Respiratory Syncytial Virus Uses CX3CR1 as a Receptor on Primary Human Airway Epithelial Cultures

Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a “non-neutralizing” monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization.     

Respiratory Syncytial Virus G Protein CX3C Motif Impairs Human Airway Epithelial and Immune Cell Responses

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children and causes disease in the elderly and persons with compromised cardiac, pulmonary, or immune systems. Despite the high morbidity rates of RSV infection, no highly effective treatment or vaccine is yet available. The RSV G protein is an important contributor to the disease process. A conserved CX3C chemokine-like motif in G likely contributes to the pathogenesis of disease. Through this motif, G protein binds to CX3CR1 present on various immune cells and affects immune responses to RSV, as has been shown in the mouse model of RSV infection. However, very little is known of the role of RSV CX3C-CX3CR1 interactions in human disease. In this study, we use an in vitro model of human RSV infection comprised of human peripheral blood mononuclear cells (PBMCs) separated by a permeable membrane from human airway epithelial cells (A549) infected with RSV with either an intact CX3C motif (CX3C) or a mutated motif (CX4C). We show that the CX4C virus induces higher levels of type I/III interferon (IFN) in A549 cells, increased IFN-α and tumor necrosis factor alpha (TNF-α) production by human plasmacytoid dendritic cells (pDCs) and monocytes, and increased IFN-γ production in effector/memory T cell subpopulations. Treatment of CX3C virus-infected cells with the F(ab′)₂ form of an anti-G monoclonal antibody (MAb) that blocks binding to CX3CR1 gave results similar to those with the CX4C virus. Our data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development.     

Respiratory syncytial virus G protein and G protein CX3C motif adversely affect CX3CR1+ T cell responses

Interactions between fractalkine (CX3CL1) and its receptor, CX3CR1, mediate leukocyte adhesion, activation, and trafficking. The respiratory syncytial virus (RSV) G protein has a CX3C chemokine motif that can bind CX3CR1 and modify CXCL1-mediated responses. In this study, we show that expression of the RSV G protein or the G protein CX3C motif during infection is associated with reduced CX3CR1+ T cell trafficking to the lung, reduced frequencies of RSV-specific, MHC class I-restricted IFN-gamma-expressing cells, and lower numbers of IL-4- and CX3CL1-expressing cells. In addition, we show that CX3CR1+ cells constitute a major component of the cytotoxic response to RSV infection. These results suggest that G protein and the G protein CX3C motif reduce the antiviral T cell response to RSV infection.     

Effect of chemokine receptor CX3CR1 deficiency in a murine model of respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory illness in infants and young children worldwide, making it a high priority for development of strategies for prevention and treatment. RSV can cause repeat infections throughout life, with serious complications in elderly and immunocompromised patients. Previous studies indicate that the RSV G protein binds through a CX3C chemokine motif to the host chemokine receptor, CX3CR1, and modulates the inflammatory immune response. In the current study, we examined the contribution of CX3CR1 to the immune response to RSV infection in mice. CX3CR1-deficient mice showed an impaired innate immune response to RSV infection, characterized by substantially decreased NK1.1(+) natural killer, CD11b(+), and RB6-8C5(+) polymorphonuclear cell trafficking to the lung and reduced IFNγ production compared with those in wildtype control mice. Leukocytes from CX3CR1-deficient mice were poorly chemotactic toward RSV G protein and CX3CL1. These results substantiate the importance of the RSV G CX3C-CX3CR1 interaction in the innate immune response to RSV infection.     

