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1.  RESPIRATORY SYNCYTIAL VIRUS INFECTION DOWNREGULATES ANTIOXIDANT ENZYME EXPRESSION BY TRIGGERING deacetylation-proteasomal DEGRADATION OF NRF2 
Free radical biology & medicine  2015;88(0 0):391-403.
Respiratory syncytial virus (RSV) is the most important cause of viral acute respiratory tract infections and hospitalizations in children, for which no vaccine or treatment is available. RSV infection in cells, mice and children leads to rapid generation of reactive oxygen species, which are associated with oxidative stress and lung damage, due to a significant decrease in the expression of airway antioxidant enzymes (AOEs). Oxidative stress plays an important role in the pathogenesis of RSV-induced lung disease, as antioxidants ameliorate clinical disease and inflammation in vivo. The aim of this study is to investigate the unknown mechanism(s) of viral-induced inhibition of AOE expression. This study shows that RSV infection induces a progressive reduction in nuclear and total cellular level of the transcription factor NF-E2-related factor 2 (NRF2), resulting in decreased binding to endogenous AOE gene promoters and decreased AOE expression. RSV induces NRF2 deacetylation and degradation via the proteasome pathway in vitro and in vivo. Histone deacetylase and proteasome inhibitors block NRF2 degradation and increase NRF2 binding to AOE endogenous promoters, resulting in increased AOE expression. Known inducers of NRF2 are able to increase NRF2 activation and subsequent AOE expression during RSV infection in vitro and in vivo, with significant amelioration of oxidative stress. This is the first study to investigate the mechanism(s) of viral-induced inhibition of AOE expression. RSV-induced inhibition of NRF2 activation, due to deacetylation and proteasomal degradation, could be targeted for therapeutic intervention aimed to increase antioxidant airway capacity during infection.
Graphical Abstract
doi:10.1016/j.freeradbiomed.2015.05.043
PMCID: PMC4628892  PMID: 26073125
Respiratory Syncytial Virus; ROS; oxidative stress; antioxidant enzymes; NRF2; acetylation; proteasome
2.  ROLE OF DIETARY ANTIOXIDANTS IN HUMAN METAPNEUMOVIRUS INFECTION 
Virus research  2015;200:19-23.
Summary
Human metapneumovirus (hMPV) is a major cause of respiratory tract infections in children, elderly and immunocompromised hosts, for which no vaccine or treatment are currently available. Oxidative stress and inflammatory responses represent important pathogenic mechanism(s) of hMPV infection. Here, we explored the potential protective role of dietary antioxidants in hMPV infection. Treatment of airway epithelial cells with resveratrol and quercetin during hMPV infection significantly reduced cellular oxidative damage, inflammatory mediator secretion and viral replication, without affecting viral gene transcription and protein synthesis, indicating that inhibition of viral replication occurred at the level of viral assembly and/or release. Modulation of proinflammatory mediator expression occurred through the inhibition of transcription factor nuclear factor (NF)-κB and interferon regulatory factor (IRF)-3 binding to their cognate site of endogenous gene promoters. Our results indicate the use of dietary antioxidants as an effective treatment approach for modulating hMPV induced lung oxidative damage and inflammation.
doi:10.1016/j.virusres.2015.01.018
PMCID: PMC5022781  PMID: 25645280
Human metapneumovirus; resveratrol; quercetin
3.  Oxidative stress in Nipah virus-infected human small airway epithelial cells 
The Journal of General Virology  2015;96(Pt 10):2961-2970.
Nipah virus (NiV) is a zoonotic emerging pathogen that can cause severe and often fatal respiratory disease in humans. The pathogenesis of NiV infection of the human respiratory tract remains unknown. Reactive oxygen species (ROS) produced by airway epithelial cells in response to viral infections contribute to lung injury by inducing inflammation and oxidative stress; however, the role of ROS in NiV-induced respiratory disease is unknown. To investigate whether NiV induces oxidative stress in human respiratory epithelial cells, we used oxidative stress markers and monitored antioxidant gene expression. We also used ROS scavengers to assess their role in immune response modulation. Oxidative stress was confirmed in infected cells and correlated with the reduction in antioxidant enzyme gene expression. Infected cells treated by ROS scavengers resulted in a significant decrease of the (F2)-8-isoprostane marker, inflammatory responses and virus replication. In conclusion, ROS are induced during NiV infection in human respiratory epithelium and contribute to the inflammatory response. Understanding how oxidative stress contributes to NiV pathogenesis is crucial for therapeutic development.
doi:10.1099/jgv.0.000243
PMCID: PMC4635479  PMID: 26297489
4.  Mitochondrial antiviral-signalling protein plays an essential role in host immunity against human metapneumovirus 
The Journal of General Virology  2015;96(Pt 8):2104-2113.
Human metapneumovirus (hMPV) is a common cause of respiratory tract infection in the paediatrics population. Recently, we and others have shown that retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) are essential for hMPV-induced cellular antiviral signalling. However, the contribution of those receptors to host immunity against pulmonary hMPV infection is largely unexplored. In this study, mice deficient in mitochondrial antiviral-signalling protein (MAVS), an adaptor of RLRs, were used to investigate the role(s) of these receptors in pulmonary immune responses to hMPV infection. MAVS deletion significantly impaired the induction of antiviral and pro-inflammatory cytokines and the recruitment of immune cells to the bronchoalveolar lavage fluid by hMPV. Compared with WT mice, mice lacking MAVS demonstrated decreased abilities to activate pulmonary dendritic cells (DCs) and abnormal primary T-cell responses to hMPV infection. In addition, mice deficient in MAVS had a higher peak of viral load at day 5 post-infection (p.i.) than WT mice, but were able to clear hMPV by day 7 p.i. similarly to WT mice. Taken together, our data indicate a role of MAVS-mediated pathways in the pulmonary immune responses to hMPV infection and the early control of hMPV replication.
