Acute effects of parainfluenza virus on epithelial electrolyte transport

Parainfluenza viruses are important causes of respiratory disease in both children and adults. In particular, they are the major cause of the serious childhood illness croup (laryngotracheobronchitis). The infections produced by parainfluenza viruses are associated with the accumulation of ions and fluid in the respiratory tract. It is not known, however, whether this accumulation is because of a direct effect of the viruses on ion and fluid transport by the respiratory epithelium. Here we show that a model parainfluenza virus (the Sendai virus), in concentrations observed during respiratory infections, activates Cl- secretion and inhibits Na+ absorption across the tracheal epithelium. It does so by binding to a neuraminidase-insensitive glycolipid, possibly asialo-GM1, triggering the release of ATP, which then acts in an autocrine fashion on apical P2Y receptors to produce the observed changes in ion transport. These findings indicate that fluid accumulation in the respiratory tract associated with parainfluenza virus infection is attributable, at least in part, to direct effects of the virus on ion transport by the respiratory epithelium.     

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.     

The transmembrane domain of the respiratory syncytial virus F protein is an orientation-independent apical plasma membrane sorting sequence

The processes that facilitate transport of integral membrane proteins though the secretory pathway and subsequently target them to particular cellular membranes are relevant to almost every field of biology. These transport processes involve integration of proteins into the membrane of the endoplasmic reticulum (ER), passage from the ER to the Golgi, and post-Golgi trafficking. The respiratory syncytial virus (RSV) fusion (F) protein is a type I integral membrane protein that is uniformly distributed on the surface of infected nonpolarized cells and localizes to the apical plasma membrane of polarized epithelial cells. We expressed wild-type or altered RSV F proteins to gain a better understanding of secretory transport and plasma membrane targeting of type I membrane proteins in polarized and nonpolarized epithelial cells. Our findings reveal a novel, orientation-independent apical plasma membrane targeting function for the transmembrane domain of the RSV F protein in polarized epithelial cells. This work provides a basis for a more complete understanding of the role of the transmembrane domain and cytoplasmic tail of viral type I integral membrane proteins in secretory transport and plasma membrane targeting in polarized and nonpolarized cells.     

Cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells

Respiratory syncytial virus (RSV) is one of the major causes of respiratory infections in children, and it is the main pathogen causing bronchiolitis in infants. The binding and entry mechanism by which RSV infects respiratory epithelial cells has not yet been determined. In this study, the earliest stages of RSV infection in normal human bronchial epithelial cells were probed by tracking virions with fluorescent lipophilic dyes in their membranes. Virions colocalized with cholesterol-containing plasma membrane microdomains, identified by their ability to bind cholera toxin subunit B. Consistent with an important role for cholesterol in RSV infection, cholesterol depletion profoundly inhibited RSV infection, while cholesterol repletion reversed this inhibition. Merger of the outer leaflets of the viral envelope and the cell membrane appeared to be triggered at these sites. Using small-molecule inhibitors, RSV infection was found to be sensitive to Pak1 inhibition, suggesting the requirement of a subsequent step of cytoskeletal reorganization that could involve plasma membrane rearrangements or endocytosis. It appears that RSV entry depends on its ability to dock to cholesterol-rich microdomains (lipid rafts) in the plasma membrane where hemifusion events begin, assisted by a Pak1-dependent process.     

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.     

Respiratory syncytial virus induces TLR3 protein and protein kinase R, leading to increased double-stranded RNA responsiveness in airway epithelial cells

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells, causing bronchiolitis, upper respiratory infections, asthma exacerbations, chronic obstructive pulmonary disease exacerbations, and pneumonia in immunocompromised hosts. A replication intermediate of RSV is dsRNA. This is an important ligand for both the innate immune receptor, TLR3, and protein kinase R (PKR). One known effect of RSV infection is the increased responsiveness of airway epithelial cells to subsequent bacterial ligands (i.e., LPS). In this study, we examined a possible role for RSV infection in increasing amounts and responsiveness of another TLR, TLR3. These studies demonstrate that RSV infection of A549 and human tracheobronchial epithelial cells increases the amounts of TLR3 and PKR in a time-dependent manner. This leads to increased NF-kappaB activity and production of the inflammatory cytokine IL-8 following a later exposure to dsRNA. Importantly, TLR3 was not detected on the cell surface at baseline but was detected on the cell surface after RSV infection. The data demonstrate that RSV, via an effect on TLR3 and PKR, sensitizes airway epithelial cells to subsequent dsRNA exposure. These findings are consistent with the hypothesis that RSV infection sensitizes the airway epithelium to subsequent viral and bacterial exposures by up-regulating TLRs and increasing their membrane localization.     

Respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species- and cell type-dependent manner

Secondary bacterial infections often complicate respiratory viral infections, but the mechanisms whereby viruses predispose to bacterial disease are not completely understood. We determined the effects of infection with respiratory syncytial virus (RSV), human parainfluenza virus 3 (HPIV-3), and influenza virus on the abilities of nontypeable Haemophilus influenzae and Streptococcus pneumoniae to adhere to respiratory epithelial cells and how these viruses alter the expression of known receptors for these bacteria. All viruses enhanced bacterial adhesion to primary and immortalized cell lines. RSV and HPIV-3 infection increased the expression of several known receptors for pathogenic bacteria by primary bronchial epithelial cells and A549 cells but not by primary small airway epithelial cells. Influenza virus infection did not alter receptor expression. Paramyxoviruses augmented bacterial adherence to primary bronchial epithelial cells and immortalized cell lines by up-regulating eukaryotic cell receptors for these pathogens, whereas this mechanism was less significant in primary small airway epithelial cells and in influenza virus infections. Respiratory viruses promote bacterial adhesion to respiratory epithelial cells, a process that may increase bacterial colonization and contribute to disease. These studies highlight the distinct responses of different cell types to viral infection and the need to consider this variation when interpreting studies of the interactions between respiratory cells and viral pathogens.     

Intracellular electrolyte concentrations in the frog skin epithelium: Effect of vasopressin and dependence on the Na concentration in the bathing media

Summary The intracellular electrolyte concentrations of the frog skin epithelium have been determined in thin freeze-dried cryosections using the technique of electron microprobe analysis. Stimulation of the transepithelial Na transport by arginine vasopressin (AVP) resulted in a marked increase in the Na concentration and a reciprocal drop in the K concentration in all epithelial cell layers. The effects of AVP were cancelled by addition of amiloride. It is concluded from these results that the primary mechanism by which AVP stimulates transepithelial Na transport is an increase in the Na permeability of the apical membrane. However, also some evidence has been obtained for an additional stimulatory effect of AVP on the Na pump. In mitochondria-rich cells and in gland cells no significant concentration changes were detected, supporting the view that these cells do not share in transepithelial Na transport. Furthermore, the dependence of the intracellular electrolyte concentrations upon the Na concentration in the outer and inner bathing solution was evaluated. Both in control and AVP-stimulated skins the intracellular Na concentration showed saturation already at low external Na concentrations, indicating that the self-inhibition of transepithelial Na transport is due to a reduction of the permeability of the apical membrane. After lowering the Na concentration in the internal bath frequently a Na increase in the outermost and a drop in the deeper epithelial layers was observed. It is concluded that partial uncoupling of the transport syncytium occurs, which may explain the inhibition of the transepithelial Na transport and blunting of the AVP response under this condition.     

Chloride and potassium channel function in alveolar epithelial cells

Electrolyte transport across the adult alveolar epithelium plays an important role in maintaining a thin fluid layer along the apical surface of the alveolus that facilitates gas exchange across the epithelium. Most of the work published on the transport properties of alveolar epithelial cells has focused on the mechanisms and regulation of Na(+) transport and, in particular, the role of amiloride-sensitive Na(+) channels in the apical membrane and the Na(+)-K(+)-ATPase located in the basolateral membrane. Less is known about the identity and role of Cl(-) and K(+) channels in alveolar epithelial cells, but studies are revealing important functions for these channels in regulation of alveolar fluid volume and ionic composition. The purpose of this review is to examine previous work published on Cl(-) and K(+) channels in alveolar epithelial cells and to discuss the conclusions and speculations regarding their role in alveolar cell transport function.     

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.     

Transforming growth factor beta enhances respiratory syncytial virus replication and tumor necrosis factor alpha induction in human epithelial cells

Asthma is characterized as a chronic inflammatory disease associated with significant tissue remodeling. Patients with asthma are more susceptible to virus-induced exacerbation, which subsequently can lead to increased rates of hospitalization and mortality. While the most common cause of asthma-related deaths is respiratory viral infections, the underlying factors in the lung environment which render asthmatic subjects more susceptible to viral exacerbation are not yet identified. Since transforming growth factor beta (TGF-beta) is a critical cytokine for lung tissue remodeling and asthma phenotype, we have focused on the effects of TGF-beta on viral replication and virus-induced inflammation. Treatment of human epithelial cells with TGF-beta increased respiratory syncytial virus (RSV) replication by approximately fourfold. Tumor necrosis factor alpha (TNF-alpha) mRNA and protein expression were also significantly increased above levels with RSV infection alone. The increase in RSV replication and TNF-alpha expression after TGF-beta treatment was concomitant with an increase in virus-induced p38 mitogen-activated protein kinase activation. Our data reveal a novel effect for TGF-beta on RSV replication and provide a potential mechanism for the exaggerated inflammatory response observed in asthmatic subjects during respiratory viral infections.     

