24 hours was isolated from cells using the RNeasy Kit (Qiagen, Valencia, CA) with optional on-column DNase digestion per the manufacturer's directions. The concentration and quality of RNA was determined by spectrophotometry and equal amounts of RNA were subjected to one-step quantitative RT-PCR using reagents and primer and probe sets (Applied Biosystems, Foster City, CA) per the manufacturer's instructions to measure mRNA levels of MxA (Hs00182073_m1), ISG56 (Hs00356631_g1), RIG-I (Hs00204833_m1), Mda5 (Hs00223420_A1), and 18SrRNA (Hs99999901_s1). The procedure was performed in an ABI Prism 7900 Sequence Detection System using universal thermal cycling parameters. Each sample was run in duplicate and each condition was performed in triplicate. Gene expression was analyzed using the comparative CT method after normalization to 18S of mock- versus hMPV-infected cells.

Cell Extracts and Immunoblotting
Approximately 90% confluent cells were mock- or hMPV-infected for 48 hours before IFN-{alpha} treatment for 30 minutes. Cells were washed with ice-cold PBS and protein was isolated from cells chilled on ice after washing with ice-cold PBS using 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 1x protease and phosphatase inhibitor cocktail (Pierce, Rockford, IL). The concentration of the protein was determined using a BCA assay (Pierce) and diluted in Laemmli buffer (Bio-Rad, Hercules, CA) containing β-mercaptoethanol (Sigma) and boiled for 10 minutes before equal amounts were loaded into a 5 to 20% Tris-HCl gel (Bio-Rad). Protein was transferred to a nitrocellulose membrane (Bio-Rad), blocked using SuperBlock T20 TBS (Pierce) and probed for STAT1, P-STAT1, STAT2, P-Tyk2, P-Jak1 (Cell Signaling), P-STAT2 (Upstate), or β-actin (Abcam Inc., Cambridge, MA) followed by incubation with a goat anti-rabbit horseradish peroxidase–conjugated antibody (Zymed, Invitrogen, Carlsbad, CA) and chemiluminescence detection (Perkin Elmer, Boston. MA).

STAT-Mediated Transactivation of ISRE
Reporter gene assays were used to assess IFN-mediated transactivation of the ISRE. Luciferase levels were detected in cells that were infected with hMPV for 24 hours and then transfected with 2 µg of the IFN-{alpha}–responsive pISRE (Stratagene, La Jolla, CA). After 18 hours of transfection, media was removed and replaced with media containing IFN-{alpha} (1,000 IU/ml). After 6 hours of incubation, cell lysates were collected using Reporter Lysis Buffer (Promega, Madison, WI) and luciferase levels were measured using the Luciferase Assay System (Promega) and luminometer (Turner Designs, Sunnyvale, CA) as previously described (25).

Flow Cytometric Analysis of IFNAR Expression
Adherent A549 cells were mock- or hMPV-infected for 48 hours and then gently removed using cell dissociation buffer (GIBCO, Invitrogen) and centrifuged at 500 x g for 5 minutes at 4°C. Cells were washed twice with PBS and resuspended in PBS, where they were blocked with FcR bocking Reagent (Miltenyi Biotec, Auburn, CA) on ice for 10 minutes. Cells were then incubated on ice in the dark for 45 minutes with a mouse monoclonal anti-human IFN-{alpha}/β R1-fluorescein–conjugated antibody (R&D Systems, Minneapolis, MN), or mouse monoclonal anti-human IFN-{alpha}/β R2 (Abcam) followed by staining with a horse anti-mouse fluorescein-conjugated antibody (Vector) or mouse IgG1 isotype control (R&D Systems). Cells were washed twice with PBS before being resuspended and fixed in 1% paraformaldehyde for 10 minutes. PBS was added to a total volume of 500 µl and surface expression of IFNAR1 and IFNAR2 was analyzed by flow cytometry using FACS Calibur (BD, Franklin Lakes, NJ).