Respiratory syncytial virus synergizes with Th2 cytokines to induce optimal levels of TARC/CCL17

Respiratory syncytial virus (RSV) is a ubiquitous virus that preferentially infects airway epithelial cells, causing asthma exacerbations and severe disease in immunocompromised hosts. Acute RSV infection induces inflammation in the lung. Thymus- and activation-regulated chemokine (TARC) recruits Th2 cells to sites of inflammation. We found that acute RSV infection of BALB/c mice increased TARC production in the lung. Immunization of BALB/c mice with individual RSV proteins can lead to the development of Th1- or Th2-biased T cell responses in the lung after RSV infection. We primed animals with a recombinant vaccinia virus expressing either the RSV fusion (F) protein or the RSV attachment (G) protein, inducing Th1- and Th2-biased pulmonary memory T cell responses, respectively. After RSV infection, TARC production significantly increased in the vaccinia virus G-primed animals only. These data suggest a positive feedback loop for TARC production between RSV infection and Th2 cytokines. RSV-infected lung epithelial cells cultured with IL-4 or IL-13 demonstrated a marked increase in the production of TARC. The synergistic effect of RSV and IL-4/IL-13 on TARC production reflected differential induction of NF kappa B and STAT6 by the two stimuli (both are in the TARC promoter). These findings demonstrate that RSV induces a chemokine TARC that has the potential to recruit Th2 cells to the lung.     

Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology

Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.     

CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung

Allergic asthma is a T helper type 2 (T(H)2)-dominated disease of the lung. In people with asthma, a fraction of CD4(+) T cells express the CX3CL1 receptor, CX3CR1, and CX3CL1 expression is increased in airway smooth muscle, lung endothelium and epithelium upon allergen challenge. Here we found that untreated CX3CR1-deficient mice or wild-type (WT) mice treated with CX3CR1-blocking reagents show reduced lung disease upon allergen sensitization and challenge. Transfer of WT CD4(+) T cells into CX3CR1-deficient mice restored the cardinal features of asthma, and CX3CR1-blocking reagents prevented airway inflammation in CX3CR1-deficient recipients injected with WT T(H)2 cells. We found that CX3CR1 signaling promoted T(H)2 survival in the inflamed lungs, and injection of B cell leukemia/lymphoma-2 protein (BCl-2)-transduced CX3CR1-deficient T(H)2 cells into CX3CR1-deficient mice restored asthma. CX3CR1-induced survival was also observed for T(H)1 cells upon airway inflammation but not under homeostatic conditions or upon peripheral inflammation. Therefore, CX3CR1 and CX3CL1 may represent attractive therapeutic targets in asthma.     

Vaccination To Induce Antibodies Blocking the CX3C-CX3CR1 Interaction of Respiratory Syncytial Virus G Protein Reduces Pulmonary Inflammation and Virus Replication in Mice

