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1.  RSV-encoded NS2 promotes epithelial cell shedding and distal airway obstruction 
The Journal of Clinical Investigation  2014;124(5):2219-2233.
Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in young children. The factors that contribute to the increased propensity of RSV-induced distal airway disease compared with other commonly encountered respiratory viruses remain unclear. Here, we identified the RSV-encoded nonstructural 2 (NS2) protein as a viral genetic determinant for initiating RSV-induced distal airway obstruction. Infection of human cartilaginous airway epithelium (HAE) and a hamster model of disease with recombinant respiratory viruses revealed that NS2 promotes shedding of infected epithelial cells, resulting in two consequences of virus infection. First, epithelial cell shedding accelerated the reduction of virus titers, presumably by clearing virus-infected cells from airway mucosa. Second, epithelial cells shedding into the narrow-diameter bronchiolar airway lumens resulted in rapid accumulation of detached, pleomorphic epithelial cells, leading to acute distal airway obstruction. Together, these data indicate that RSV infection of the airway epithelium, via the action of NS2, promotes epithelial cell shedding, which not only accelerates viral clearance but also contributes to acute obstruction of the distal airways. Our results identify RSV NS2 as a contributing factor for the enhanced propensity of RSV to cause severe airway disease in young children and suggest NS2 as a potential therapeutic target for reducing the severity of distal airway disease.
doi:10.1172/JCI72948
PMCID: PMC4001550  PMID: 24713657
2.  Effects of Vitamin D on Airway Epithelial Cell Morphology and Rhinovirus Replication 
PLoS ONE  2014;9(1):e86755.
Vitamin D has been linked to reduced risk of viral respiratory illness. We hypothesized that vitamin D could directly reduce rhinovirus (RV) replication in airway epithelium. Primary human bronchial epithelial cells (hBEC) were treated with vitamin D, and RV replication and gene expression were evaluated by quantitative PCR. Cytokine/chemokine secretion was measured by ELISA, and transepithelial resistance (TER) was determined using a voltohmmeter. Morphology was examined using immunohistochemistry. Vitamin D supplementation had no significant effects on RV replication, but potentiated secretion of CXCL8 and CXCL10 from infected or uninfected cells. Treatment with vitamin D in the form of 1,25(OH)2D caused significant changes in cell morphology, including thickening of the cell layers (median of 46.5 µm [35.0–69.0] vs. 30 µm [24.5–34.2], p<0.01) and proliferation of cytokeratin-5-expressing cells, as demonstrated by immunohistochemical analysis. Similar effects were seen for 25(OH)D. In addition to altering morphology, higher concentrations of vitamin D significantly upregulated small proline-rich protein (SPRR1β) expression (6.3 fold-induction, p<0.01), suggestive of squamous metaplasia. Vitamin D treatment of hBECs did not alter repair of mechanically induced wounds. Collectively, these findings indicate that vitamin D does not directly affect RV replication in airway epithelial cells, but can influence chemokine synthesis and alters the growth and differentiation of airway epithelial cells.
doi:10.1371/journal.pone.0086755
PMCID: PMC3901706  PMID: 24475177
3.  Molecular Organization of the Mucins and Glycocalyx Underlying Mucus Transport Over Mucosal Surfaces of the Airways 
Mucosal immunology  2012;6(2):379-392.
Mucus, with its burden of inspired particulates, and pathogens, is cleared from mucosal surfaces of the airways by cilia beating within the periciliary layer (PCL). The PCL is held to be ‘watery’ and free of mucus by thixotropic-like forces arising from beating cilia. With radii of gyration ~250 nm, however, polymeric mucins should reptate readily into the PCL, so we assessed the glycocalyx for barrier functions. The PCL stained negative for MUC5AC and MUC5B, but it was positive for keratan sulfate, a glycosaminoglycan commonly associated with glycoconjugates. Shotgun proteomics showed keratan sulfate-rich fractions from mucus containing abundant tethered mucins, MUC1, MUC4, and MUC16, but no proteoglycans. Immuno-histology by light and electron microscopy localized MUC1 to microvilli, MUC4 and MUC20 to cilia, and MUC16 to goblet cells. Electron and atomic force microscopy revealed molecular lengths of 190–1,500 nm for tethered mucins, and a finely textured glycocalyx matrix filling interciliary spaces. Adenoviral particles were excluded from glycocalyx of the microvilli, while the smaller AAV penetrated, but were trapped within. Hence, tethered mucins organized as a space-filling glycocalyx function as a selective barrier for the PCL, broadening their role in innate lung defense and offering new molecular targets for conventional and gene therapies.