RT-PCR Detection of hMPV and β-Actin
Total RNA was collected using the RNeasy Kit (Qiagen) with optional on-column DNase digestion following the manufacturer's recommendations and subjected to reverse transcription using an M-MLV reverse transcriptase kit (Invitrogen) and oligo dT primers (Roche, Indianapolis, IN). The mRNA of the hMPV fusion protein was detected by PCR using forward (5'-GCCAATACACCACCAGCAGTTC) and reverse (5'-TGATCAGTCCCGCATA-AGGTG) primers specific to the CAN97-83 strain of hMPV annealed at 65°C. β-actin mRNA was detected as previously described (26).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Lung Epithelial Cells Are Permissive to hMPV Infection
The permissivity of hMPV in lung epithelia has not been well established, with some reports finding poor or no growth in lung epithelial cell lines (1, 27), whereas others have reported efficient replication in a human bronchiolar cell line (28). To fully understand the mechanisms of viral pathogenesis, it is crucial to study respiratory viruses in cells that most closely represent the cells of natural infection as opposed to studying viruses in the cells that are used for propagation. Therefore, the ability of hMPV to infect and replicate in the human lung epithelial A549 cells and human Clara cell–like, H441 cells was assessed and compared with Vero E6 and LLC-MK2, cell lines commonly used for propagation. Vero E6, LLC-MK2, A549, and H441 cells were infected with hMPV strain CAN97-83 at an MOI of 0.5 for 1 hour. After 48 hours of infection, cell culture media from each cell type was collected and used to infect Vero E6 cells and infection was analyzed by immunofluorescence (IF) microscopy and by RT-PCR after an additional 48 hours of incubation. The presence of hMPV in the media was confirmed by infecting Vero E6 cells for all cell types (data not shown), indicating that hMPV is able to infect and replicate in human lung A549 and H441 cell lines. All four cell types readily stained for hMPV as assessed by IF microscopy (Figure 1A), indicating that hMPV is able to infect human lung cell lines in addition to cells lines commonly used for viral propagation. In contrast, no viral matrix protein was detected in mock-infected cells (data not shown).


Figure 1
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  		Figure 1. Lung epithelial cells are permissive to human metapneumovirus (hMPV) and resistant to IFN-{alpha}–mediated viral clearance. (A) Vero E6, LLC-MK2, H441, and A549 cells were infected with hMPV at a multiplicity of infection (MOI) of 0.5 for 48 hours, fixed, permeablized, and probed for the matrix protein of hMPV followed by Cy3-conjugated secondary antibody. (B) A549 cells were pretreated with IFN-{alpha} for 20 hours and then infected with hMPV or VSV. Supernatants were collected and virus levels were quantified by viral plaque assay in LLC-MK2 cells at 6, 24, 48, and 72 hours after infection. The percentage of virus yield as compared with non–IFN-{alpha} treatment controls was calculated for each independent IFN-{alpha} treatment (3 ± 6 replicates per virus strain). Shown are the mean and SEM of the log-transformed values of the virus yields.

 
hMPV Is Not Sensitive to IFN-{alpha}–Mediated Viral Clearance
Type I IFNs serve to initiate viral clearance mechanisms in multiple cell types. However, numerous viruses, including paramyxoviruses, encode various mechanisms to subvert these mechanisms. A feature of many viruses that encode immunomodulatory proteins is the ability to resist clearance by exogenous IFN-{alpha} treatment. To investigate the sensitivity of hMPV to IFN-{alpha}, A549 cells were pretreated with IFN-{alpha} for 20 hours before viral infection. Supernatant from virally infected cells was collected at 6, 24, 48, and 72 hours after infection and infectious virus yield was determined by viral titration. Pretreatment of A549 cells with IFN-{alpha} did not have a significant impact on hMPV infection or virus yield (Figure 1B). Conversely, IFN-{alpha} significantly reduced the virus yield of the IFN-{alpha}–sensitive virus, VSV (Figure 1B). Subsequent experiments have found that hMPV infection is not significantly impacted by IFN-{alpha} concentrations at 1 to 5,000 U/ml. These results indicate that hMPV can resist the antiviral effects of IFN-{alpha}.

IFN-{alpha}–Mediated Signal Transduction Is Inhibited by hMPV Infection
A hallmark feature of viruses able to inhibit IFN-{alpha} signaling is the ability to prevent the IFN-{alpha}–mediated transactivation of the ISRE and the subsequent induction of ISGs by IFN-{alpha}/β (29). To initially test the ability of hMPV to subvert IFN-{alpha}–mediated responses, an IFN-{alpha}–responsive ISRE luciferase reporter assay was used to assess the global transactivation of ISRE genes in response to IFN-{alpha} in the presence or absence of hMPV infection. A549 cells were either mock- or hMPV-infected at an MOI of 0.5 for 24 hours and transfected with the ISRE luciferase and β-gal constructs, followed by subsequent stimulation with cell culture media or cell culture media containing IFN-{alpha}. In mock-infected cells, IFN-{alpha} treatment resulted in robust induction of ISRE luciferase activity (Figure 2A). IFN-{alpha}–mediated induction of ISRE luciferase activity was completely abolished in hMPV-infected A549 cells (Figure 2A), suggesting that hMPV-infected cells are unable to respond to IFN-{alpha}.