Bidirectional virus secretion and nonciliated cell tropism following Andes virus infection of primary airway epithelial cell cultures

Hantavirus pulmonary syndrome (HPS) is an acute disease resulting from infection with any one of a number of New World hantaviruses. HPS has a mortality rate of 40% and, unlike many other severe respiratory diseases, often occurs in young, healthy adults. Infection is usually initiated after inhalation of rodent excreta containing virus particles, but human-to-human transmission has been documented. Postmortem tissue samples show high levels of viral antigen within the respiratory endothelium, but it is not clear how the virus can traverse the respiratory epithelium in order to initiate infection in the microvasculature. We have utilized Andes virus infection of primary, differentiated airway epithelial cells to investigate the ability of the virus to interact with and cross the respiratory epithelium. Andes virus infects the Clara and goblet cell populations but not the ciliated cells, and this infection pattern corresponds to the expression of beta(3) integrin, the viral receptor. The virus can infect via the apical or basolateral membrane, and progeny virus particles are secreted bidirectionally. There is no obvious cytopathology associated with infection, and beta(3) integrins do not appear to be critical for respiratory epithelial cell monolayer integrity. Our data suggest that hantavirus infection of the respiratory epithelium may play an important role in the early or prodrome phase of disease as well as serving as a source of virus involved in transmission.     

Digging through the Obstruction: Insight into the Epithelial Cell Response to Respiratory Virus Infection in Patients with Cystic Fibrosis

Respiratory virus infections are common but generally self-limiting infections in healthy individuals. Although early clinical studies reported low detection rates, the development of molecular diagnostic techniques by PCR has led to an increased recognition that respiratory virus infections are associated with morbidity and acute exacerbations of chronic lung diseases, such as cystic fibrosis (CF). The airway epithelium is the first barrier encountered by respiratory viruses following inhalation and the primary site of respiratory viral replication. Here, we describe how the airway epithelial response to respiratory viral infections contributes to disease progression in patients with CF and other chronic lung diseases, including the role respiratory viral infections play in bacterial acquisition in the CF patient lung.     

Late activation of the Raf/MEK/ERK pathway is required for translocation of the respiratory syncytial virus F protein to the plasma membrane and efficient viral replication

Activation of the Raf/MEK/ERK cascade is required for efficient propagation of several RNA and DNA viruses, including human respiratory syncytial virus (RSV). In RSV infection, activation of the Raf/MEK/ERK cascade is biphasic. An early induction within minutes after infection is associated with viral attachment. Subsequently, a second activation occurs with, so far, unknown function in the viral life cycle. In this study, we aimed to characterise the role of Raf/MEK/ERK‐mediated signalling during ongoing RSV infection. Our data show that inhibition of the kinase MEK after the virus has been internalised results in a reduction of viral titers. Further functional investigations revealed that the late‐stage activation of ERK is required for a specific step in RSV replication, namely, the secretory transport of the RSV fusion protein F. Thus, MEK inhibition resulted in impaired surface accumulation of the F protein. F protein surface expression is essential for efficient replication as it is involved in viral filament formation, cell fusion, and viral transmission. In summary, we provide detailed insights of how host cell signalling interferes with RSV replication and identified the Raf/MEK/ERK kinase cascade as potential target for novel anti‐RSV strategies.     

Hsp90 Inhibitors Exhibit Resistance-Free Antiviral Activity against Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) is a major cause of respiratory illness in young children, leading to significant morbidity and mortality worldwide. Despite its medical importance, no vaccine or effective therapeutic interventions are currently available. Therefore, there is a pressing need to identify novel antiviral drugs to combat RSV infections. Hsp90, a cellular protein-folding factor, has been shown to play an important role in the replication of numerous viruses. We here demonstrate that RSV requires Hsp90 for replication. Mechanistic studies reveal that inhibition of Hsp90 during RSV infection leads to the degradation of a viral protein similar in size to the RSV L protein, the viral RNA-dependent RNA polymerase, implicating it as an Hsp90 client protein. Accordingly, Hsp90 inhibitors exhibit antiviral activity against laboratory and clinical isolates of RSV in both immortalized as well as primary differentiated airway epithelial cells. Interestingly, we find a high barrier to the emergence of drug resistance to Hsp90 inhibitors, as extensive growth of RSV under conditions of Hsp90 inhibition did not yield mutants with reduced sensitivity to these drugs. Our results suggest that Hsp90 inhibitors may present attractive antiviral therapeutics for treatment of RSV infections and highlight the potential of chaperone inhibitors as antivirals exhibiting high barriers to development of drug resistance.     

