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1.  Respiratory Viral Infections and Subversion of Cellular Antioxidant Defenses 
Reactive oxygen species (ROS) formation is part of normal cellular aerobic metabolism, due to respiration and oxidation of nutrients in order to generate energy. Low levels of ROS are involved in cellular signaling and are well controlled by the cellular antioxidant defense system. Elevated levels of ROS generation due to pollutants, toxins and radiation exposure, as well as infections, are associated with oxidative stress causing cellular damage. Several respiratory viruses, including respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and influenza, induce increased ROS formation, both intracellularly and as a result of increased inflammatory cell recruitment at the site of infection. They also reduce antioxidant enzyme (AOE) levels and/or activity, leading to unbalanced oxidative-antioxidant status and subsequent oxidative cell damage. Expression of several AOE is controlled by the activation of the nuclear transcription factor NF-E2-related factor 2 (Nrf2), through binding to the antioxidant responsive element (ARE) present in the AOE gene promoters. While exposure to several pro-oxidant stimuli usually leads to Nrf2 activation and upregulation of AOE expression, respiratory viral infections are associated with inhibition of AOE expression/activity, which in the case of RSV and hMPV is associated with reduced Nrf2 nuclear localization, decreased cellular levels and reduced ARE-dependent gene transcription. Therefore, administration of antioxidant mimetics or Nrf2 inducers represents potential viable therapeutic approaches to viral-induced diseases, such as respiratory infections and other infections associated with decreased cellular antioxidant capacity.
PMCID: PMC4288774  PMID: 25584194
Respiratory syncytial virus; Oxidative stress; Nrf2; ROS; Free radicals
2.  MyD88 controls human metapneumovirus-induced pulmonary immune responses and disease pathogenesis 
Virus research  2013;176(0):10.1016/j.virusres.2013.06.014.
Human metapneumovirus (hMPV) is a common cause of lung and airway infections in infants and young children. Recently, we and others have shown that hMPV infection induces Toll-like receptor (TLR)-dependent cellular signaling. However, the contribution of TLR-mediated signaling in host defenses against pulmonary hMPV infection and associated disease pathogenesis has not been elucidated. In this study, mice deficient in MyD88, a common adaptor of TLRs, was used to investigate the contribution of TLRs to in vivo pulmonary response to hMPV infection. MyD88−/− mice have significantly reduced pulmonary inflammation and associated disease compared with wild-type (WT) C57BL/6 mice after intranasal infection with hMPV. hMPV-induced cytokines and chemokines in bronchoalveolar lavage fluid (BALF) and isolated lung conventional dendritic cells (cDC) are also significantly impaired by MyD88 deletion. In addition, we found that MyD88 is required for the recruitment of DC, T cells, and other immune cells to the lungs, and for the functional regulation of DC and T cells in response to hMPV infection. Taken together, our data indicate that MyD88-mediated pathways are essential for the pulmonary immune and pathogenic responses to this viral pathogen.
doi:10.1016/j.virusres.2013.06.014
PMCID: PMC3819944  PMID: 23845303
hMPV; MyD88; Cytokines/chemokines; Pulmonary immune response
3.  Paramyxovirus Infection Regulates T Cell Responses by BDCA-1+ and BDCA-3+ Myeloid Dendritic Cells 
PLoS ONE  2014;9(6):e99227.
Respiratory syncytial virus (RSV) and human Metapneumovirus (hMPV), viruses belonging to the family Paramyxoviridae, are the most important causes of lower respiratory tract infection in young children. Infections with RSV and hMPV are clinically indistinguishable, and both RSV and hMPV infection have been associated with aberrant adaptive immune responses. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping adaptive immune responses during infection; however, few studies have examined how interactions of RSV and hMPV with individual mDC subsets (BDCA-1+ and BDCA-3+ mDCs) affect the outcome of anti-viral responses. To determine whether RSV and hMPV induce virus-specific responses from each subset, we examined co-stimulatory molecules and cytokines expressed by BDCA-1+ and BDCA-3+ mDCs isolated from peripheral blood after infection with hMPV and RSV, and examined their ability to stimulate T cell proliferation and differentiation. Our data show that RSV and hMPV induce virus-specific and subset-specific patterns of co-stimulatory molecule and cytokine expression. RSV, but not hMPV, impaired the capacity of infected mDCs to stimulate T cell proliferation. Whereas hMPV-infected BDCA-1+ and BDCA-3+ mDCs induced expansion of Th17 cells, in response to RSV, BDCA-1+ mDCs induced expansion of Th1 cells and BDCA-3+ mDCs induced expansion of Th2 cells and Tregs. These results demonstrate a virus-specific and subset-specific effect of RSV and hMPV infection on mDC function, suggesting that these viruses may induce different adaptive immune responses.
doi:10.1371/journal.pone.0099227
PMCID: PMC4053357  PMID: 24918929
4.  CDK9-Dependent Transcriptional Elongation in the Innate Interferon-Stimulated Gene Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells 
Journal of Virology  2013;87(12):7075-7092.
Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus responsible for lower respiratory tract infections. During infection, the presence of double-stranded RNA (dsRNA) activates the interferon (IFN) regulatory factor 3 (IRF3) transcription factor, an event triggering expression of immediate early, IFN-stimulated genes (ISGs). We examine the role of transcriptional elongation in control of IRF3-dependent ISG expression. RSV infection induces ISG54, ISG56, and CIG5 gene expression in an IRF3-dependent manner demonstrated by IRF3 small interfering RNA (siRNA) silencing in both A549 epithelial cells and IRF3−/− MEFs. ISG expression was mediated by the recruitment of IRF3, CDK9, polymerase II (Pol II), and phospho-Ser2 carboxy-terminal domain (CTD) Pol II to the IFN-stimulated response element (ISRE) binding sites of the IRF3-dependent ISG promoters in native chromatin. We find that RSV infection enhances the activated fraction of cyclin-dependent kinase 9 (CDK9) by promoting its association with bromodomain 4 (BRD4) and disrupting its association with the inhibitory 7SK small nuclear RNA. The requirement of CDK9 activity for ISG expression was shown by siRNA-mediated silencing of CDK9 and by a selective CDK9 inhibitor in A549 cells. In contrast, RSV-induced beta interferon (IFN-β) expression is not influenced by CDK9 inhibition. Using transcript-selective quantitative real-time reverse transcription-PCR (Q-RT-PCR) assays for the ISG54 gene, we observed that RSV induces transition from short to fully spliced mRNA transcripts and that this transition is blocked by CDK9 inhibition in both A549 and primary human small airway epithelial cells. These data indicate that transcription elongation plays a major role in RSV-induced ISG expression and is mediated by IRF3-dependent recruitment of activated CDK9. CDK9 activity may be a target for immunomodulation in RSV-induced lung disease.
doi:10.1128/JVI.03399-12
PMCID: PMC3676079  PMID: 23596302
5.  Host-Viral Interactions: Role of Pattern Recognition Receptors (PRRs) in Human Pneumovirus Infections 
Pathogens  2013;2(2):10.3390/pathogens2020232.
Acute respiratory tract infection (RTI) is a leading cause of morbidity and mortality worldwide and the majority of RTIs are caused by viruses, among which respiratory syncytial virus (RSV) and the closely related human metapneumovirus (hMPV) figure prominently. Host innate immune response has been implicated in recognition, protection and immune pathological mechanisms. Host-viral interactions are generally initiated via host recognition of pathogen-associated molecular patterns (PAMPs) of the virus. This recognition occurs through host pattern recognition receptors (PRRs) which are expressed on innate immune cells such as epithelial cells, dendritic cells, macrophages and neutrophils. Multiple PRR families, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs), contribute significantly to viral detection, leading to induction of cytokines, chemokines and type I interferons (IFNs), which subsequently facilitate the eradication of the virus. This review focuses on the current literature on RSV and hMPV infection and the role of PRRs in establishing/mediating the infection in both in vitro and in vivo models. A better understanding of the complex interplay between these two viruses and host PRRs might lead to efficient prophylactic and therapeutic treatments, as well as the development of adequate vaccines.
doi:10.3390/pathogens2020232
PMCID: PMC3826451  PMID: 24244872
PRRs; RSV; hMPV; TLR; RLR; NLR; PAMP; IFN; innate immunity
6.  Critical Role of TLR4 in Human Metapneumovirus Mediated Innate Immune Responses and Disease Pathogenesis 
PLoS ONE  2013;8(10):e78849.
Human metapneumovirus (hMPV) is one of the main causes of acute respiratory tract infections in children, elderly and immunocompromised patients. The mammalian Toll-like receptors (TLR) were identified as critical regulators of innate immunity to a variety of microbes, including viruses. We have recently shown that hMPV-induced cytokine, chemokine and type I interferon secretion in dendritic cells occurs via TLR4, however, its role in hMPV-induced disease is unknown. In this study, wild-type(WT) and TLR4-deficient mice (TLR4−/−) were infected with hMPV and examined for clinical disease parameters, such as body weight loss and airway obstruction, viral clearance, lung inflammation, dendritic cell maturation, T-cell proliferation and antibody production. Our results demonstrate that absence of TLR4 in hMPV-infected mice significantly reduced the inflammatory response as well as disease severity, shown by reduced body weight loss and airway obstruction and hyperresponsiveness (AHR), compared to WT mice. Levels of cytokines and chemokines were also significantly lower in the TLR4−/− mice. Accordingly, recruitment of inflammatory cells in the BAL, lungs, as well as in lymph nodes, was significantly reduced in the TLR4−/− mice, however, viral replication and clearance, as well as T-cell proliferation and neutralizing antibody production, were not affected. Our findings indicate that TLR4 is important for the activation of the innate immune response to hMPV, however it does play a role in disease pathogenesis, as lack of TLR4 expression is associated with reduced clinical manifestations of hMPV disease, without affecting viral protection.
doi:10.1371/journal.pone.0078849
PMCID: PMC3812158  PMID: 24205331
7.  Host-Viral Interactions: Role of Pattern Recognition Receptors (PRRs) in Human Pneumovirus Infections 
Pathogens  2013;2(2):232-263.