Figure 2
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  		Figure 2. IFN-{alpha}–mediated gene expression is inhibited by hMPV infection. (A) The levels of luciferase were measured from A549 cells that were mock- or hMPV-infected at an MOI of 4.0 for 24 hours and then transfected with an ISRE-luciferase promoter construct and stimulated for 8 hours with IFN-{alpha} (1,000 U/ml). Luciferase levels were normalized to co-transfected β-gal. Levels of mock-infected and IFN-{alpha}–stimulated cells were set to 100 percent. A549 cells were infected at an MOI of 0.5 for 24 hours and then treated with 1,000 U/ml of IFN-{alpha} for 24 hours. Total RNA was then isolated and subjected to one-step quantitative RT-PCR to measure mRNA levels of classically IFN-{alpha}–induced ISGs, ISG56, and MxA (B) and constitutively expressed Mda5 and RIG-I (C). mRNA levels were normalized to 18S rRNA and are expressed as fold induction compared with virally infected and untreated cells. Error bars represent SEM. *P value < 0.001, ANOVA.

 
To further assess IFN-{alpha} signal transduction in hMPV-infected cells, the ability of IFN-{alpha} to up-regulate ISGs was analyzed. The mRNA levels of ISGs were measured by quantitative RT-PCR in cells infected with hMPV for 24 hours and subsequently treated with IFN-{alpha} for 24 hours. In mock-infected A549 cells, mRNA of ISG56 and MxA, two genes highly responsive to IFN-{alpha} (30), was markedly up-regulated by greater than 200-fold over control with IFN-{alpha} treatment (Figure 2B). Conversely, in hMPV-infected A549 cells, ISG56 and MxA were not induced by IFN-{alpha} treatment (Figure 2B). Similarly, mRNA levels of RIG-I and Mda-5, two constitutively expressed genes that are also up-regulated in response to IFN-{alpha} (31), were significantly reduced in hMPV-infected lung epithelial cells treated with IFN-{alpha} as compared with mock-infected IFN-{alpha}–treated lung epithelial cells (Figure 2C). To ensure that the lack of induction of ISGs was not due to the global reduction of all mRNAs by hMPV infection, semi-quantitative RT-PCR was used assess the levels of β-actin and the transcription factor C/EBP-β. The mRNA levels of both of these genes were similar in mock- and hMPV-infected cells (data not shown). Together these results suggest that hMPV is able to inhibit IFN-{alpha}–mediated transcriptional induction of ISGs.

IFN-{alpha}–Mediated Phosphorylation of STAT1 Is Inhibited in hMPV-Infected Cells
Multiple members of the Paramyxovirus family inhibit IFN-{alpha} signaling by interfering with the normal activities of STAT1 in the Jak/STAT signal transduction pathway. Therefore, steady-state protein levels and phosphorylation of STAT1 in response to IFN-{alpha} in human lung epithelial cells was assessed in hMPV-infected lung epithelial cells. Despite evidence that some paramyxoviruses diminish STAT protein levels through degradation mechanisms, STAT1 levels were markedly increased in hMPV-infected cells (Figure 3A). STAT1 phosphorylation was assessed using phosphorylated STAT1-specific antibodies to determine the activation of STAT1 in response to IFN-{alpha}. In mock-infected lung epithelial cells, IFN-{alpha} induced a rapid and easily discernable STAT1 phosphorylation, as assessed by immunoblotting with a phosphorylated STAT1 specific antibody. STAT1 phosphorylation was detectible 5 minutes after IFN-{alpha} administration and was further increased at 15 minutes, but was not detectable in mock-infected, untreated lung epithelial cells (Figure 3B). Infection with hMPV itself did not result in phosphorylation of STAT1 despite published reports of high levels of IFN-{alpha} production during hMPV infection (32–34). Moreover, infection with hMPV before IFN-{alpha} treatment completely eliminated the induction of STAT1 phosphorylation at 5 and 15 minutes after treatment with exogenous IFN-{alpha} (Figure 3B).


Figure 3
Figure 3
Figure 3
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  		Figure 3. hMPV infection prevented IFN-{alpha}–mediated phosphorylation of STAT1. (A) STAT1 and β-actin protein levels were measured by immunoblotting in mock-infected and hMPV-infected (MOI 0.5) A549 cells 48 hours after infection. (B) A549 cells were infected at an MOI of 0.5 with hMPV for 48 hours and then stimulated with 1,000 U/ml of IFN-{alpha} for 5 or 15 minutes. Tyrosine 701 phosphorylation of STAT1 was analyzed by immunobloting. β-actin was used a control for equal loading of protein. (C) The inhibition of phosphorylation of STAT1 in response to 30 minutes of IFN-{alpha} (1,000 U/ml) stimulation and total levels of STAT1 protein were assessed in mock- or hMPV-infected (MOIs of 0.05, 0.01, 0.5, 1.0, and 5.0) A549 cells. (D) Phosphorylated STAT1 and β-actin protein levels were detected by immunoblotting in cells infected with live or ultraviolet-killed hMPV for 48 hours at an MOI of 0.5 and treated with IFN-{alpha} (1,000 U/ml) for 30 minutes before cell harvesting. (E) Increasing MOIs of hMPV were used to infect A549 cells for 6, 12, 24, and 48 hours. Tyrosine phosphorylated STAT1, total STAT1, and β-actin levels were analyzed by immunoblotting after IFN-{alpha} (1,000 U/ml) treatment for 30 minutes (F) A549 cells were infected with hMPV for 48 hours at an MOI of 0.5 and treated with IFN-{alpha} (1,000 U/ml) or IFN-{gamma} (10 ng/ml) for 30 minutes before cell harvesting. Phosphorylated STAT1 and β-actin protein levels were detected by immunoblotting.