Respiratory syncytial virus (RSV) infection causes substantial morbidity and some deaths in the young and elderly worldwide. There is no safe and effective vaccine available, although it is possible to reduce the hospitalization rate for high-risk children by anti-RSV antibody prophylaxis. RSV has been shown to modify the immune response to infection, a feature linked in part to RSV G protein CX3C chemokine mimicry. This study determined if vaccination with G protein polypeptides or peptides spanning the central conserved region of the G protein could induce antibodies that blocked G protein CX3C-CX3CR1 interaction and disease pathogenesis mediated by RSV infection. The results show that mice vaccinated with G protein peptides or polypeptides containing the CX3C motif generate antibodies that inhibit G protein CX3C-CX3CR1 binding and chemotaxis, reduce lung virus titers, and prevent body weight loss and pulmonary inflammation. The results suggest that RSV vaccines that induce antibodies that block G protein CX3C-CX3CR1 interaction may offer a new, safe, and efficacious RSV vaccine strategy.Human respiratory syncytial virus (RSV) is an important and ubiquitous respiratory virus causing serious lower respiratory tract diseases in infants and young children and substantial morbidity and mortality in the elderly and immunocompromised (7, 11, 20, 21). Despite substantial efforts to develop safe and effective RSV vaccines, none have been successful. The first RSV candidate vaccine, a formalin-inactivated alum-precipitated RSV (FI-RSV) preparation, did not confer protection and was associated with a greater risk of serious disease with subsequent natural infection (9, 60). Live attenuated and inactivated whole virus vaccine candidates have also failed to protect, as they were either insufficiently attenuated or demonstrated the potential for enhanced pulmonary disease upon subsequent RSV infection (6, 37, 39, 41, 45). Similarly, subunit vaccine candidates, such as purified F protein and a prokaryotically expressed fusion protein comprising a fragment of the RSV G protein (residues 130 to 230) fused by its N terminus to the albumin binding domain of streptococcal protein G (designated BBG2Na), have been shown to be inadequate (8, 33, 37, 41). The specific reasons for RSV vaccine failure remain to be answered but could be related to RSV-mediated circumvention of immunity and, more broadly, to the lack of durable immunity elicited in response to natural RSV infection, as people of all ages may experience repeated infections and disease throughout life (3, 41, 45).Evidence indicates that the RSV F protein is important in inducing protective immunity (19, 38), but studies evaluating a BBG2Na vaccine candidate in combination with different adjuvants and by different routes of administration have shown a role for G protein in protection against RSV in rodents (4, 10, 17, 32, 43, 44, 49, 51). The structural elements of the G protein fragment in the BBG2Na vaccine candidate implicated in protective efficacy were mapped, and five different B-cell epitopes were determined, i.e., residues 145 to 159, 164 to 176, 171 to 187, 172 to 187, and 190 to 204 (44, 48). Interestingly, immunogenicity of peptides with residues 145 to 159 was dependent on the orientation of the covalent peptide coupling to the carrier proteins, as mice vaccinated with C-terminally coupled peptides developed protective antibody titers, whereas mice vaccinated with N-terminal peptides did not. The focus of the BBG2Na vaccine studies centered on development of protective neutralizing antibodies, and the studies showed that vaccination or priming with the G protein fragment in BBG2Na did not induce signs of enhanced pulmonary pathology (17, 42, 46, 50).Despite the strong evidence that G protein peptides and polypeptides can induce protective immunity, the G protein has also been implicated in disease pathogenesis (30, 40, 41, 54). One of the disease mechanisms linked to the G protein is CX3C chemokine mimicry (56). RSV G protein has marked similarities to fractalkine, the only known CX3C chemokine, including similarities in structural features (56). Both G protein and fractalkine exist as membrane-bound and secreted forms, and both contain a CX3C chemokine motif that can bind to the fractalkine receptor, CX3CR1 (15, 27). Fractalkine functions to recruit immune cells to sites of inflammation, in particular, CX3CR1⁺ leukocytes, which include subsets of NK cells and CD4⁺ and CD8⁺ T cells (23). RSV G protein has been shown to have fractalkine-like leukocyte chemotactic activity in vitro (56). In vivo, RSV G protein acts as a fractalkine antagonist, modulating the immune response to infection by inhibiting fractalkine-mediated responses by altering the trafficking of CX3CR1⁺ cells and modifying the magnitude and cadence of cytokine and chemokine expression (23, 55). Infection of mice with a mutant RSV lacking the CX3C motif leads to a substantial increase of pulmonary NK cells and CD4⁺ and CD8⁺ cells compared to infection with wild-type RSV (23). This suggests that G protein CX3C-CX3CR1 interaction contributes to immune evasion and may contribute to disease pathogenesis. Thus, G protein CX3C interaction with CX3CR1 is an important target for disease intervention strategies against RSV infection.In the present study, we investigated a new RSV vaccine strategy, using G protein polypeptide and peptide vaccination to generate antibodies reactive to the central conserved cysteine noose region of the G protein to block G protein CX3C motif interaction with CX3CR1. We hypothesize that vaccines inducing G protein-CX3CR1 blocking antibodies will prevent much of the RSV G protein-mediated immune modulation and disease pathogenesis. Our results show that antibodies induced by the central conserved noose region of the G protein block G protein binding to CX3CR1, prevent body weight loss indicative of disease pathogenesis, decrease pulmonary inflammation, and decrease lung virus titers compared to antibodies reactive to N- and C-terminal regions of the G protein. These results suggest that a vaccine strategy to induce G protein CX3C-CX3CR1 blocking antibodies may be useful to prevent G protein-mediated immune modulation and disease pathogenesis.     