doi:10.1038/mi.2012.81
PMCID: PMC3637662  PMID: 22929560
lung; airways epithelium; mucosal innate immunity; mucus; mucin; mucociliary clearance
4.  Human Parainfluenza Virus Serotypes Differ in Their Kinetics of Replication and Cytokine Secretion in Human Tracheobronchial Airway Epithelium 
Virology  2012;433(2):320-328.
Human parainfluenza viruses (PIVs) cause acute respiratory illness in children, the elderly, and immunocompromised patients. PIV3 is a common cause of bronchiolitis and pneumonia, whereas PIV1 and 2 are frequent causes of upper respiratory tract illness and croup. To assess how PIV1, 2, and 3 differ with regard to replication and induction of type I interferons, interleukin-6, and relevant chemokines, we infected primary human airway epithelium (HAE) cultures from the same tissue donors and examined replication kinetics and cytokine secretion. PIV1 replicated to high titer yet did not induce cytokine secretion until late in infection, while PIV2 replicated less efficiently but induced an early cytokine peak. PIV3 replicated to high titer but induced a slower rise in cytokine secretion. The T cell chemoattractants CXCL10 and CXCL11 were the most abundant chemokines induced. Differences in replication and cytokine secretion might explain some of the differences in PIV serotype-specific pathogenesis and epidemiology.
doi:10.1016/j.virol.2012.08.027
PMCID: PMC3469718  PMID: 22959894
Human parainfluenza virus; Human airway epithelium; cytokines; chemokines; interferon; pathogenesis
5.  Comparison of Differing Cytopathic Effects in Human Airway Epithelium of Parainfluenza Virus 5 (W3A), Parainfluenza Virus Type 3, and Respiratory Syncytial Virus 
Virology  2011;421(1):67-77.
Parainfluenza virus 5 (PIV5) infects a wide range of animals including dogs, pigs, cats, and humans; however, its association with disease in humans remains controversial. In contrast to parainfluenza virus 3 (PIV3) or respiratory syncytial virus (RSV), PIV5 is remarkably non-cytopathic in monolayer cultures of immortalized epithelial cells. To compare the cytopathology produced by these viruses in a relevant human tissue, we infected an in vitro model of human ciliated airway epithelium and measured outcomes of cytopathology. PIV5, PIV3 and, RSV all infected ciliated cells, and PIV5 and PIV3 infection was dependent on sialic acid residues. Only PIV5-infected cells formed syncytia. PIV5 infection resulted in a more rapid loss of infected cells by shedding of infected cells into the lumen. These studies revealed striking differences in cytopathology of PIV5 versus PIV3 or RSV and indicate the extent of cytopathology determined in cell-lines does not predict events in differentiated airway cells.
doi:10.1016/j.virol.2011.08.020
PMCID: PMC3208758  PMID: 21986028
Parainfluenza virus; respiratory syncytial virus; airway epithelium; cytopathic effect; viral pathogenesis; syncytia; ciliated cell shedding; viral persistence; multi-potent progenitor cells; 3-dimensional (3-D) image reconstruction
6.  Limited Effects of Muc1 Deficiency on Mouse Adenovirus Type 1 Respiratory Infection 
Virus research  2011;160(1-2):351-359.