 
Infection with increasing MOIs of hMPV was used to determine at what level of infection inhibition of IFN-{alpha}–mediated STAT1 phosphorylation occurred. Lung epithelial cells were infected with increasing MOIs of hMPV followed by stimulation with IFN-{alpha} for 30 minutes. Total levels of STAT1 were increased in A549 cells by hMPV in a dose-dependent manner that was unrelated to treatment with IFN-{alpha}. The lowest MOI of 0.05 of hMPV was sufficient to cause increased levels of STAT1 protein in lung epithelial cells (Figure 3C). However, the phosphorylation of STAT1 in response to IFN-{alpha} was decreased in a dose-dependent manner, consistent with previous findings. Infection with MOIs of 0.05 and 0.1 resulted in a partial decrease in STAT1 phosphorylation when compared with mock-infected and IFN-{alpha}–stimulated cells, whereas infection with MOIs of 0.5 or higher resulted in complete inhibition of STAT1 phosphorylation (Figure 3C).

To examine the importance of viral gene expression on STAT1 regulation, the phosphorylation status of STAT1 was analyzed using ultraviolet-inactivated hMPV. Inhibition of hMPV infection and viral gene expression by ultraviolet treatment was confirmed by RT-PCR and IF microscopy (data not shown). Consistent with previous findings, phosphorylated STAT1 was undetectable in untreated mock-infected A549 cells, but readily detectable after 30 minutes of IFN-{alpha} treatment (Figure 3D). Furthermore, infection with hMPV resulted in increased total levels of STAT1 and prevention of IFN-{alpha}–mediated phosphorylation of STAT1, as shown previously (Figure 3D). However, inactivation of hMPV by ultraviolet treatment restored the ability of IFN-{alpha} to phosphorylate STAT1 (Figure 3D). Importantly, ultraviolet-inactivated hMPV inoculum contained all vehicle ingredients of viral propagation, suggesting that the inhibition of STAT1 phosphorylation is not due to a cellular product produced during viral propagation.

To gain better insight into the mechanism of hMPV inhibition of IFN-{alpha}–mediated STAT1 phosphorylation, a temporal analysis of STAT1 inhibition was conducted. Lung epithelial cells were infected with increasing MOIs of hMPV for 6, 12, 24, and 48 hours before assessment of STAT1 phosphorylation after treatment with IFN-{alpha} for 30 minutes. Phosphorylation of STAT1 by IFN-{alpha} treatment was not inhibited by hMPV infection at MOIs of 0.01, 0.5, or 1.0 at 6 or 12 hours of infection (Figure 3E). However, infection with hMPV at an MOI of 0.5 or 1.0 for 24 or 48 hours was capable of inhibiting IFN-{alpha}–meditated STAT1 phosphorylation (Figure 3E). Consistent with previous observations, total levels of STAT1 were significantly increased with 24 and 48 hours of hMPV infection (Figure 3E). However, infection of A549 cells with hMPV for 12 hours or less did not result in increased levels of total STAT1 (Figure 3E). Taken together, these results suggest that hMPV is able to prevent the IFN-{alpha}–mediated phosphorylation of STAT1 in a dose-dependent manner despite increased levels of total STAT1, and that viral gene expression and/or replicating virus is required for the hMPV-mediated prevention of STAT1 phosphorylation by IFN-{alpha}.

To determine if inhibition of STAT1 phosphorylation by hMPV was specific to the type I IFN signaling pathway, IFN-{gamma}–mediated STAT1 phosphorylation was analyzed. Mock- and hMPV-infected lung epithelial cells were treated with IFN-{gamma} (10 ng/ml) for 30 minutes. Total cell lysates were collected and STAT1 phosphorylation was assessed by immunoblotting. IFN-{alpha}–mediated phosphorylation of STAT1 was inhibited in lung epithelial cells infected with hMPV; however, IFN-{gamma}–mediated STAT1 phosphorylation was unaffected by hMPV infection (Figure 3F). These results suggest that the inhibition of STAT1 phosphorylation by hMPV is limited to type I IFN signaling.

