Nerve growth factor modulates human rhinovirus infection in airway epithelial cells by controlling ICAM-1 expression

Human rhinoviruses (HRV) are the most common agent of upper respiratory infections and an important cause of lower respiratory tract symptoms. Our previous research with other viral pathogens has shown that virus-induced airway inflammation and hyperreactivity involve neurotrophic pathways that also affect tropism and severity of the infection. The goals of this study were to analyze systematically the expression of key neurotrophic factors and receptors during HRV-16 infection of human airway epithelial cells and to test the hypothesis that neurotrophins modulate HRV infection by controlling the expression of a major cellular receptor for this virus, the intercellular adhesion molecule 1 (ICAM-1). Neurotrophins and ICAM-1 expression were analyzed at the mRNA level by real-time PCR and at the protein level by flow cytometry and immunocytochemistry. A small inhibitory RNA (siRNA) or a specific blocking antibody was utilized to suppress nerve growth factor (NGF) expression and measure its effects on viral replication and virus-induced cell death. Nasal and bronchial epithelial cells were most susceptible to HRV-16 infection at 33°C and 37°C, respectively, and a significant positive relationship was noted between expression of NGF and tropomyosin-related kinase A (TrkA) and virus copy number. ICAM-1 expression was dose dependently upregulated by exogenous NGF and significantly downregulated by NGF inhibition with corresponding decrease in HRV-16 replication. NGF inhibition also increased apoptotic death of infected cells. Our results suggest that HRV upregulates the NGF-TrkA pathway in airway epithelial cells, which in turn amplifies viral replication by increasing HRV entry via ICAM-1 receptors and by limiting apoptosis.     

Rhinovirus-induced MMP-9 expression is dependent on Fra-1, which is modulated by formoterol and dexamethasone

Matrix metalloproteinase-9 is implicated in airway inflammation and airway remodeling in asthma. We have previously confirmed that human rhinovirus-16 (HRV-16) infection increases MMP-9 expression both in vivo and in vitro. However, the role of the AP-1 sites within the MMP-9 promoter and the effect of commonly used asthma pharmacotherapies in modulating human rhinovirus (HRV)-induced MMP-9 production have not yet been elucidated. Experiments were performed in vitro in the human bronchial epithelial (HBE) cell line BEAS-2B and in primary HBE cells obtained from non-transplanted lungs. Using site-directed mutagenesis approaches, AP-1 sites were found to be necessary for HRV-induced MMP-9 promoter drive. EMSAs and supershift assays identified complexes consisting of Fos-related Ag-1 (Fra-1) in addition to other AP-1 subunits. Small interfering RNA approaches indicated that Fra-1 was induced upon HRV-16 infection in BEAS-2B cells and was necessary for MMP-9 expression in both BEAS-2B and primary HBE cells. Inhibition of MEK1/2 activity using PD98059 and U0126 reduced Fra-1 expression, DNA binding, MMP-9 promoter drive, and MMP-9 protein production. The long-acting β(2)-agonist formoterol and the glucocorticoid dexamethasone significantly reduced HRV-induced ERK phosphorylation, Fra-1, and MMP-9 expression in BEAS-2B cells. These data indicate that HRV-induced activation of the MEK/ERK MAPK pathway and Fra-1 expression are necessary for the upregulation of MMP-9 and can be modulated by two distinct but commonly used asthma pharmacotherapies. Together, these results offer insights into the mechanisms by which long-acting β(2)-agonists and glucocorticoids might reduce HRV-related asthma exacerbations.     

Cigarette smoke modulates expression of human rhinovirus-induced airway epithelial host defense genes

Human rhinovirus (HRV) infections trigger acute exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. The human airway epithelial cell is the primary site of HRV infection and responds to infection with altered expression of multiple genes, the products of which could regulate the outcome to infection. Cigarette smoking aggravates asthma symptoms, and is also the predominant risk factor for the development and progression of COPD. We, therefore, examined whether cigarette smoke extract (CSE) modulates viral responses by altering HRV-induced epithelial gene expression. Primary cultures of human bronchial epithelial cells were exposed to medium alone, CSE alone, purified HRV-16 alone or to HRV-16+ CSE. After 24 h, supernatants were collected and total cellular RNA was isolated. Gene array analysis was performed to examine mRNA expression. Additional experiments, using real-time RT-PCR, ELISA and/or western blotting, validated altered expression of selected gene products. CSE and HRV-16 each induced groups of genes that were largely independent of each other. When compared to gene expression in response to CSE alone, cells treated with HRV+CSE showed no obvious differences in CSE-induced gene expression. By contrast, compared to gene induction in response to HRV-16 alone, cells exposed to HRV+CSE showed marked suppression of expression of a number of HRV-induced genes associated with various functions, including antiviral defenses, inflammation, viral signaling and airway remodeling. These changes were not associated with altered expression of type I or type III interferons. Thus, CSE alters epithelial responses to HRV infection in a manner that may negatively impact antiviral and host defense outcomes.     