Muc1 (MUC1 in humans) is a membrane-tethered mucin that exerts anti-inflammatory effects in the lung during bacterial infection. Muc1 and other mucins are also likely to form a protective barrier in the lung. We used mouse adenovirus type 1 (MAV-1, also known as MAdV-1) to determine the role of Muc1 in the pathogenesis of an adenovirus in its natural host. Following intranasal inoculation of wild type mice, we detected increased TNF-α, a cytokine linked to Muc1 production, but no consistent changes in the production of lung Muc1, Muc5ac or overall lung mucus production. Viral loads were modestly higher in the lungs of Muc1−/− mice compared to Muc1+/+ mice at several early time points but decreased to similar levels by 14 days post infection in both groups. However, cellular inflammation and the expression of CXCL1, CCL5, and CCL2 did not significantly differ between Muc1−/− and Muc1+/+ mice. Our data therefore suggest that Muc1 may contribute to a physical barrier that protects against MAV-1 respiratory infection. However, our data do not reveal an anti-inflammatory effect of Muc1 that contributes to MAV-1 pathogenesis..
doi:10.1016/j.virusres.2011.07.012
PMCID: PMC3163747  PMID: 21816184
mucin; Muc1; adenovirus
7.  AAV-6 mediated efficient transduction of mouse lower airways 
Virology  2011;417(2):327-333.
AAV1 and AAV6 are two closely related AAV serotypes. In the present study, we found AAV6 was more efficient in transducing mouse lower airway epithelia in vitro and in vivo than AAV1. To further explore the mechanism of this difference, we found that significantly more AAV1 bound to mouse airway epithelia than AAV6, yet transduction by AAV6 was far superior. Lectin competition assays demonstrated that both AAV1 and AAV6 similarly utilize α-2, 3-, and to a lesser extend α-2, 6- linked sialic acids as the receptors for transduction. Furthermore, the rates of AAV endocytosis could not account for the transduction differences of AAV1 and AAV6. Finally, it was revealed that AAV6 was less susceptible to ubiquitin/proteasome-mediated blocks than AAV1 when transducing mouse airway epithelia. Thus compared with AAV1, AAV6 has a unique ability to escape proteasome-mediated degradation, which is likely responsible for its higher transduction efficiency in mouse airway epithelium.
doi:10.1016/j.virol.2011.06.009
PMCID: PMC3163804  PMID: 21752418
8.  Coupled Nucleotide and Mucin Hypersecretion from Goblet-Cell Metaplastic Human Airway Epithelium 
Adenosine triphosphate (ATP) and its metabolite adenosine regulate airway mucociliary clearance via activation of purinoceptors. In this study, we investigated the contribution of goblet cells to airway epithelial ATP release. Primary human bronchial epithelial (HBE) cultures, typically dominated by ciliated cells, were induced to develop goblet cell metaplasia by infection with respiratory syncytial virus (RSV) or treatment with IL-13. Under resting conditions, goblet-cell metaplastic cultures displayed enhanced mucin secretion accompanied by increased rates of ATP release and mucosal surface adenosine accumulation as compared with nonmetaplastic control HBE cultures. Intracellular calcium chelation [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester] or disruption of the secretory pathways (nocodazole, brefeldin A, and N-ethylmaleimide) decreased mucin secretion and ATP release in goblet-cell metaplastic HBE cultures. Conversely, stimuli that triggered calcium-regulated mucin secretion (e.g., ionomycin or UTP) increased luminal ATP release and adenyl purine accumulation in control and goblet-cell metaplastic HBE cultures. Goblet cell–associated ATP release was not blocked by the connexin/pannexin hemichannel inhibitor carbenoxolone, suggesting direct nucleotide release from goblet cell vesicles rather than the hemichannel insertion. Collectively, our data demonstrate that nucleotide release is increased by goblet cell metaplasia, reflecting, at least in part, a mechanism tightly associated with goblet cell mucin secretion. Increased goblet cell nucleotide release and resultant adenosine accumulation provide compensatory mechanisms to hydrate mucins by paracrine stimulation of ciliated cell ion and water secretion and maintain mucociliary clearance, and to modulate inflammatory responses.
doi:10.1165/rcmb.2010-0253OC
PMCID: PMC3175555  PMID: 20935191
goblet cell metaplasia; ATP release; mucin; airway epithelia; RSV
9.  NLRX1 protein attenuates inflammatory responses to virus infection by interfering with the RIG-I-MAVS signaling pathway and TRAF6 ubiquitin ligase 
Immunity  2011;34(6):854-865.
SUMMARY
The nucleotide-binding domain and leucine-rich repeat containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed Nlrx1−/− mice exhibited increased expression of antiviral signaling molecules IFN-β, STAT2, OAS1 and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1−/− mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS-activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.
doi:10.1016/j.immuni.2011.03.026
PMCID: PMC3166771  PMID: 21703540
10.  α-Fetoprotein Gene Delivery to the Nasal Epithelium of Nonhuman Primates by Human Parainfluenza Viral Vectors 
Human Gene Therapy  2010;21(12):1657-1664.