doi:10.1099/vir.0.000178
PMCID: PMC4681063  PMID: 25953917
5.  Alveolar Macrophages Contribute to the Pathogenesis of Human Metapneumovirus Infection while Protecting against Respiratory Syncytial Virus Infection 
Human metapneumovirus (hMPV) and respiratory syncytial virus (RSV) are leading causes of upper and lower respiratory tract infections in young children and among elderly and immunocompromised patients. The pathogenesis of hMPV-induced lung disease is poorly understood. The lung macrophage population consists of alveolar macrophages (AMs) residing at the luminal surface of alveoli and interstitial macrophages present within the parenchymal lung interstitium. The involvement of AMs in innate immune responses to virus infections remains elusive. In this study, BALB/c mice depleted of AMs by intranasal instillation of dichloromethylene bisphosphonate (L-CL2MBP) liposomes were examined for disease, lung inflammation, and viral replication after infection with hMPV or RSV. hMPV-infected mice lacking AMs exhibited improved disease in terms of body weight loss, lung inflammation, airway obstruction, and hyperresponsiveness compared with AM-competent mice. AM depletion was associated with significantly reduced hMPV titers in the lungs, suggesting that hMPV required AMs for early entry and replication in the lung. In contrast, AM depletion in the context of RSV infection was characterized by an increase in viral replication, worsened disease, and inflammation, with increased airway neutrophils and inflammatory dendritic cells. Overall, lack of AMs resulted in a broad-spectrum disruption in type I IFN and certain inflammatory cytokine production, including TNF and IL-6, while causing a virus-specific alteration in the profile of several immunomodulatory cytokines, chemokines, and growth factors. Our study demonstrates that AMs have distinct roles in the context of human infections caused by members of the Paramyxoviridae family.
doi:10.1165/rcmb.2013-0414OC
PMCID: PMC4189482  PMID: 24749674
human metapneumovirus; respiratory syncytial virus; viral infection; pathogenesis; alveolar macrophages
6.  Role of Hydrogen Sulfide in Paramyxovirus Infections 
Journal of Virology  2015;89(10):5557-5568.
ABSTRACT
Hydrogen sulfide (H2S) is an endogenous gaseous mediator that has gained increasing recognition as an important player in modulating acute and chronic inflammatory diseases. However, its role in virus-induced lung inflammation is currently unknown. Respiratory syncytial virus (RSV) is a major cause of upper and lower respiratory tract infections in children for which no vaccine or effective treatment is available. Using the slow-releasing H2S donor GYY4137 and propargylglycin (PAG), an inhibitor of cystathionine-γ-lyase (CSE), a key enzyme that produces intracellular H2S, we found that RSV infection led to a reduced ability to generate and maintain intracellular H2S levels in airway epithelial cells (AECs). Inhibition of CSE with PAG resulted in increased viral replication and chemokine secretion. On the other hand, treatment of AECs with the H2S donor GYY4137 reduced proinflammatory mediator production and significantly reduced viral replication, even when administered several hours after viral absorption. GYY4137 also significantly reduced replication and inflammatory chemokine production induced by human metapneumovirus (hMPV) and Nipah virus (NiV), suggesting a broad inhibitory effect of H2S on paramyxovirus infections. GYY4137 treatment had no effect on RSV genome replication or viral mRNA and protein synthesis, but it inhibited syncytium formation and virus assembly/release. GYY4137 inhibition of proinflammatory gene expression occurred by modulation of the activation of the key transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3) at a step subsequent to their nuclear translocation. H2S antiviral and immunoregulatory properties could represent a novel treatment strategy for paramyxovirus infections.
IMPORTANCE RSV is a global health concern, causing significant morbidity and economic losses as well as mortality in developing countries. After decades of intensive research, no vaccine or effective treatment, with the exception of immunoprophylaxis, is available for this infection as well as for other important respiratory mucosal viruses. This study identifies hydrogen sulfide as a novel cellular mediator that can modulate viral replication and proinflammatory gene expression, both important determinants of lung injury in respiratory viral infections, with potential for rapid translation of such findings into novel therapeutic approaches for viral bronchiolitis and pneumonia.
doi:10.1128/JVI.00264-15
PMCID: PMC4442521  PMID: 25740991
7.  Exosomes and Their Role in the Life Cycle and Pathogenesis of RNA Viruses 
Viruses  2015;7(6):3204-3225.
Exosomes are membrane-enclosed vesicles actively released into the extracellular space, whose content reflect the physiological/pathological state of the cells they originate from. These vesicles participate in cell-to-cell communication and transfer of biologically active proteins, lipids, and RNAs. Their role in viral infections is just beginning to be appreciated. RNA viruses are an important class of pathogens and affect millions of people worldwide. Recent studies on Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), human T-cell lymphotropic virus (HTLV), and Dengue Virus (DENV) have demonstrated that exosomes released from infected cells harbor and deliver many regulatory factors including viral RNA and proteins, viral and cellular miRNA, and other host functional genetic elements to neighboring cells, helping to establish productive infections and modulating cellular responses. Exosomes can either spread or limit an infection depending on the type of pathogen and target cells, and can be exploited as candidates for development of antiviral or vaccine treatments. This review summarizes recent progress made in understanding the role of exosomes in RNA virus infections with an emphasis on their potential contribution to pathogenesis.
doi:10.3390/v7062770
PMCID: PMC4488737  PMID: 26102580
exosomes; microvesicles; RNA virus; pathogenesis; miRNA; infection
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
21.  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
22.  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
23.  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
24.  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
25.  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

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