Acute respiratory tract infection (RTI) is a leading cause of morbidity and mortality worldwide and the majority of RTIs are caused by viruses, among which respiratory syncytial virus (RSV) and the closely related human metapneumovirus (hMPV) figure prominently. Host innate immune response has been implicated in recognition, protection and immune pathological mechanisms. Host-viral interactions are generally initiated via host recognition of pathogen-associated molecular patterns (PAMPs) of the virus. This recognition occurs through host pattern recognition receptors (PRRs) which are expressed on innate immune cells such as epithelial cells, dendritic cells, macrophages and neutrophils. Multiple PRR families, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs), contribute significantly to viral detection, leading to induction of cytokines, chemokines and type I interferons (IFNs), which subsequently facilitate the eradication of the virus. This review focuses on the current literature on RSV and hMPV infection and the role of PRRs in establishing/mediating the infection in both in vitro and in vivo models. A better understanding of the complex interplay between these two viruses and host PRRs might lead to efficient prophylactic and therapeutic treatments, as well as the development of adequate vaccines.
doi:10.3390/pathogens2020232
PMCID: PMC3826451  PMID: 24244872
PRRs; RSV; hMPV; TLR; RLR; NLR; PAMP; IFN; innate immunity
8.  Human Metapneumovirus Glycoprotein G Disrupts Mitochondrial Signaling in Airway Epithelial Cells 
PLoS ONE  2013;8(4):e62568.
Human metapneumovirus (hMPV) is a recently identified RNA virus belonging to the Paramyxoviridae family. It is a common cause of respiratory tract infections in children, adults, and immunocompromised patients, for which no specific treatment or vaccine is available. Recent investigations in our lab identified hMPV glycoprotein G as an important virulence factor, as a recombinant virus lacking the G protein (rhMPV-ΔG) exhibited enhanced production of important immune and antiviral mediators, such as cytokines, chemokines and type I interferon (IFN) in airway epithelial cells, and expression of G protein alone inhibits cellular signaling dependent on retinoic induced gene (RIG)-I, a RNA helicase with a fundamental role in initiating hMPV-induced cellular responses. In this study, we have further investigated the mechanism underlying the inhibitory role of hMPV G protein on RIG-I-dependent signaling. We found that the interaction of hMPV G with RIG-I occurs primarily through the CARD domains of RIG-I N-terminus, preventing RIG-I association with the adaptor protein MAVS (mitochondrial antiviral signaling protein), recruitment of RIG-I to mitochondria, as well as the interaction between mitochondria and mitochondria-associated membrane (MAM) component of the endoplasmic reticulum (ER), which contains STINGS, an important part of the viral-induced RIG-I/MAVS signaling pathway, leading in the end to the inhibition of cytokine, chemokine and type I IFN expression. Mutagenesis analysis showed that hMPV G protein cytoplasmic domain played a major role in the observed inhibitory activity, and recombinant viruses expressing a G protein with amino acid substitution in position 2 and 3 recapitulated most of the phenotype observed with rhMPV-ΔG mutant upon infection of airway epithelial cells.
doi:10.1371/journal.pone.0062568
PMCID: PMC3633857  PMID: 23626834
9.  Respiratory Syncytial Virus Infection: Mechanisms of Redox Control and Novel Therapeutic Opportunities 
Antioxidants & Redox Signaling  2013;18(2):186-217.
Abstract
Respiratory syncytial virus (RSV) is one of the most important causes of upper and lower respiratory tract infections in infants and young children, for which no effective treatment is currently available. Although the mechanisms of RSV-induced airway disease remain incompletely defined, the lung inflammatory response is thought to play a central pathogenetic role. In the past few years, we and others have provided increasing evidence of a role of reactive oxygen species (ROS) as important regulators of RSV-induced cellular signaling leading to the expression of key proinflammatory mediators, such as cytokines and chemokines. In addition, RSV-induced oxidative stress, which results from an imbalance between ROS production and airway antioxidant defenses, due to a widespread inhibition of antioxidant enzyme expression, is likely to play a fundamental role in the pathogenesis of RSV-associated lung inflammatory disease, as demonstrated by a significant increase in markers of oxidative injury, which correlate with the severity of clinical illness, in children with RSV infection. Modulation of ROS production and oxidative stress therefore represents a potential novel pharmacological approach to ameliorate RSV-induced lung inflammation and its long-term consequences. Antioxid. Redox Signal. 18, 186–217.