Trehalose-Mediated Autophagy Impairs the Anti-Viral Function of Human Primary Airway Epithelial Cells

Human rhinovirus (HRV) is the most common cause of acute exacerbations of chronic lung diseases including asthma. Impaired anti-viral IFN-λ1 production and increased HRV replication in human asthmatic airway epithelial cells may be one of the underlying mechanisms leading to asthma exacerbations. Increased autophagy has been shown in asthmatic airway epithelium, but the role of autophagy in anti-HRV response remains uncertain. Trehalose, a natural glucose disaccharide, has been recognized as an effective autophagy inducer in mammalian cells. In the current study, we used trehalose to induce autophagy in normal human primary airway epithelial cells in order to determine if autophagy directly regulates the anti-viral response against HRV. We found that trehalose-induced autophagy significantly impaired IFN-λ1 expression and increased HRV-16 load. Inhibition of autophagy via knockdown of autophagy-related gene 5 (ATG5) effectively rescued the impaired IFN-λ1 expression by trehalose and subsequently reduced HRV-16 load. Mechanistically, ATG5 protein interacted with retinoic acid-inducible gene I (RIG-I) and IFN-β promoter stimulator 1 (IPS-1), two critical molecules involved in the expression of anti-viral interferons. Our results suggest that induction of autophagy in human primary airway epithelial cells inhibits the anti-viral IFN-λ1 expression and facilitates HRV infection. Intervention of excessive autophagy in chronic lung diseases may provide a novel approach to attenuate viral infections and associated disease exacerbations.     

Nitric Oxide Inhibits Rhinovirus-Induced Cytokine Production and Viral Replication in a Human Respiratory Epithelial Cell Line

To better understand the early biochemical events that occur in human rhinovirus (HRV) infections, we examined the kinetics and mechanisms of interleukin-8 (IL-8) and IL-6 production from infected epithelial cells. Several HRV strains caused IL-8 and IL-6 production, but HRV-16 induced maximal IL-8 and IL-6 mRNA expression and protein production more rapidly than did HRV-14, despite similar rates of replication of the two viral strains. Viral induction of cytokine mRNA does not require new protein synthesis, since it was unaffected by cycloheximide treatment. The potent glucocorticoid budesonide did not affect viral replication or cytokine mRNA induction but modestly inhibited cytokine protein production. Interestingly, the nitric oxide donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (NONOate) inhibited both rhinovirus replication and cytokine production in a dose-dependent fashion without reducing levels of cytokine mRNA. The NONOate effects were due to release of nitric oxide, because NONOate that had been depleted of its nitric oxide content had no effect. Thus, nitric oxide may play an important anti-inflammatory and antiviral role in colds and nitric oxide donors may represent a novel therapeutic approach.     

A MicroRNA Network Dysregulated in Asthma Controls IL-6 Production in Bronchial Epithelial Cells

MicroRNAs are short non-coding single stranded RNAs that regulate gene expression. While much is known about the effects of individual microRNAs, there is now growing evidence that they can work in co-operative networks. MicroRNAs are known to be dysregulated in many diseases and affect pathways involved in the pathology. We investigated dysregulation of microRNA networks using asthma as the disease model. Asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness and airway remodelling. The airway epithelium is a major contributor to asthma pathology and has been shown to produce an excess of inflammatory and pro-remodelling cytokines such as TGF-β, IL-6 and IL-8 as well as deficient amounts of anti-viral interferons. After performing microRNA arrays, we found that microRNAs -18a, -27a, -128 and -155 are down-regulated in asthmatic bronchial epithelial cells, compared to cells from healthy donors. Interestingly, these microRNAs are predicted in silico to target several components of the TGF-β, IL-6, IL-8 and interferons pathways. Manipulation of the levels of individual microRNAs in bronchial epithelial cells did not have an effect on any of these pathways. Importantly, knock-down of the network of microRNAs miR-18a, -27a, -128 and -155 led to a significant increase of IL-8 and IL-6 expression. Interestingly, despite strong in silico predictions, down-regulation of the pool of microRNAs did not have an effect on the TGF-β and Interferon pathways. In conclusion, using both bioinformatics and experimental tools we found a highly relevant potential role for microRNA dysregulation in the control of IL-6 and IL-8 expression in asthma. Our results suggest that microRNAs may have different roles depending on the presence of other microRNAs. Thus, interpretation of in silico analysis of microRNA function should be confirmed experimentally in the relevant cellular context taking into account interactions with other microRNAs when studying disease.     