Zhang and colleagues examine the efficacy of a replication-competent parainfluenza virus (PIV)-based vector for airway gene transfer applications. Using an in vitro model of rhesus airway epithelium, the authors demonstrate that PIV mediates efficient gene transfer in rhesus epithelium. In vivo experiments revealed that intranasal administration of a PIV vector expressing rhesus macaque α-fetoprotein (rhAFP) results in the transient secretion of rhAFP in both mucosal and serosal compartments.
Abstract
Over the last two decades, enormous effort has been focused on developing virus-based gene delivery vectors to target the respiratory airway epithelium as a potential treatment for cystic fibrosis (CF) lung disease. However, amongst other problems, the efficiency of gene delivery to the differentiated airway epithelial cells of the lung has been too low for clinical benefit. Although not a target for CF therapy, the nasal epithelium exhibits cellular morphology and composition similar to that of the lower airways, thus representing an accessible and relevant tissue target for evaluating novel and improved gene delivery vectors. We previously reported that replication-competent human parainfluenza virus (PIV)-based vectors efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to sufficient numbers of cultured CF airway epithelial cells to completely correct the bioelectric function of CF cells to normal levels, resulting in restoration of mucus transport. Here, using an in vitro model of rhesus airway epithelium, we demonstrate that PIV mediates efficient gene transfer in rhesus epithelium as in the human counterpart. Naive rhesus macaques were inoculated intranasally with a PIV vector expressing rhesus macaque α-fetoprotein (rhAFP), and expression was monitored longitudinally. rhAFP was detected in nasal lavage fluid and in serum samples, indicating that PIV-mediated gene transfer was effective and that rhAFP was secreted into both mucosal and serosal compartments. Although expression was transient, lasting up to 10 days, it paralleled virus replication, suggesting that as PIV was cleared, rhAFP expression was lost. No adverse reactions or signs of discomfort were noted, and only mild, transient elevations of a small number of inflammatory cytokines were measured at the peak of virus replication. In summary, rhAFP proved suitable for monitoring in vivo gene delivery over time, and PIV vectors appear to be promising airway-specific gene transfer vehicles that warrant further development.
doi:10.1089/hum.2010.065
PMCID: PMC2999572  PMID: 20735256
11.  Infection of human mucosal tissue by Pseudomonas aeruginosa requires sequential and mutually dependent virulence factors and a novel pilus-associated adhesin 
Cellular microbiology  2010;12(8):1158-1173.
Summary
Tissue damage predisposes humans to life-threatening disseminating infection by the opportunistic pathogen Pseudomonas aeruginosa. Bacterial adherence to host tissue is a critical first step in this infection process. It is well established that P. aeruginosa attachment to host cells involves type IV pili (TFP), which are retractile surface fibers. The molecular details of attachment and the identity of the bacterial adhesin and host receptor remain controversial. Using a mucosal epithelium model system derived from primary human tissue, we show that the pilus-associated protein PilY1 is required for bacterial adherence. We establish that P. aeruginosa preferentially binds to exposed basolateral host cell surfaces, providing a mechanistic explanation for opportunistic infection of damaged tissue. Further, we demonstrate that invasion and fulminant infection of intact host tissue requires the coordinated and mutually dependent action of multiple bacterial factors, including pilus fiber retraction and the host cell intoxication system, termed type III secretion. Our findings offer new and important insights into the complex interactions between a pathogen and its human host and provide compelling evidence that PilY1 serves as the principal P. aeruginosa adhesin for human tissue and that it specifically recognizes a host receptor localized or enriched on basolateral epithelial cell surfaces.
doi:10.1111/j.1462-5822.2010.01461.x
PMCID: PMC2906647  PMID: 20331639
12.  AAV Exploits Subcellular Stress Associated with Inflammation, Endoplasmic Reticulum Expansion, and Misfolded Proteins in Models of Cystic Fibrosis 
PLoS Pathogens  2011;7(5):e1002053.