I. Introduction
II. Redox-Sensitive Transcription Factors in RSV Infection
A. Nuclear factor-IL6
B. Nuclear factor-kappa B
C. Activator protein-1
D. Interferon regulatory factor
E. Signal transducers and activators of transcription
F. Hypoxia-inducible factor
III. ROS in RSV-Induced Cellular Signaling and Oxidative Stress
A. ROS generation in RSV infection
B. ROS as mediators of cellular signaling in RSV infection
1. NF-κB/NF-IL6/AP-1 activation
2. IRF/STAT activation
C. RSV and oxidative stress
D. Potential regulatory mechanisms of AOE gene expression in RSV infection
IV. Role of Reactive Nitrogen Species in RSV Infection
A. NO production and iNOS expression in RSV infection
B. Effect of NO on RSV replication, cellular signaling, and lung disease
V. Potential Therapeutic Approaches
A. Vitamin A
B. Vitamin D
C. Melatonin
D. Thiols
E. Polyphenols
F. SOD and SOD mimetics
G. Nrf2-inducing agents
1. Triterpenoids
2. Sulforaphane and other isothiocyanates
3. Polyphenols
4. Other classes of Nrf2 inducers
VI. Conclusions
doi:10.1089/ars.2011.4307
PMCID: PMC3513983  PMID: 22799599
10.  Human Metapneumovirus Antagonism of Innate Immune Responses 
Viruses  2012;4(12):3551-3571.
Human metapneumovirus (hMPV) is a recently identified RNA virus belonging to the Paramyxoviridae family, which includes several major human and animal pathogens. Epidemiological studies indicate that hMPV is a significant human respiratory pathogen with worldwide distribution. It is associated with respiratory illnesses in children, adults, and immunocompromised patients, ranging from upper respiratory tract infections to severe bronchiolitis and pneumonia. Interferon (IFN) represents a major line of defense against virus infection, and in response, viruses have evolved countermeasures to inhibit IFN production as well as IFN signaling. Although the strategies of IFN evasion are similar, the specific mechanisms by which paramyxoviruses inhibit IFN responses are quite diverse. In this review, we will present an overview of the strategies that hMPV uses to subvert cellular signaling in airway epithelial cells, the major target of infection, as well as in primary immune cells.
doi:10.3390/v4123551
PMCID: PMC3528279  PMID: 23223197
metapneumovirus; viral proteins; innate immune system; interferon antagonism
11.  TAK1 REGULATES NF-κB AND AP-1 ACTIVATION IN AIRWAY EPITHELIAL CELLS FOLLOWING RSV INFECTION 
Virology  2011;418(2):93-101.
Respiratory syncytial virus (RSV) is the most common cause of epidemic respiratory diseases in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-κB (NF-κB) and AP-1. In this study, we have investigated the signaling pathway leading to activation of these two transcription factors in response to RSV infection. Our results show that IKKβ plays a key role in viral-induced NF-κB activation, while JNK regulates AP-1-dependent gene transcription, as demonstrated by using kinase inactive proteins and chemical inhibitors of the two kinases. Inhibition of TAK1 activation, by overexpression of kinase inactive TAK1 or using cells lacking TAK1 expression, significantly reduced RSV-induced NF-κB and AP-1 nuclear translocation and DNA-binding activity, as well as NF-κB-dependent gene expression, identifying TAK1 as an important upstream signaling molecule regulating RSV-induced NF-κB and AP-1 activation.
doi:10.1016/j.virol.2011.07.007
PMCID: PMC3164748  PMID: 21835421
RSV; airway epithelial cells; NF-κB; inflammation
12.  A novel mechanism for the inhibition of interferon regulatory factor-3-dependent gene expression by human respiratory syncytial virus NS1 protein 
The Journal of General Virology  2011;92(Pt 9):2153-2159.
Human respiratory syncytial virus (RSV), a leading cause of respiratory tract infections in infants, inhibits type I interferon (IFN)-dependent signalling, as well as IFN synthesis. RSV non-structural protein NS1 plays a significant role in this inhibition; however, the mechanism(s) responsible is not fully known. The transcription factor interferon regulatory factor (IRF)-3 is essential for viral-induced IFN-β synthesis. In this study, we found that NS1 protein inhibits IRF-3-dependent gene transcription in constitutively active IRF-3 overexpressing cells, demonstrating that NS1 directly targets IRF-3. Our data also demonstrate that NS1 associates with IRF-3 and its transcriptional coactivator CBP, leading to disrupted association of IRF-3 to CBP and subsequent reduced binding of IRF-3 to the IFN-β promoter without blocking viral-induced IRF-3 phosphorylation, nuclear translocation and dimerization, thereby identifying a novel molecular mechanism by which RSV inhibits IFN-β synthesis.