Oxidant tone regulates RANTES gene expression in airway epithelial cells infected with respiratory syncytial virus. Role in viral-induced interferon regulatory factor activation

Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part, through its ability to induce chemokine synthesis in infected airway epithelial cells. RANTES (regulated upon activation, normal T-cells expressed and secreted) is a CC chemokine which recruits and activates monocytes, lymphocytes, and eosinophils, all cell types present in the lung inflammatory infiltrate induced by RSV infection. In this study we investigated the role of reactive oxygen species in the induction of RANTES gene expression in human type II alveolar epithelial cells (A549), following RSV infection. Our results indicate that RSV infection of airway epithelial cells rapidly induces reactive oxygen species production, prior to RANTES expression, as measured by oxidation of 2',7'-dichlorofluorescein. Pretreatment of airway epithelial cells with the antioxidant butylated hydroxyanisol (BHA), as well a panel of chemically unrelated antioxidants, blocks RSV-induced RANTES gene expression and protein secretion. This effect is mediated through the ability of BHA to inhibit RSV-induced interferon regulatory factor binding to the RANTES promoter interferon-stimulated responsive element, that is absolutely required for inducible RANTES promoter activation. BHA inhibits de novo interferon regulator factor (IRF)-1 and -7 gene expression and protein synthesis, and IRF-3 nuclear translocation. Together, these data indicates that a redox-sensitive pathway is involved in RSV-induced IRF activation, an event necessary for RANTES gene expression.     

Respiratory syncytial virus up-regulates TLR4 and sensitizes airway epithelial cells to endotoxin

Airway epithelial cells are unresponsive to endotoxin (lipopolysaccharide (LPS)) exposure under normal conditions. This study demonstrates that respiratory syncytial virus (RSV) infection results in increased sensitivity to this environmental exposure. Infection with RSV results in increased expression of Toll-like receptor (TLR) 4 mRNA, protein, and increased TLR4 membrane localization. This permits significantly enhanced LPS binding to the epithelial monolayer that is blocked by disruption of the Golgi. The increased TLR4 results in an LPS-induced inflammatory response as demonstrated by increased mitogen-activated protein (MAP) kinase activity, IL-8 production, and tumor necrosis factor alpha production. RSV infection also allowed for tumor necrosis factor alpha production subsequent to TLR4 cross-linking with an immobilized antibody. These data suggest that RSV infection sensitizes airway epithelium to a subsequent environmental exposure (LPS) by altered expression and membrane localization of TLR4. The increased interaction between airway epithelial cells and LPS has the potential to profoundly alter airway inflammation.     

Cutting edge: Protective effect of CX3CR1+ dendritic cells in a vaccinia virus pulmonary infection model

The protective host immune response to viral infections requires both effective innate and adaptive immune responses. Cross-talk between the two responses is coordinated by the chemokine network and professional APCs such as dendritic cells (DCs). In mice, subpopulations of myeloid DCs in peripheral tissues such as lungs and in blood express CX3CR1 depending on the inflammation state. We thus examined the host response of mice deficient in the chemokine receptor CX3CR1 to an intranasal vaccinia virus infection. CX3CR1-deficient mice displayed significantly more severe morbidity and mortality compared with control wild-type mice within 10 d following vaccinia virus infection. CX3CR1(-/-) mice had increased viral loads and a reduced T cell response compared with wild-type mice. Finally, an adoptive transfer of CX3CR1(+/+) DCs completely protected CX3CR1(-/-) mice to a previously lethal infection. This study therefore opens up the possibility of novel antiviral therapeutics targeting lung DC recruitment.     

Respiratory syncytial virus decreases p53 protein to prolong survival of airway epithelial cells