Barriers to infection act at multiple levels to prevent viruses, bacteria, and parasites from commandeering host cells for their own purposes. An intriguing hypothesis is that if a cell experiences stress, such as that elicited by inflammation, endoplasmic reticulum (ER) expansion, or misfolded proteins, then subcellular barriers will be less effective at preventing viral infection. Here we have used models of cystic fibrosis (CF) to test whether subcellular stress increases susceptibility to adeno-associated virus (AAV) infection. In human airway epithelium cultured at an air/liquid interface, physiological conditions of subcellular stress and ER expansion were mimicked using supernatant from mucopurulent material derived from CF lungs. Using this inflammatory stimulus to recapitulate stress found in diseased airways, we demonstrated that AAV infection was significantly enhanced. Since over 90% of CF cases are associated with a misfolded variant of Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR), we then explored whether the presence of misfolded proteins could independently increase susceptibility to AAV infection. In these models, AAV was an order of magnitude more efficient at transducing cells expressing ΔF508-CFTR than in cells expressing wild-type CFTR. Rescue of misfolded ΔF508-CFTR under low temperature conditions restored viral transduction efficiency to that demonstrated in controls, suggesting effects related to protein misfolding were responsible for increasing susceptibility to infection. By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. The presence of misfolded proteins did not affect cell surface attachment of virus or influence expression levels from promoter transgene cassettes in plasmid transfection studies, indicating exploitation occurs at the level of virion trafficking or processing. Thus, we surmised that factors enlisted to process misfolded proteins such as ΔF508-CFTR in the secretory pathway also act to restrict viral infection. In line with this hypothesis, we found that AAV trafficked to the microtubule organizing center and localized near Golgi/ER transport proteins. Moreover, AAV infection efficiency could be modulated with siRNA-mediated knockdown of proteins involved in processing ΔF508-CFTR or sorting retrograde cargo from the Golgi and ER (calnexin, KDEL-R, β-COP, and PSMB3). In summary, our data support a model where AAV exploits a compromised secretory system and, importantly, underscore the gravity with which a stressed subcellular environment, under internal or external insults, can impact infection efficiency.
Author Summary
Misfolded proteins have been associated with a variety of disorders such as cystic fibrosis, diabetes insipidus, alpha-antitrypsin deficiency, Parkinson's disease, and cancer. In this study, by using cellular models of events in cystic fibrosis lung disease we have revealed an effect of misfolded proteins on increasing susceptibility to infection with a parvovirus. Infection efficiency was an order of magnitude higher in cells expressing misfolded Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutant proteins than in cells expressing the correctly folded protein. During infection, virus capsids accumulated near cellular factors that normally process misfolded proteins and are involved in retrograde trafficking from the Golgi to endoplasmic reticulum. Furthermore, we have demonstrated that infection efficiency can be attenuated by restoring correct protein folding or augmented by siRNA-mediated knockdown of secretory pathway components. Taken together our results indicate that converging cellular systems operate to clear misfolded proteins and virus capsids from an infected cell. We raise the possibility that parvoviruses and perhaps other viruses exploit congested cellular secretory pathways during entry, and that viral infection could be a contributing factor in the progression of diseases associated with misfolded proteins.
doi:10.1371/journal.ppat.1002053
PMCID: PMC3098238  PMID: 21625534
13.  Growth restriction of an experimental live attenuated human parainfluenza virus type 2 vaccine in human ciliated airway epithelium in vitro parallels attenuation in African green monkeys 
Vaccine  2010;28(15):2788-2798.
Human parainfluenza viruses (HPIVs) are common causes of severe pediatric respiratory viral disease. We characterized wild-type HPIV2 infection in an in vitro model of human airway epithelium (HAE) and found that the virus replicates to high titer, sheds apically, targets ciliated cells, and induces minimal cytopathology. Replication of an experimental, live attenuated HPIV2 vaccine strain, containing both temperature sensitive (ts) and non-ts attenuating mutations, was restricted >30-fold compared to rHPIV2-WT in HAE at 32°C and exhibited little productive replication at 37°C. This restriction paralleled attenuation in the upper and lower respiratory tract of African green monkeys, supporting the HAE model as an appropriate and convenient system for characterizing HPIV2 vaccine candidates.
doi:10.1016/j.vaccine.2010.01.050
PMCID: PMC2844349  PMID: 20139039
Human parainfluenza virus; Live attenuated vaccine candidate; Human ciliated airway epithelium
14.  Human parainfluenza virus type 2 V protein inhibits interferon production and signaling and is required for replication in non-human primates 
Virology  2009;397(2):285.