doi:10.1099/vir.0.032987-0
PMCID: PMC3353388  PMID: 21632562
13.  HUMAN METAPNEUMOVIRUS GLYCOPROTEIN G INHIBITS TLR4-DEPENDENT SIGNALING IN MONOCYTE-DERIVED DENDRITIC CELLS2 
Human metapneumovirus (hMPV) is a major cause of upper and lower respiratory infections in children and adults. Recent work from our group demonstrated that hMPV G glycoprotein is an important virulence factor, responsible for inhibiting innate immune responses in airway epithelial cells. Myeloid dendritic cells are potent antigen presenting cells and play a major role in initiating and modulating the innate and adaptive immune responses. In this study, we found that TLR4 plays a major role in hMPV-induced activation of monocyte-derived dendritic cells (moDCs), as downregulation of its expression by siRNA significantly blocked hMPV-induced chemokine and type I interferon expression. Similar results were found in bone marrow derived-dendritic cells (BM-DCs) from TLR4 deficient mice. MoDCs infected with a virus lacking G protein expression (rhMPV-ΔG) produced higher levels of cytokines and chemokines, compared to cells infected with wild-type virus (rhMPV-WT), suggesting that G protein plays an inhibitory role in viral-induced cellular responses. Specifically, G protein affects TLR4-dependent signaling, as rhMPV-ΔG infection of moDCs inhibited LPS-induced production of cytokine and chemokines significantly less than rhMPV-WT, and treatment of moDCs with purified G protein resulted in a similar inhibition of LPS-dependent signaling. Our results demonstrate that hMPV G protein plays an important role in inhibiting host innate immune responses, likely affecting adaptive responses too.
doi:10.4049/jimmunol.1002589
PMCID: PMC3119724  PMID: 21632720
14.  Viral-mediated Inhibition of Antioxidant Enzymes Contributes to the Pathogenesis of Severe Respiratory Syncytial Virus Bronchiolitis 
Rationale: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in children, for which no specific treatment or vaccine is currently available. We have previously shown that RSV induces reactive oxygen species in cultured cells and oxidative injury in the lungs of experimentally infected mice. The mechanism(s) of RSV-induced oxidative stress in vivo is not known.
Objectives: To measure changes of lung antioxidant enzymes expression/activity and activation of NF-E2-related factor 2 (Nrf2), a transcription factor that regulates detoxifying and antioxidant enzyme gene expression, in mice and in infants with naturally acquired RSV infection.
Methods: Superoxide dismutase 1 (SOD 1), SOD 2, SOD 3, catalase, glutathione peroxidase, and glutathione S-transferase, as well as Nrf2 expression, were measured in murine bronchoalveolar lavage, cell extracts of conductive airways, and/or in human nasopharyngeal secretions by Western blot and two-dimensional gel electrophoresis. Antioxidant enzyme activity and markers of oxidative cell injury were measured in either murine bronchoalveolar lavage or nasopharyngeal secretions by colorimetric/immunoassays.
Measurements and Main Results: RSV infection induced a significant decrease in the expression and/or activity of SOD, catalase, glutathione S-transferase, and glutathione peroxidase in murine lungs and in the airways of children with severe bronchiolitis. Markers of oxidative damage correlated with severity of clinical illness in RSV-infected infants. Nrf2 expression was also significantly reduced in the lungs of viral-infected mice.
Conclusions: RSV infection induces significant down-regulation of the airway antioxidant system in vivo, likely resulting in lung oxidative damage. Modulation of oxidative stress may pave the way toward important advances in the therapeutic approach of RSV-induced acute lung disease.
doi:10.1164/rccm.201010-1755OC
PMCID: PMC3137144  PMID: 21471094
respiratory syncytial virus; airways; antioxidant enzymes; oxidative stress
15.  IKKε MODULATES RSV-INDUCED NF-κB-DEPENDENT GENE TRANSCRIPTION 
Virology  2010;408(2):224-231.
Respiratory syncytial virus (RSV), a negative-strand RNA virus, is the most common cause of epidemic respiratory disease in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-κB (NF-κB). In this study we have investigated the role of the non canonical IκB kinase (IKK)ε in modulating RSV-induced NF-κB activation. Our results show that inhibition of IKKε activation results in significant impairment of viral-induced NF-κB-dependent gene expression, through a reduction in NF-κB transcriptional activity, without changes in nuclear translocation or DNA-binding activity. Absence of IKKε results in a significant decrease of RSV-induced NF-κB phosphorylation on serine 536, a post-translational modification important for RSV-induced NF-κB-dependent gene expression, known to regulate NF-κB transcriptional activity without affecting nuclear translocation. This study identifies a novel mechanism by which IKKε regulates viral-induced cellular signaling.
doi:10.1016/j.virol.2010.09.016
PMCID: PMC2975836  PMID: 20961594
RSV; airway epithelial cells; NF-κB; inflammation
16.  Human Metapneumovirus Inhibits IFN-β Signaling by Downregulating Jak1 and Tyk2 Cellular Levels 
PLoS ONE  2011;6(9):e24496.