Integumental amino acid uptake in a carnivorous predator mollusc (Sepia officinalis, Cephalopoda)

The epithelial cells of the integument of body, arms and tentacles of Sepia officinalis present on their apical membrane a well-organised brush border and show the morphological and histochemical characteristics of a typical absorptive epithelium. The ability of the integument to absorb amino acids was investigated both in the arms incubated in vitro and in a purified preparation of brush border membrane vesicles (BBMV). Autoradiographic pictures of the integument after incubation of the arms in sea-water with or without sodium, showed that proline intake was Na+-dependent, whereas leucine intake appeared to be a largely cation-independent process. Time course experiments of labelled leucine, proline and lysine uptakes in BBMV evidenced that these amino acids are accumulated within the vesicles in the presence of an inwardly directed sodium gradient. The sodium-driven accumulation proves that cationic and neutral amino acids are taken up by the apical membrane of the epithelium of Sepia integument through a secondary active mechanism. For leucine, a 90% inhibition of the uptake was recorded in the presence of a large excess of the substrate. In agreement with the autoradiography results, an analysis of the cation specificity transport in BBMV showed that leucine uptake had a low cation specificity, whereas lysine and proline uptakes were Na+-dependent. An excess of lysine and proline, which share with alanine two different transport systems in the gill epithelium of marine bivalves, reduced eucine uptake. The possible role of the absorptive ability of the integument in a carnivorous mollusc is discussed.     

Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules

A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E- cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.     

Influenza virus inhibits ENaC and lung fluid clearance

Fluid-free alveolar space is critical for normal gas exchange. Influenza virus alters fluid transport across respiratory epithelia producing rhinorrhea, middle ear effusions, and alveolar flooding. However, the mechanism of fluid retention remains unclear. We investigated influenza virus strain A/PR/8/34, which can attach and enter mammalian cells but is incapable of viral replication and productive infection in mammalian epithelia, on epithelial sodium channels (ENaC) in rat alveolar type II (ATII) cells. In parallel, we determined the effects of virus on amiloride-sensitive (i.e., ENaC-mediated) fluid clearance in rat lungs in vivo. Although influenza virus did not change the inulin permeability of ATII monolayers, it rapidly reduced the net volume transport across monolayers. Virus reduced the open probability of single ENaC channels in apical cell-attached patches. U-73122, a phospholipase C (PLC) inhibitor, and PP2, a Src inhibitor, blocked the effect of virus on ENaC. GF-109203X, a protein kinase C (PKC) inhibitor, also blocked the effect, suggesting a PKC-mediated mechanism. In parallel, intratracheal administration of influenza virus produced a rapid inhibition of amiloride-sensitive (i.e., ENaC-dependent) lung fluid transport. Together, these results show that influenza virus rapidly inhibits ENaC in ATII cells via a PLC- and Src-mediated activation of PKC but does not increase epithelial permeability in this same rapid time course. We speculate that this rapid inhibition of ENaC and formation of edema when the virus first attaches to the alveolar epithelium might facilitate subsequent influenza infection and may exacerbate influenza-mediated alveolar flooding that can lead to acute respiratory failure and death.     

Direct Inhibition of Cellular Fatty Acid Synthase Impairs Replication of Respiratory Syncytial Virus and Other Respiratory Viruses

Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a fatty acid utilized for synthesis of more complex fatty acids, plasma membrane structure, and post-translational palmitoylation of host and viral proteins. We have developed a potent inhibitor of FASN (TVB-3166) that reduces the production of respiratory syncytial virus (RSV) progeny in vitro from infected human lung epithelial cells (A549) and in vivo from mice challenged intranasally with RSV. Addition of TVB-3166 to the culture medium of RSV-infected A549 cells reduces viral spread without inducing cytopathic effects. The antiviral effect of the FASN inhibitor is a direct consequence of reducing de novo palmitate synthesis; similar doses are required for both antiviral activity and inhibition of palmitate production, and the addition of exogenous palmitate to TVB-3166-treated cells restores RSV production. TVB-3166 has minimal effect on RSV entry but significantly reduces viral RNA replication, protein levels, viral particle formation and infectivity of released viral particles. TVB-3166 substantially impacts viral replication, reducing production of infectious progeny 250-fold. In vivo, oral administration of TVB-3166 to RSV-A (Long)-infected BALB/c mice on normal chow, starting either on the day of infection or one day post-infection, reduces RSV lung titers 21-fold and 9-fold respectively. Further, TVB-3166 also inhibits the production of RSV B, human parainfluenza 3 (PIV3), and human rhinovirus 16 (HRV16) progeny from A549, HEp2 and HeLa cells respectively. Thus, inhibition of FASN and palmitate synthesis by TVB-3166 significantly reduces RSV progeny both in vitro and in vivo and has broad-spectrum activity against other respiratory viruses. FASN inhibition may alter the composition of regions of the host cell membrane where RSV assembly or replication occurs, or change the membrane composition of RSV progeny particles, decreasing their infectivity.