High Basal Expression of Interferon-Stimulated Genes in Human Bronchial Epithelial (BEAS-2B) Cells Contributes to Influenza A Virus Resistance

Respiratory epithelial cells play a key role in influenza A virus (IAV) pathogenesis and host innate response. Transformed human respiratory cell lines are widely used in the study of IAV−host interactions due to their relative convenience, and inherent difficulties in working with primary cells. Transformed cells, however, may have altered susceptibility to virus infection. Proper characterization of different respiratory cell types in their responses to IAV infection is therefore needed to ensure that the cell line chosen will provide results that are of relevance in vivo. We compared replication kinetics of human H1N1 (A/USSR/77) IAVs in normal primary human bronchial epithelial (NHBE) and two commonly used respiratory epithelial cell lines namely BEAS-2B and A549 cells. We found that IAV replication was distinctly poor in BEAS-2B cells in comparison with NHBE, A549 and Madin-Darby canine kidney (MDCK) cells. IAV resistance in BEAS-2B cells was accompanied by an activated antiviral state with high basal expression of interferon (IFN) regulatory factor-7 (IRF-7), stimulator of IFN genes (STING) and IFN stimulated genes (ISGs). Treatment of BEAS-2B cells with a pan-Janus-activated-kinase (JAK) inhibitor decreased IRF-7 and ISG expression and resulted in increased IAV replication. Therefore, the use of highly resistant BEAS-2B cells in IAV infection may not reflect the cytopathogenicity of IAV in human epithelial cells in vivo.     

MicroRNA-221 modulates RSV replication in human bronchial epithelium by targeting NGF expression

Background

Early-life infection by respiratory syncytial virus (RSV) is associated with aberrant expression of the prototypical neurotrophin nerve growth factor (NGF) and its cognate receptors in human bronchial epithelium. However, the chain of events leading to this outcome, and its functional implications for the progression of the viral infection, has not been elucidated. This study sought to test the hypothesis that RSV infection modulates neurotrophic pathways in human airways by silencing the expression of specific microRNAs (miRNAs), and that this effect favors viral growth by interfering with programmed death of infected cells.

Methodology

Human bronchial epithelial cells infected with green fluorescent protein-expressing RSV (rgRSV) were screened with multiplex qPCR arrays, and miRNAs significantly affected by the virus were analyzed for homology with mRNAs encoding neurotrophic factors or receptors. Mimic sequences of selected miRNAs were transfected into non-infected bronchial cells to confirm the role of each of them in regulating neurotrophins expression at the gene and protein level, and to study their influence on cell cycle and viral replication.

Principal Findings

RSV caused downregulation of 24 miRNAs and upregulation of 2 (p<0.01). Homology analysis of microarray data revealed that 6 of those miRNAs exhibited a high degree of complementarity to NGF and/or one of its cognate receptors TrKA and p75^NTR. Among the selected miRNAs, miR-221 was significantly downregulated by RSV and its transfection in bronchial epithelial cells maximally inhibited gene and protein expression of NGF and TrKA, increased apoptotic cell death, and reduced viral replication and infectivity.

Conclusions/Significance

Our data suggest that RSV upregulates the NGF-TrKA axis in human airways by silencing miR-221 expression, and this favors viral replication by interfering with the apoptotic death of infected cells. Consequently, the targeted delivery of exogenous miRNAs to the airways may provide a new strategy for future antiviral therapies based on RNA interference.     

Coordination between terminal variation of the viral genome and insect microRNAs regulates rice stripe virus replication in insect vectors

Maintenance of a balance between the levels of viral replication and selective pressure from the immune systems of insect vectors is one of the prerequisites for efficient transmission of insect-borne propagative phytoviruses. The mechanism regulating the adaptation of RNA viruses to insect vectors by genomic variation remains unknown. Our previous study demonstrated an extension of the 3’-untranslated terminal region (UTR) of two genomic segments of rice stripe virus (RSV). In the present study, a reverse genetic system for RSV in human cells and an insect vector, the small brown planthopper Laodelphax striatellus, was used to demonstrate that the 3’-terminal extensions suppressed viral replication in vector insects by inhibiting promoter activity due to structural interference with the panhandle structure formed by viral 3’- and 5’-UTRs. The extension sequence in the viral RNA1 segment was targeted by an endogenous insect microRNA, miR-263a, which decreased the inhibitory effect of the extension sequence on viral promoter activity. Surprisingly, the expression of miR-263a was negatively regulated by RSV infection. This elaborate coordination between terminal variation of the viral genome and endogenous insect microRNAs controls RSV replication in planthopper, thus reflecting a distinct strategy of adaptation of phytoviruses to insect vectors.     