Human metapneumovirus (hMPV), a leading cause of respiratory tract infections in infants, inhibits type I interferon (IFN) signaling by an unidentified mechanism. In this study, we showed that infection of airway epithelial cells with hMPV decreased cellular level of Janus tyrosine kinase (Jak1) and tyrosine kinase 2 (Tyk2), due to enhanced proteosomal degradation and reduced gene transcription. In addition, hMPV infection also reduced the surface expression of type I IFN receptor (IFNAR). These inhibitory mechanisms are different from the ones employed by respiratory syncytial virus (RSV), which does not affect Jak1, Tyk2 or IFNAR expression, but degrades downstream signal transducer and activator of transcription proteins 2 (STAT2), although both viruses are pneumoviruses belonging to the Paramyxoviridae family. Our study identifies a novel mechanism by which hMPV inhibits STAT1 and 2 activation, ultimately leading to viral evasion of host IFN responses.
doi:10.1371/journal.pone.0024496
PMCID: PMC3176284  PMID: 21949722
17.  Respiratory Syncytial Virus Induces Oxidative Stress by Modulating Antioxidant Enzymes 
Oxidative stress plays an important role in the pathogenesis of lung inflammation. Respiratory syncytial virus (RSV) infection induces reactive oxygen species (ROS) production in vitro and oxidative injury in lungs in vivo; however, the mechanism of RSV-induced cellular oxidative stress has not been investigated. Therefore, we determined whether RSV infection of airway epithelial cells modified the expression and/or activities of antioxidant enzymes (AOE). A549 cells, a human alveolar type II–like epithelial cell line, and small airway epithelial (SAE) cells, normal human cells derived from terminal bronchioli, were infected with RSV and harvested at various time points to measure F2-8 isoprostanes by enzyme-linked immunosorbent assay and total and reduced glutathione (GSH and GSSG) by colorimetric assay. Superoxide dismutase (SOD) 1, 2, and 3, catalase, glutathione peroxidase (GPx), and glutathione S-transferase (GST) expression was determined by quantitative real-time PCR and Western blot, and their activity was measured by colorimetric assays. RSV infection induced a significant increase of lipid peroxidation products as well as a significant decrease in the GSH/GSSG ratio. There was a significant decrease in SOD 1, SOD 3, catalase, and GST expression with a concomitant increase of SOD 2 in RSV-infected cells, compared with uninfected cells. Total SOD activity was increased, but catalase, GPx, and GST activities were decreased, after RSV infection. Our findings suggest that RSV-induced cellular oxidative damage is the result of an imbalance between ROS production and antioxidant cellular defenses. Modulation of oxidative stress represents a potential novel pharmacologic approach to ameliorate RSV-induced acute lung inflammation.
doi:10.1165/rcmb.2008-0330OC
PMCID: PMC2742754  PMID: 19151318
RSV; airway epithelial cells; antioxidant enzymes; oxidative stress
18.  SUBVERSION OF PULMONARY DENDRITIC CELL FUNCTION BY PARAMYXOVIRUS INFECTIONS1 
Lower respiratory tract infections caused by the paramyxoviruses human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are characterized by short-lasting virus-specific immunity and often long term airway morbidity, both of which may be the result of alterations in the antigen presenting function of the lung which follow these infections. In this study, we investigated whether hMPV and RSV experimental infections alter the phenotype and function of dendritic cells (DC) subsets which are recruited to the lung. Characterization of lung DC trafficking demonstrated a differential recruitment of plasmacytoid DC (pDC), conventional DC (cDC) and interferon-producing killer DC (IKDC) to the lung and draining lymph nodes after hMPV and RSV infection. In vitro infection of lung DC indicated that in pDC, production of IFN-α, TNF-α, and CCL5 was induced only by hMPV while CCL3 and CCL4 were induced by both viruses. In cDC, a similar repertoire of cytokines was induced by hMPV and RSV, except for IFN-β, which was not induced by RSV. The function of lung pDC was altered following hMPV or RSV infection in vivo, as we demonstrated a reduced capacity of lung pDC to produce IFN-α as well as other cytokines including IL-6, TNF-α, CCL2, CCL3 and CCL4 in response to TLR9 agonist. Moreover, we observed an impaired capacity of cDC from infected mice to present Ag to CD4+ T cells, an effect that lasted beyond the acute phase of infection. Our findings suggest that acute paramyxovirus infections can alter the long term immune function of pulmonary DC.
doi:10.4049/jimmunol.0802262
PMCID: PMC2865244  PMID: 19234204
Dendritic cells; Lung; Viral; Cytokines; Cell trafficking
19.  EFFECT OF NMSO3 TREATMENT IN A MURINE MODEL OF HUMAN METAPNEUMOVIRUS INFECTION 
The Journal of general virology  2008;89(Pt 11):2709-2712.