Respiratory syncytial virus (RSV) is a clinically important pathogen. It preferentially infects airway epithelial cells causing bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and life-threatening pneumonia in the immunosuppressed. The p53 protein is a tumor suppressor protein that promotes apoptosis and is tightly regulated for optimal cell growth and survival. A critical negative regulator of p53 is murine double minute 2 (Mdm2), an E3 ubiquitin ligase that targets p53 for proteasome degradation. Mdm2 is activated by phospho-Akt, and we previously showed that RSV activates Akt and delays apoptosis in primary human airway epithelial cells. In this study, we explore further the mechanism by which RSV regulates p53 to delay apoptosis but paradoxically enhance inflammation. We found that RSV activates Mdm2 1-6 h after infection resulting in a decrease in p53 6-24 h after infection. The p53 down-regulation correlates with increased airway epithelial cell longevity. Importantly, inhibition of the PI3K/Akt pathway blocks the activation of Mdm2 by RSV and preserves the p53 response. The effects of RSV infection are antagonized by Nutlin-3, a specific chemical inhibitor that prevents the Mdm2/p53 association. Nutlin-3 treatment increases endogenous p53 expression in RSV infected cells, causing earlier cell death. This same increase in p53 enhances viral replication and limits the inflammatory response as measured by IL-6 protein. These findings reveal that RSV decreases p53 by enhancing Akt/Mdm2-mediated p53 degradation, thereby delaying apoptosis and prolonging survival of airway epithelial cells.     

Blocking intercellular adhesion molecule-1 on human epithelial cells decreases respiratory syncytial virus infection

The respiratory syncytial virus (RSV) causes potentially fatal lower respiratory tract infection in infants. The molecular mechanism of RSV infection is unknown. Our data show that RSV colocalizes with intercellular adhesion molecule-1 (ICAM-1) on the HEp-2 epithelial cell surface. Furthermore, a neutralizing anti-ICAM-1 mAb significantly inhibits RSV infection and infection-induced secretion of proinflammatory chemokine RANTES and mediator ET-1 in HEp-2 cells. Similar decrease in RSV infection is also observed in A549, a type-2 alveolar epithelial cell line, and NHBE, the normal human bronchial epithelial cell line when pretreated with anti-ICAM-1 mAb prior to RSV infection. Incubation of virus with soluble ICAM-1 also significantly decreases RSV infection of epithelial cells. Binding studies using ELISA indicate that RSV binds to ICAM-1, which can be inhibited by an antibody to the fusion F protein and also the recombinant F protein can bind to soluble ICAM-1, suggesting that RSV interaction with ICAM-1 involves the F protein. It is thus concluded that ICAM-1 facilitates RSV entry and infection of human epithelial cells by binding to its F protein, which is important to viral replication and infection and may lend itself as a therapeutic target.     

miRNA-34b/c regulates mucus secretion in RSV-infected airway epithelial cells by targeting FGFR1

Respiratory syncytial virus (RSV) infection in airway epithelial cells is the main cause of bronchiolitis in children. Excessive mucus secretion is one of the primary symbols in RSV related lower respiratory tract infections (RSV-related LRTI). However, the pathological processes of mucus hypersecretion in RSV-infected airway epithelial cells remains unclear. The current study explores the involvement of miR-34b/miR-34c in mucus hypersecretion in RSV-infected airway epithelial cells by targeting FGFR1. First, miR-34b/miR-34c and FGFR1 mRNA were quantified by qPCR in throat swab samples and cell lines, respectively. Then, the luciferase reporters’ assay was designed to verify the direct binding between FGFR1 and miR-34b/miR-34c. Finally, the involvement of AP-1 signalling was assessed by western blot. This study identified that miR-34b/miR-34c was involved in c-Jun-regulated MUC5AC production by targeting FGFR1 in RSV-infected airway epithelial cells. These results provide some useful insights into the molecular mechanisms of mucus hypersecretion which may also bring new potential strategies to improve mucus hypersecretion in RSV disease.     

Enhanced disease and pulmonary eosinophilia associated with formalin-inactivated respiratory syncytial virus vaccination are linked to G glycoprotein CX3C-CX3CR1 interaction and expression of substance P

Vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine or RSV G glycoprotein results in enhanced pulmonary disease after live RSV infection. Enhanced pulmonary disease is characterized by pulmonary eosinophilia and is associated with a substantial inflammatory response. We show that the absence of the G glycoprotein or G glycoprotein CX3C motif during FI-RSV vaccination or RSV challenge of FI-RSV-vaccinated mice, or treatment with anti-substance P or anti-CX3CR1 antibodies, reduces or eliminates enhanced pulmonary disease, modifies T-cell receptor Vbeta usage, and alters CC and CXC chemokine expression. These data suggest that the G glycoprotein, and in particular the G glycoprotein CX3C motif, is key in the enhanced inflammatory response to FI-RSV vaccination, possibly through the induction of substance P.     