Nuclear Translocation of STAT1 in Response to IFN-{alpha} Is Blocked in hMPV-Infected Lung Epithelial Cells
Nuclear localization of phosphorylated STAT1 is a key cellular event in mediating IFN-{alpha}–transduced signaling. Therefore, IF microscopy was used to further characterize the cellular localization of STAT1 during hMPV infection and in response to IFN-{alpha} signaling. In mock-infected A549 cells, STAT1 is predominantly present in the cytoplasm as indicated by indirect IF microscopy (Figure 4). However, in mock-infected cells, treatment with IFN-{alpha} for 30 minutes resulted in marked redistribution of STAT1 to the nucleus as observed by colocalization of STAT1 and Hoechst-stained nuclei in nearly 100 percent of cells visualized (Figure 4). Infection of A549 cells with hMPV, as confirmed by IF staining of the matrix protein, alone did not noticeably alter the cellular distribution of STAT1 compared with mock-infected cells. Consistent with the phosphorylation data obtained by immunoblotting analysis, treatment of hMPV-infected A549 cells with IFN-{alpha} did not result in redistribution of STAT1 into the nucleus as was seen in the mock-infected and IFN-{alpha}–treated cells (Figure 4). However, when hMPV was inactivated by ultraviolet treatment before infection, the ability of STAT1 to translocate to the nucleus in response to IFN-{alpha} treatment was unaltered (Figure 4). These results further confirm that IFN-{alpha}–mediated signaling is interrupted by hMPV through inhibition of STAT1 phosphorylation and nuclear translocation.


Figure 4
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  		Figure 4. IFN-{alpha}–mediated STAT1 nuclear localization prevented by hMPV infection. Cellular localization of STAT1 in mock-, hMPV-, or ultraviolet-killed hMPV-infected A549 cells was analyzed by immunofluorescence microscopy in untreated or IFN-{alpha} (1,000 U/ml for 30 min)–treated cells. Nuclei (blue) were stained with Hoechst (DAPI), STAT1 (green) was detected with a rabbit anti-STAT1 antibody followed by incubation with a goat anti-rabbit–conjugated Cy3 antibody, while hMPV (red) infection was detected using a mouse anti-matrix (hMPV M) protein antibody and a goat anti-mouse Cy5 antibody. Magnification: x400.

 
The Expression of the IFNAR Is Unaltered by hMPV Infection
Some viruses, such as herpes simplex virus, can alter cell-surface abundance of IFN-{alpha} receptors, thus reducing type I IFN-mediated signaling (35). Therefore, the expression level of the IFNAR was assessed in mock and hMPV-infected human lung epithelial cells. IFNAR1 and IFNAR2 were readily detectable on A549 cells, as assessed by FACS analysis. Flow cytometric analysis found no quantitative difference of IFNAR1 (Figure 5A) or IFNAR2 (Figure 5B) surface expression between mock-infected controls and hMPV-infected cells after 48 hours of hMPV infection. These results indicate that the expression levels of the IFNAR are unaffected by hMPV infection and suggest that the inhibition of STAT1 phosphorylation is not due to IFN-{alpha} ligand receptor interactions.


Figure 5
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  		Figure 5. Cell surface expression of the IFNAR is unchanged by hMPV infection. Adherent A549 cells that were either mock-infected (solid black line) or infected with an MOI 0.5 of hMPV (dashed black line) for 48 hours were removed using cell dissociation buffer (Gibco) and were stained for the IFNAR1 (A) or IFNAR2 (B) and measured by flow cytometry. Isotype control is shown with gray shading.

 
Phosphorylation of STAT2, Tyk2, and Jak1 Is Not Affected by hMPV
To establish whether events upstream of STAT1 phosphorylation are altered in hMPV infection, STAT2, Tyk2, and Jak1 were evaluated for abundance and phosphorylation. STAT2 serves as a docking site for STAT1 and must be phosphorylated before recruitment and subsequent phosphorylation of STAT1 (36). RSV prevents STAT1 phosphorylation by targeting STAT2 for proteasomal degradation (18). Consequently, immunoblot analysis was used to determine STAT2 levels in hMPV-infected lung epithelial cells. STAT2 levels were low, but detectable, in mock-infected A549 cells. However, STAT2 levels were markedly increased and easily detectable in hMPV-infected cells (Figure 6A). Therefore, in contrast to RSV, hMPV does not target STAT2 for proteasomal degradation. STAT2 phosphorylation was assessed using phospho-specific STAT2 antibodies after IFN-{alpha} stimulation of mock- and hMPV-infected cells. Very low levels of phosphorylated STAT2 were detectable in unstimulated and mock-infected cells; however, infection with hMPV or stimulation with IFN-{alpha} for 15 minutes significantly increased levels of phosphorylated STAT2 (Figure 6B). Furthermore, in IFN-{alpha}–stimulated and hMPV-infected cells, phosphorylated STAT2 was readily detectable. In contrast to STAT1, these results indicate that hMPV infection in lung epithelial cells does not prevent STAT2 phosphorylation.