SUMMARY
BALB/c mice, infected with human metapneumovirus (hMPV), were treated with NMSO3, one dose of 50 mg/kg given at the time of infection. NMSO3 significantly reduced viral replication in the lungs, as well as hMPV-induced body weight loss, pulmonary inflammation and cytokine production, suggesting that antiviral treatment initiated at the beginning of viral infection can modify hMPV-induced disease.
doi:10.1099/vir.0.2008/003301-0
PMCID: PMC2800786  PMID: 18931066
20.  Cigarette Smoke Condensate Enhances Respiratory Syncytial Virus–Induced Chemokine Release by Modulating NF-kappa B and Interferon Regulatory Factor Activation 
Toxicological Sciences  2008;106(2):509-518.
Exposure to cigarette smoke is a risk factor contributing to the severity of respiratory tract infections associated with respiratory syncytial virus (RSV). Stimulation of airway epithelial cells by either RSV or cigarette smoke condensate (CSC) has been shown to induce secretion of the proinflammatory chemokines. However, the effect of coexposure of airway epithelial cells to CSC and RSV on inducible chemokine production has not been previously investigated. The results of this study indicate that CSC costimulation significantly increased RSV-induced interleukin-8 (IL-8) and monocyte chemoattactant protein-1 gene and protein expression when compared with each stimulus alone. Promoter deletion studies identified the interferon stimulatory response element (ISRE) of the IL-8 promoter as a critical region responsible for the synergistic increase of IL-8 gene transcription during mixed exposure. CSC costimulation enhanced RSV-induced activation of interferon regulatory factor (IRF)-1 and IRF-7, which bind to the ISRE site. CSC also furthered RSV-induced activation of the transcription factor nuclear factor kappa B (NF-κB), as shown by increased NF-κB DNA binding to its specific site of the IL-8 promoter and increased NF-κB–driven gene transcription. Therefore, our data demonstrate that a combined exposure to CSC and RSV synergistically increases chemokine expression in airway epithelial cells, suggesting that CSC contributes to an exuberant immune response to RSV by stimulating overlapping signal transduction pathways.
doi:10.1093/toxsci/kfn175
PMCID: PMC2721674  PMID: 18723827
RSV; cigarette smoke condensate; chemokines; IRF; NF-κB
21.  INHIBITION OF RESPIRATORY SYNCYTIAL VIRUS INFECTION IN CELL CULTURES AND IN MICE WITH MORPHOLINO OLIGOMERS 
Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants, young children, and high-risk adults. Currently, there is no vaccine for the prevention of RSV infection, and available therapeutics are of limited utility. Peptide conjugated phosphorodiamidate morpholino oligomers (PPMO) are a class of antisense agents that can enter cells readily and interfere with viral protein expression through steric blocking of complementary RNA. Two antisense PPMO, designed to target sequence that includes the 5′ terminal- and translation start site-regions of RSV L mRNA, were tested for anti-RSV activity in cultures of two human airway cell lines. Both PPMO showed minimal cytotoxicity, and one of them (AUG-2), reduced viral titers by more than 2.0 log10. Intranasal treatment of BALB/c mice with AUG-2 PPMO prior to RSV inoculation produced a reduction in viral titer of 1.2 log10 in lung tissue at day 5 post-infection, and attenuated pulmonary inflammation at day 7 post-infection. These data show that the AUG-2 PPMO possessed potent anti-RSV activity and is worthy of further investigation as a candidate for therapeutic development.
doi:10.1038/mt.2008.81
PMCID: PMC2782410  PMID: 18443602
RSV; morpholino oligomers; antisense; PPMO; antiviral agents
22.  T Lymphocytes Contribute to Antiviral Immunity and Pathogenesis in Experimental Human Metapneumovirus Infection▿  
Journal of Virology  2008;82(17):8560-8569.
Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is a leading cause of lower respiratory tract infections in children, the elderly, and immunocompromised patients. Virus- and host-specific mechanisms of pathogenesis and immune protection are not fully understood. By an intranasal inoculation model, we show that hMPV-infected BALB/c mice developed clinical disease, including airway obstruction and hyperresponsiveness (AHR), along with histopathologic evidence of lung inflammation and viral replication. hMPV infection protected mice against subsequent viral challenge, as demonstrated by undetectable viral titers, lack of body weight loss, and a significant reduction in the level of lung inflammation. No cross-protection with other paramyxoviruses, such as respiratory syncytial virus, was observed. T-lymphocyte depletion studies showed that CD4+ and CD8+ T cells cooperate synergistically in hMPV eradication during primary infection, but CD4+ more than CD8+ T cells also enhanced clinical disease and lung pathology. Concurrent depletion of CD4+ and CD8+ T cells completely blocked airway obstruction as well as AHR. Despite impaired generation of neutralizing anti-hMPV antibodies in the absence of CD4+ T cells, mice had undetectable viral replication after hMPV challenge and were protected from clinical disease, suggesting that protection can be provided by an intact CD8+ T-cell compartment. Whether these findings have implications for naturally acquired human infections remains to be determined.
doi:10.1128/JVI.00699-08
PMCID: PMC2519650  PMID: 18562525
23.  Human Metapneumovirus Small Hydrophobic Protein Inhibits NF-κB Transcriptional Activity▿  
Journal of Virology  2008;82(16):8224-8229.