In wild-type human parainfluenza virus type 2 (WT HPIV2), one gene (the P/V gene) encodes both the polymerase-associated phosphoprotein (P) and the accessory V protein. We generated a HPIV2 virus (rHPIV2-Vko) in which the P/V gene encodes only the P protein to examine the role of V in replication in vivo and as a potential live attenuated virus vaccine. Preventing expression of V protein severely impaired virus recovery from cDNA and growth in vitro, particularly in IFN-competent cells. rHPIV2-Vko, unlike WT HPIV2, strongly induced IFN-β and permitted IFN signaling, leading to establishment of a robust antiviral state. rHPIV2-Vko infection induced extensive syncytia and cytopathicity that was due to both apoptosis and necrosis. Replication of rHPIV2-Vko was highly restricted in the respiratory tract of African green monkeys and in differentiated primary human airway epithelial (HAE) cultures, suggesting that V protein is essential for efficient replication of HPIV2 in organized epithelial cells and that rHPIV2-Vko is over-attenuated for use as a live attenuated vaccine.
doi:10.1016/j.virol.2009.11.018
PMCID: PMC2822077  PMID: 19969320
15.  Respiratory Syncytial Virus Engineered To Express the Cystic Fibrosis Transmembrane Conductance Regulator Corrects the Bioelectric Phenotype of Human Cystic Fibrosis Airway Epithelium In Vitro▿  
Journal of Virology  2010;84(15):7770-7781.
Cystic fibrosis (CF) is the most common lethal recessive genetic disease in the Caucasian population. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that is normally expressed in ciliated airway epithelial cells and the submucosal glands of the lung. Since the CFTR gene was first characterized in 1989, a major goal has been to develop an effective gene therapy for CF lung disease, which has the potential to ameliorate morbidity and mortality. Respiratory syncytial virus (RSV) naturally infects the ciliated cells in the human airway epithelium. In addition, the immune response mounted against an RSV infection does not prevent subsequent infections, suggesting that an RSV-based vector might be effectively readministered. To test whether the large 4.5-kb CFTR gene could be expressed by a recombinant RSV and whether infectious virus could be used to deliver CFTR to ciliated airway epithelium derived from CF patients, we inserted the CFTR gene into four sites in a recombinant green fluorescent protein-expressing RSV (rgRSV) genome to generate virus expressing four different levels of CFTR protein. Two of these four rgRSV-CFTR vectors were capable of expressing CFTR with little effect on viral replication. rgRSV-CFTR infection of primary human airway epithelial cultures derived from CF patients resulted in expression of CFTR protein that was properly localized at the luminal surface and corrected the chloride ion channel defect in these cells.
doi:10.1128/JVI.00346-10
PMCID: PMC2897634  PMID: 20504917
16.  Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium 
Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium, we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants but not the parental AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.
doi:10.1038/mt.2009.155
PMCID: PMC2801879  PMID: 19603002
17.  Receptor Binding Profiles of Avian Influenza Virus Hemagglutinin Subtypes on Human Cells as a Predictor of Pandemic Potential ▿ ‖  
Journal of Virology  2010;85(4):1875-1880.
The host adaptation of influenza virus is partly dependent on the sialic acid (SA) isoform bound by the viral hemagglutinin (HA). Avian influenza viruses preferentially bind the α-2,3 SA and human influenza viruses the α-2,6 isoform. Each isoform is predominantly associated with different surface epithelial cell types of the human upper airway. Using recombinant HAs and human tracheal airway epithelial cells in vitro and ex vivo, we show that many avian HA subtypes do not adhere to this canonical view of SA specificity. The propensity of avian viruses to adapt to human receptors may thus be more widespread than previously supposed.
doi:10.1128/JVI.01822-10
PMCID: PMC3028872  PMID: 21106732
18.  Normal and Cystic Fibrosis Airway Surface Liquid Homeostasis 
The Journal of biological chemistry  2005;280(42):35751-35759.