Sustained Protein Kinase D Activation Mediates Respiratory Syncytial Virus-Induced Airway Barrier Disruption

Understanding the regulation of airway epithelial barrier function is a new frontier in asthma and respiratory viral infections. Despite recent progress, little is known about how respiratory syncytial virus (RSV) acts at mucosal sites, and very little is known about its ability to influence airway epithelial barrier function. Here, we studied the effect of RSV infection on the airway epithelial barrier using model epithelia. 16HBE14o- bronchial epithelial cells were grown on Transwell inserts and infected with RSV strain A2. We analyzed (i) epithelial apical junction complex (AJC) function, measuring transepithelial electrical resistance (TEER) and permeability to fluorescein isothiocyanate (FITC)-conjugated dextran, and (ii) AJC structure using immunofluorescent staining. Cells were pretreated or not with protein kinase D (PKD) inhibitors. UV-irradiated RSV served as a negative control. RSV infection led to a significant reduction in TEER and increase in permeability. Additionally it caused disruption of the AJC and remodeling of the apical actin cytoskeleton. Pretreatment with two structurally unrelated PKD inhibitors markedly attenuated RSV-induced effects. RSV induced phosphorylation of the actin binding protein cortactin in a PKD-dependent manner. UV-inactivated RSV had no effect on AJC function or structure. Our results suggest that RSV-induced airway epithelial barrier disruption involves PKD-dependent actin cytoskeletal remodeling, possibly dependent on cortactin activation. Defining the mechanisms by which RSV disrupts epithelial structure and function should enhance our understanding of the association between respiratory viral infections, airway inflammation, and allergen sensitization. Impaired barrier function may open a potential new therapeutic target for RSV-mediated lung diseases.     

Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene

Respiratory syncytial virus (RSV) infection is one of the major causes of respiratory tract infection for which no vaccine or antiviral treatment is available. The RSV NS1 protein seems to antagonize the host interferon (IFN) response; however, its mechanism is unknown. Here, we used a plasmid-borne small interfering RNA targeting the NS1 gene (siNS1) to examine the role of NS1 in modulating RSV infection. RSV replication was reduced in A549 cells, but not IFN-deficient Vero cells, transfected with siNS1. siNS1 induced upregulated expression of IFN-beta and IFN-inducible genes in A549 cells. siNS1-transfected human dendritic cells, upon RSV infection, produced elevated type-1 IFN and induced differentiation of naive CD4+ T cells to T helper type 1 (TH1) cells. Mice treated intranasally with siNS1 nanoparticles before or after infection with RSV showed substantially decreased virus titers in the lung and decreased inflammation and airway reactivity compared to controls. Thus, siNS1 nanoparticles may provide an effective inhibition of RSV infection in humans.     

Respiratory syncytial virus matrix protein induces lung epithelial cell cycle arrest through a p53 dependent pathway

Respiratory syncytial virus (RSV) is the major cause of viral respiratory infections in children. Our previous study showed that the RSV infection induced lung epithelial cell cycle arrest, which enhanced virus replication. To address the mechanism of RSV-induced cell cycle arrest, we examined the contribution of RSV-matrix (RSV-M) protein. In this report, we show that in both the A549 cell line and primary human bronchial epithelial (PHBE) cells, transfection with RSV-M protein caused the cells to proliferate at a slower rate than in control cells. The cell cycle analysis showed that RSV-M protein induced G1 phase arrest in A549 cells, and G1 and G2/M phase arrest in PHBE cells. Interestingly, RSV-M expression induced p53 and p21 accumulation and decreased phosphorylation of retinoblastoma protein (Rb). Further, induction of cell cycle arrest by RSV-M was not observed in a p53-deficient epithelial cell line (H1299). However, cell cycle arrest was restored after transfection of p53 cDNA into H1299 cells. Taken together, these results indicate that RSV-M protein regulates lung epithelial cell cycle through a p53-dependent pathway, which enhances RSV replication.