Human metapneumovirus, a leading cause of respiratory tract infections in infants, encodes a small hydrophobic (SH) protein of unknown function. In this study, we showed that infection of airway epithelial cells or mice with recombinant human metapneumovirus lacking SH expression (rhMPV-ΔSH) enhanced secretion of proinflammatory mediators, including interleukin 6 (IL-6) and IL-8, encoded by two NF-kB-dependent genes, compared to infection with wild-type rhMPV. RhMPV-ΔSH infection resulted in enhanced NF-kB-dependent gene transcription and in increased levels of phosphorylated and acetylated NF-kB without affecting its nuclear translocation, identifying a possible novel mechanism by which paramyxovirus SH proteins modulate NF-kB activation.
doi:10.1128/JVI.02584-07
PMCID: PMC2519579  PMID: 18550666
24.  Effect of NMSO3 treatment in a murine model of human metapneumovirus infection 
The Journal of General Virology  2008;89(Pt 11):2709-2712.
BALB/c mice infected with human metapneumovirus (hMPV) were treated with the sulfated sialyl lipid NMSO3 (one dose of 50 mg kg−1) given at the time of infection. NMSO3 significantly reduced viral replication in the lungs, as well as hMPV-induced body weight loss, pulmonary inflammation and cytokine production, suggesting that antiviral treatment initiated at the beginning of viral infection can modify hMPV-induced disease.
doi:10.1099/vir.0.2008/003301-0
PMCID: PMC2800786  PMID: 18931066
25.  Human Metapneumovirus Glycoprotein G Inhibits Innate Immune Responses 
PLoS Pathogens  2008;4(5):e1000077.
Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infection in infants, as well as in the elderly and immunocompromised patients. No effective treatment or vaccine for hMPV is currently available. A recombinant hMPV lacking the G protein (rhMPV-ΔG) was recently developed as a potential vaccine candidate and shown to be attenuated in the respiratory tract of a rodent model of infection. The mechanism of its attenuation, as well as the role of G protein in modulation of hMPV-induced cellular responses in vitro, as well as in vivo, is currently unknown. In this study, we found that rhMPV-ΔG-infected airway epithelial cells produced higher levels of chemokines and type I interferon (IFN) compared to cells infected with rhMPV-WT. Infection of airway epithelial cells with rhMPV-ΔG enhanced activation of transcription factors belonging to the nuclear factor (NF)-κB and interferon regulatory factor (IRF) families, as revealed by increased nuclear translocation and/or phosphorylation of these transcription factors. Compared to rhMPV-WT, rhMPV-ΔG also increased IRF- and NF-κB-dependent gene transcription, which was reversely inhibited by G protein expression. Since RNA helicases have been shown to play a fundamental role in initiating viral-induced cellular signaling, we investigated whether retinoic induced gene (RIG)-I was the target of G protein inhibitory activity. We found that indeed G protein associated with RIG-I and inhibited RIG-I-dependent gene transcription, identifying an important mechanism by which hMPV affects innate immune responses. This is the first study investigating the role of hMPV G protein in cellular signaling and identifies G as an important virulence factor, as it inhibits the production of important immune and antiviral mediators by targeting RIG-I, a major intracellular viral RNA sensor.
Author Summary
Human metapneumovirus (hMPV), a member of the Paramyxoviridae family, is an important cause of respiratory morbidity throughout life. The contribution of viral-specific proteins to the pathogenesis of hMPV infection and immune evasion is largely unknown. Previous work has suggested that the glycoprotein G of hMPV is not necessary for the process of viral fusion and attachment to host cells, and a recombinant hMPV lacking the G protein (rhMPV-ΔG) shows an attenuated phenotype in the respiratory tract of animal models of infection. Airway epithelial cells, a major component of the innate immune system, are a primary target of hMPV infection. In this study, we show that hMPV G protein functions as a major inhibitory factor of the host antiviral response by blocking production of inducible chemokines and IFN-α/β. A major finding of this work is the demonstration that hMPV G protein interacts with RIG-I, a cytoplasmic viral sensor. As result, hMPV G protein inhibits RIG-I-dependent signaling pathways, including activation of NF-κB and IRF-3, two transcription factors necessary for the synthesis of inflammatory and antiviral cytokines. Understanding the function of hMPV proteins is critical for the future design of effective antiviral therapies and rationale design of vaccine candidates.
doi:10.1371/journal.ppat.1000077
PMCID: PMC2386556  PMID: 18516301

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