Figure 6
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  		Figure 6. The phopshorylation of STAT2, Tyk2, and Jak1 in response to IFN-{alpha} treatment is unaltered by hMPV infection. (A) A549 cells infected with hMPV (MOI of 0.5) for 48 hours were lysed and total levels of STAT2 and β-actin were detected by immunoblot. (B) Mock- and hMPV-infected (MOI 0.5) A549 cells were stimulated with IFN-{alpha} (1,000 U/ml) for 5 or 15 minutes and the protein levels of phosphoryled and total STAT2 levels and β-actin were measured by immunoblotting. (C) Phosphorylation of Tyk2 and Jak1 in mock- and hMPV-infected (MOI of 0.5) A549 cells was measured after 5 or 15 minutes of stimulation with IFN-{alpha} (1,000 U/ml).

 
Tyk2 and Jak1 are IFN receptor–associated kinases that initiate the signal transduction upon IFN receptor-ligand binding. To assess whether hMPV can alter Tyk2 or Jak1 phosphorylation, immunoblotting with phospho-Tyk2– or phospho-Jak1–specific antibodies was performed. In mock-infected and untreated cells, both Tyk2 and Jak1 displayed a basal level of phosphorylation (Figures 6C and 6D). This is probably due to activation by one of the many signaling cascades that use these two kinases (37). However, stimulation with IFN-{alpha} for 5 or 15 minutes was sufficient to induce increased phosphorylation of both proteins (Figures 6C and 6D). In addition, neither the phosphorylation of Tyk2 nor Jak1 by IFN-{alpha} was obstructed by hMPV infection (Figure 6C). Further evidence of Tyk2 and Jak1 phosphorylation is the previous observation that STAT2 is phosphorylated. Collectively, these results demonstrate that IFN-{alpha} signaling upstream of STAT1 is intact, and reveal that hMPV is able to prevent IFN-{alpha}–mediated signaling downstream of Tyk2, Jak1, and STAT2 phosphorylation.

IFN-{alpha}–Mediated STAT1 Phosphorylation Is Inhibited by hMPV in Primary NHBE Cells
To further ascertain the ability of hMPV to inhibit IFN-{alpha} signaling in lung epithelial cells, STAT1 phosphorylation after treatment with IFN-{alpha} was measured in primary normal bronchial epithelial cells (NHBE). The capacity of hMPV to infect NHBE cells was established by detecting hMPV fusion protein mRNA by RT-PCR and matrix protein by IF microscopy after 24 hours of infection with hMPV at an MOI of 1.0. The mRNA of the fusion protein of hMPV was not detected in mock-infected NHBE cells, but was readily detectible in hMPV-infected cells (Figure 7A). Conversely, mRNA from the housekeeping gene β-actin was detectible in both mock and hMPV-infected NHBE (Figure 7A). Additional confirmation of the ability of hMPV to infect NHBE cells was obtained by IF microscopic detection of the hMPV matrix protein in hMPV-infected but not mock-infected cells (Figure 7B). Phosphorylated and total levels of STAT1 in NHBE cells were analyzed in mock- and hMPV-infected cells. Consistent with previous findings in A549 cells, treatment with IFN-{alpha} for 30 minutes resulted in phosphorylation of STAT1 in mock-infected cells (Figure 7C). However, in hMPV-infected cells, exogenous treatment with IFN-{alpha} did not cause increased phosphorylation of STAT1 (Figure 7C). Also consistent with previous findings in A549 cells, total STAT1 levels were significantly increased in hMPV-infected NHBE cells when compared with mock-infected cells (Figure 7C). In contrast to A549 cells (data not shown), at 24 hours after infection with hMPV (MOI 1.0) not all NHBE cells were infected (Figure 7B), thus explaining the residual levels of phosphorylated STAT1 by immunoblotting (Figure 7C). The ability of hMPV to inhibit IFN-{alpha}–mediated STAT1 phosphorylation in primary lung epithelial cells provides further evidence of hMPV-mediated regulation of STAT signaling.