Mammalian airways normally regulate the volume of a thin liquid layer, the periciliary liquid (PCL), to facilitate the mucus clearance component of lung defense. Studies under standard (static) culture conditions revealed that normal airway epithelia possess an adenosine-regulated pathway that blends Na+ absorption and Cl− secretion to optimize PCL volume. In cystic fibrosis (CF), the absence of CF transmembrane conductance regulator results in a failure of adenosine regulation of PCL volume, which is predicted to initiate mucus stasis and infection. However, under conditions that mimic the phasic motion of the lung in vivo, ATP release into PCL was increased, CF ion transport was rebalanced, and PCL volume was restored to levels adequate for lung defense. This ATP signaling system was vulnerable, however, to insults that trigger CF bacterial infections, such as viral (respiratory syncitial virus) infections, which up-regulated extracellular ATPase activity and abolished motion-dependent ATP regulation of CF PCL height. These studies demonstrate (i) how the normal coordination of opposing ion transport pathways to maintain PCL volume is disrupted in CF, (ii) the hitherto unknown role of phasic motion in regulating key aspects of normal and CF innate airways defense, and (iii) that maneuvers directed at increasing motion-induced nucleotide release may be therapeutic in CF patients.
doi:10.1074/jbc.M505832200
PMCID: PMC2924153  PMID: 16087672
19.  The NLRP3 Inflammasome Mediates in vivo Innate Immunity to Influenza A Virus through Recognition of Viral RNA 
Immunity  2009;30(4):556-565.
SUMMARY
NLR genes mediate host immunity to various pathogenic stimuli. However, in vivo evidence for NLR involvement in viral sensing has not been widely investigated and remains controversial. As an ultimate test of the physiologic role of NLRP3 during RNA viral infection, this work explores the in vivo role of NLRP3 inflammasome components during influenza virus infection. Mice lacking Nlrp3, ASC, or Caspase-1, but not Nlrc4, exhibit dramatically increased mortality but reduced immune response following influenza virus exposure. Utilizing analogs of dsRNA (poly(I:C)) and ssRNA (ssRNA40), we demonstrate that NLRP3-mediated response can be activated by RNA species. Mechanistically, NLRP3 inflammasome activation by influenza virus is dependent upon lysosomal maturation and reactive oxygen species. Inhibition of ROS induction eliminated IL-1β production in animals during influenza infection. Together, these data place the NLRP3 inflammasome as an essential component in host defense against influenza infection through the sensing of viral RNA.
doi:10.1016/j.immuni.2009.02.005
PMCID: PMC2803103  PMID: 19362020
Cryopyrin; ASC; IPAF; NLR; NALP3; TUCAN; Caspase-1; IL-1β
20.  Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated Attachment Protein That Alters Its Infectivity and Dependence on Glycosaminoglycans ▿  
Journal of Virology  2009;83(20):10710-10718.
Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.
doi:10.1128/JVI.00986-09
PMCID: PMC2753119  PMID: 19656891
21.  Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge 
Virology  2008;383(2):348-361.
We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/ΔF-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/ΔF-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface, the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/ΔF-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV.
doi:10.1016/j.virol.2008.09.030
PMCID: PMC2649782  PMID: 19010509
22.  Tracheobronchial air-liquid interface cell culture: a model for innate mucosal defense of the upper airways? 
Human tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces “mucus” with a protein composition similar to that of in vivo, induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.
doi:10.1152/ajplung.90388.2008
PMCID: PMC2636953  PMID: 18931053
mucus; mucin; innate immunity; proteomics; human tracheobronchial epithelial cell culture
23.  Mutations in H5N1 Influenza Virus Hemagglutinin that Confer Binding to Human Tracheal Airway Epithelium 
PLoS ONE  2009;4(11):e7836.