Figure 7
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  		Figure 7. IFN-{alpha}–mediated STAT1 phosphorylation is inhibited by hMPV in primary NHBE cells. (A) NHBE cells were infected with hMPV (MOI 1.0) for 24 hours and then total RNA was isolated and subjected to RT-PCR, followed by detection of the mRNA of the fusion protein of hMPV and β-actin. (B) Immunofluorescence microscopy was used 24 hours after infection of hMPV (MOI 1.0) in NHBE cells to detect the matrix protein and confirm viral infection and protein expression. (C) Total NHBE cell lysates were probed for tyrosine phosphorylated and total STAT1 and β-actin by immunoblot 24 hours after infection with hMPV (MOI 1.0) and after treatment with IFN-{alpha} (1,000 IU/ml) for 30 minutes.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
It is clear that most, if not all, viruses have evolved mechanisms to avert the interferon response mounted by cells during infection. These mechanisms are needed to ensure that the virus has sufficient time to successfully replicate, package, and release from host cells. The findings presented here that hMPV can infect and replicate in lung epithelial cells with a competent IFN system and is not susceptible to exogenous IFN-{alpha} suggests that hMPV likewise possess mechanisms for evading type I IFNs. Herein, we show that hMPV was able to inhibit IFN-{alpha}–mediated signal transduction and the resulting induction of ISGs after treatment with IFN-{alpha}. Infection with hMPV in human lung epithelial cells prevented IFN-{alpha}–mediated phosphorylation and subsequent nuclear translocation of STAT1. Further investigation of the upstream signaling components in the IFN-{alpha} signal cascade revealed that the expression of the IFNAR1 and IFNAR2, and the ability of Tyk2, Jak1, and STAT2 to be phosphorylated in response to IFN-{alpha} stimulation were unchanged by hMPV infection. These results suggest that hMPV is able to abolish type I IFN signaling through mechanisms regulating STAT1 phosphorylation, and demonstrate that hMPV, despite the lack of obvious immunomodulatory nonstructural proteins, is able to subvert viral clearance mechanisms.

The inhibition of STAT1 phosphorylation by hMPV is limited to type I IFN signaling, as IFN-{gamma}–mediated STAT1 phosphorylation was not affected by hMPV infection. RSV has similarly been shown to inhibit IFN-{alpha}–mediated, but not IFN-{gamma}–mediated STAT1 phosphorylation (12). Type I and type II IFN signaling cascades use common and distinct signaling proteins. Type II IFN signaling is different from type I IFN signaling, as it employs distinct receptors, IFNGR1 and IFNGR2, it uses Jak2 instead of Tyk2 as an adaptor kinase of the receptor, and its lack of involvement of STAT2 as a docking site for STAT1 phosphorylation. As shown in this report, the inhibition of STAT1 phosphorylation in type I IFN signaling by hMPV occurs after STAT2 phosphorylation, as STAT2 phosphorylation was not altered by hMPV. However, hMPV-mediated regulation may involve the recruitment and docking of STAT1 to the phosphorylated STAT2. Thus, the inability of hMPV to inhibit IFN-{gamma}–mediated STAT1 phosphorylation suggests involvement of these components unique to the type I IFN signaling cascade.

Inhibition of IFN pathways by paramyxoviruses is well documented. Interestingly, members of the Paramyxovirus family have evolved separate and distinct mechanisms to target the same components of the IFN pathways. Specifically, SV5, mumps, and type II human parainfluenza viruses (PIV-2) use their V and C proteins to target STAT1 and STAT2 for proteasome degradation by polyubiquitination (19, 38). Similarly, the NS2 protein of RSV inhibits STAT1 phosphorylation by targeting STAT2 for degradation via the proteasome pathway (12, 18, 39). As a result of the proteasomal degradation the levels of the targeted STAT protein are markedly decreased or undetectable in RSV-infected cells. Clearly, hMPV does not use a similar mechanism, as the total levels of both STAT1 and STAT2 were increased after viral infection. Multiple paramyxoviruses prevent IFN signaling not by causing degradation of the STAT proteins, but instead by binding, sequestering, and preventing either their phosphorylation or nuclear translocation. Nipah and Hendra viruses are able to sequester STAT1 and STAT2 in high-molecular-mass cytoplasmic complexes, preventing their translocation into the nucleus via binding by their V proteins (22, 23). Likewise, measles virus causes a portion of STAT1 and STAT2 to be redistributed to cytoplasmic aggregates (40) and can inhibit both IFN-{alpha}– and IFN-{gamma}–dependent STAT1 and STAT2 nuclear localization (24). The C protein of Sendai virus has been shown to bind STAT1 (41) and inhibit both STAT1 and STAT2 phosphorylation (42). It is possible that in a similar fashion hMPV is preventing the IFN-{alpha}–mediated phosphorylation of STAT1.

As hMPV does not posses genes homologous to known immunomodulatory genes in other paramyxoviruses, further investigation into the precise mechanism of IFN signaling inhibition is needed. It is reasonable to hypothesize that hMPV encodes a protein or proteins that are responsible for the inhibititory effects, since this has been shown with multiple other viruses. The immunomodulatory C proteins of the paramyxoviruses from the subfamily Paramyxovirinae are encoded and translated from an overlapping ORF located within the P gene (43). In addition, viruses in the subfamily Paramyxovirinae also use RNA editing to insert G residues at a specific position in the mRNA, causing a frameshift that yields proteins with shared N-terminal domains, but different C-terminal domains (44). Many of the proteins produced in this manner by paramyxoviruses possess immunomodulatory functions (45). It is possible that hMPV encodes a novel protein that has not yet been identified that possesses these inhibitory functions from either an overlapping ORF found within the identified nine genes of hMPV or by RNA editing. Indeed, we have identified putative overlapping ORFs in hMPV (data not shown); however, these putative proteins are quite small and most are not conserved throughout the strains sequenced to date. It is also possible that the IFN signaling inhibition is due to the activity of an already defined hMPV protein, as many viral proteins, including paramyxovirus proteins, possess multiple functions.