The emergence in 2009 of a swine-origin H1N1 influenza virus as the first pandemic of the 21st Century is a timely reminder of the international public health impact of influenza viruses, even those associated with mild disease. The widespread distribution of highly pathogenic H5N1 influenza virus in the avian population has spawned concern that it may give rise to a human influenza pandemic. The mortality rate associated with occasional human infection by H5N1 virus approximates 60%, suggesting that an H5N1 pandemic would be devastating to global health and economy. To date, the H5N1 virus has not acquired the propensity to transmit efficiently between humans. The reasons behind this are unclear, especially given the high mutation rate associated with influenza virus replication. Here we used a panel of recombinant H5 hemagglutinin (HA) variants to demonstrate the potential for H5 HA to bind human airway epithelium, the predominant target tissue for influenza virus infection and spread. While parental H5 HA exhibited limited binding to human tracheal epithelium, introduction of selected mutations converted the binding profile to that of a current human influenza strain HA. Strikingly, these amino-acid changes required multiple simultaneous mutations in the genomes of naturally occurring H5 isolates. Moreover, H5 HAs bearing intermediate sequences failed to bind airway tissues and likely represent mutations that are an evolutionary “dead end.” We conclude that, although genetic changes that adapt H5 to human airways can be demonstrated, they may not readily arise during natural virus replication. This genetic barrier limits the likelihood that current H5 viruses will originate a human pandemic.
doi:10.1371/journal.pone.0007836
PMCID: PMC2775162  PMID: 19924306
24.  CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium 
PLoS Biology  2009;7(7):e1000155.
Delivering CFTR to ciliated cells of cystic fibrosis (CF) patients fully restores ion and fluid transport to the lumenal surface of airway epithelium and returns mucus transport rates to those of non-CF airways.
Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl− and Na+ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.
Author Summary
The ciliated epithelium that lines the conducting airways of the lung normally functions to transport hydrated mucus secretions out of the airways to maintain respiratory sterility. Cystic fibrosis (CF) lung disease results from reduced airway surface hydration leading to decreased mucus clearance that precipitates bacterial infection and progressive obstructive lung disease. CF is a genetic disease, and the mutant protein is a chloride ion channel (CFTR) that normally regulates ion and fluid transport on the airway surface. Restoration of corrected CFTR function to the airway epithelium of CF patients by delivering a new CFTR gene to airway epithelial cells has long been envisioned as a therapeutic strategy for CF lung disease. Towards this goal, we use a novel viral vector to deliver CFTR to a culture model that represents the ciliated airway epithelium of CF patients and show that this strategy restores airway surface hydration and mucus transport to levels of that in non-CF individuals. This study demonstrates efficient and efficacious CFTR delivery to CF ciliated airway epithelium and that CFTR delivered to approximately 25% of the surface epithelial cells restores normal levels of airway surface hydration and mucus transport. These studies serve as a benchmark for the efficiency of CFTR gene delivery to CF airways for future CF gene therapy studies in vivo.
doi:10.1371/journal.pbio.1000155
PMCID: PMC2705187  PMID: 19621064
25.  Systematic Assembly of a Full-Length Infectious Clone of Human Coronavirus NL63▿ †  
Journal of Virology  2008;82(23):11948-11957.
Historically, coronaviruses were predominantly associated with mild upper respiratory disease in humans. More recently, three novel coronaviruses associated with severe human respiratory disease were found, including (i) the severe acute respiratory syndrome coronavirus, associated with a significant atypical pneumonia and 10% mortality; (ii) HKU-1, associated with chronic pulmonary disease; and (iii) NL63, associated with both upper and lower respiratory tract disease in children and adults worldwide. These discoveries establish coronaviruses as important human pathogens and underscore the need for continued research toward the development of platforms that will enable genetic manipulation of the viral genome, allowing for rapid and rational development and testing of candidate vaccines, vaccine vectors, and therapeutics. In this report, we describe a reverse genetics system for NL63, whereby five contiguous cDNAs that span the entire genome were used to generate a full-length cDNA. Recombinant NL63 viruses which contained the expected marker mutations replicated as efficiently as the wild-type NL63 virus. In addition, we engineered the heterologous green fluorescent protein gene in place of open reading frame 3 (ORF3) of the NL63 clone, simultaneously creating a unique marker for NL63 infection and demonstrating that the ORF3 protein product is nonessential for the replication of NL63 in cell culture. The availability of the NL63 and NL63gfp clones and recombinant viruses provides powerful tools that will help advance our understanding of this important human pathogen.
doi:10.1128/JVI.01804-08
PMCID: PMC2583659  PMID: 18818320

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