An intriguing finding herein was the increased levels of both STAT1 and STAT2 during hMPV infection. Similar to hMPV, RSV infection causes increased levels of STAT1 (18). Increased levels of STAT proteins during hMPV infection have recently been shown by other laboratories (46). However, from both our studies and those of Ramaswamy and coworkers (18) it is unclear what mechanisms cause these increased levels and what, if any, biological relevance the increased STAT levels may have. Although activation of STAT1 is inhibited by both RSV and hMPV despite the increased levels of STAT1, elucidation of the cause of the increased levels may prove beneficial in explaining how the inhibition is occurring or provide important insight into the interactions of these viruses and the innate immune response. One potential explanation that accounts for the increased STAT levels is that STATs are themselves ISGs and the up-regulation observed here could be due to IFN-{alpha} signaling early in infection, before inhibition of the IFN-{alpha} signaling cascade by hMPV has occurred. This potential explanation is supported by the finding that IFN-{alpha}–mediated STAT1 phosphorylation is not inhibited by 6 or 12 hours of hMPV infection (Figure 3E), suggesting that IFN-{alpha} signaling early in infection is intact. Furthermore, hMPV infection of interferon incompetent Vero E6 cells does not result in detectable increased levels of STAT proteins (data not shown). A second mechanism to explain this phenomenon is that the expression of the STATs is up-regulated in response to hMPV infection in an IFN-independent manner. Viruses have been shown to up-regulate ISGs in an IFN-{alpha}–independent manner by activation of IRF3 (47). A component of the hMPV virion could be recognized by a pathogen-associated molecular pattern receptor, which then causes STAT protein levels to be increased in an IFN-independent manner. This idea is supported by the fact that STAT1 levels increase in ultraviolet-inactivated hMPV treated cells, similar to what has been found in ultraviolet-inactivated hantavirus (48). Conversely, a third explanation is that the normal degradation of both STAT1 and STAT2 is inhibited. Since the STAT proteins have a relatively long biological half-life of two or three days (49), the increased levels shown here could be attributed to a gradual buildup of STAT1 and STAT2 during the course of these experiments. An intriguing finding of these studies is that the majority of STAT1 remains in the cytoplasm of hMPV-unstained cells that are neighboring infected hMPV positively stained cells when treated with IFN-{alpha}. This observation has been consistently noted and is currently being further explored in the laboratory to determine if these cells may also been infected, but below the limit of detection by fluorescence microscopy.

Contradictory to the findings here, a recent publication showed that hMPV infection of A549 cells induces STAT1 phosphorylation (46). It is unclear why these different studies have produced different findings. The different findings may be explained by differences in the strain of hMPV used in the experiments. Furthermore, different experimental conditions and different phosphorylated STAT1 detection methods were used in the two studies and may also explain the discrepancies. Further studies will assist in clarifying these observations.

The consequences of the inhibition of type I IFN signaling by hMPV are not completely understood, but likely play a role in pathogenesis and severity of infection and could impact the adaptive immune response. Previous reports suggest that hMPV is able to persist in the lungs of infected mice (13, 14). The ability of hMPV to inhibit IFN signaling likely plays an important role in facilitating this persistence. Furthermore, inhibition of IFN signaling may help explain why long-term protective immunity is not seen with hMPV infection (9). Proper signaling by IFN-{alpha} and IFN-{gamma} is vital for clearance of viral pathogens because of their immunoregulatory functions that affect both innate and adaptive immunity (50). Thus, inhibition of IFN signaling by hMPV may alter the host's ability to develop proper adaptive immunity leaving the host susceptible to reinfection.

In conclusion, we report the initial findings that hMPV is able to inhibit type I IFN signaling and elucidate the regulation of STAT1 phosphorylation as the mechanism. Our findings suggest that hMPV encodes unique mechanisms to inhibit this pathway. Additional understanding of how hMPV inhibits the type I IFN pathway and the consequences with regard to the innate and adaptive immune responses will be crucial in improving approaches for treatment and the development of vaccines.


   Acknowledgments
 
The authors thank Dr. Ralph Tripp (University of Georgia) who generously supplied us with the stock of hMPV and hMPV antibodies that were initially used to propagate and titer hMPV, respectively. The authors also thank Dr. Guy Boivin (University of Quebec), who initially isolated the hMPV used in these studies, and Dr. Al Senft for critical reading and helpful comments.


   Footnotes
 
Originally Published in Press as DOI: 10.1165/rcmb.2007-0285OC on January 24, 2008

Conflict of Interest Statement: Neither author has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form July 26, 2007

Accepted in final form December 20, 2007


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 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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