Amino acid changes in proteins 2B and 3A mediate rhinovirus type 39 growth in mouse cells

Many steps of viral replication are dependent on the interaction of viral proteins with host cell components. To identify rhinovirus proteins involved in such interactions, human rhinovirus 39 (HRV39), a virus unable to replicate in mouse cells, was adapted to efficient growth in mouse cells producing the viral receptor ICAM-1 (ICAM-L cells). Amino acid changes were identified in the 2B and 3A proteins of the adapted virus, RV39/L. Changes in 2B were sufficient to permit viral growth in mouse cells; however, changes in both 2B and 3A were required for maximal viral RNA synthesis in mouse cells. Examination of infected HeLa cells by electron microscopy demonstrated that human rhinoviruses induced the formation of cytoplasmic membranous vesicles, similar to those observed in cells infected with other picornaviruses. Vesicles were also observed in the cytoplasm of HRV39-infected mouse cells despite the absence of viral RNA replication. Synthesis of picornaviral nonstructural proteins 2C, 2BC, and 3A is known to be required for formation of membranous vesicles. We suggest that productive HRV39 infection is blocked in ICAM-L cells at a step posttranslation and prior to the formation of a functional replication complex. The observation that changes in HRV39 2B and 3A proteins lead to viral growth in mouse cells suggests that one or both of these proteins interact with host cell proteins to promote viral replication.     

HepG2 cells expressing microRNA miR-122 support the entire hepatitis C virus life cycle

The liver-specific microRNA miR-122 is required for efficient hepatitis C virus (HCV) RNA replication both in cell culture and in vivo. In addition, nonhepatic cells have been rendered more efficient at supporting this stage of the HCV life cycle by miR-122 expression. This study investigated how miR-122 influences HCV replication in the miR-122-deficient HepG2 cell line. Expression of this microRNA in HepG2 cells permitted efficient HCV RNA replication and infectious virion production. When a missing HCV receptor is also expressed, these cells efficiently support viral entry and thus the entire HCV life cycle.     

Liver-specific microRNA miR-122 enhances the replication of hepatitis C virus in nonhepatic cells

The liver-specific microRNA miR-122 has been shown to be required for the replication of hepatitis C virus (HCV) in the hepatoma cell line Huh7. The aim of this study was to test if HCV replication can be modulated by exogenously expressed miR-122 in human embryonic kidney epithelial cells (HEK-293). Our results demonstrate that miR-122 enhances the colony formation efficiency of the HCV replicon and increases the steady-state level of HCV RNA in HEK-293 cells. Therefore, we conclude that although miR-122 is not absolutely required, it greatly enhances HCV replication in nonhepatic cells.     

Absence of DICER in Monocytes and Its Regulation by HIV-1

MicroRNAs (miRNAs) are a class of small RNA molecules that function to control gene expression and restrict viral replication in host cells. The production of miRNAs is believed to be dependent upon the DICER enzyme. Available evidence suggests that in T lymphocytes, HIV-1 can both suppress and co-opt the host''s miRNA pathway for its own benefit. In this study, we examined the state of miRNA production in monocytes and macrophages as well as the consequences of viral infection upon the production of miRNA. Monocytes in general express low amounts of miRNA-related proteins, and DICER in particular could not be detected until after monocytes were differentiated into macrophages. In the case where HIV-1 was present prior to differentiation, the expression of DICER was suppressed. MicroRNA chip results for RNA isolated from transfected and treated cells indicated that a drop in miRNA production coincided with DICER protein suppression in macrophages. We found that the expression of DICER in monocytes is restricted by miR-106a, but HIV-1 suppressed DICER expression via the viral gene Vpr. Additionally, analysis of miRNA expression in monocytes and macrophages revealed evidence that some miRNAs can be processed by both DICER and PIWIL4. Results presented here have implications for both the pathology of viral infections in macrophages and the biogenesis of miRNAs. First, HIV-1 suppresses the expression and function of DICER in macrophages via a previously unknown mechanism. Second, the presence of miRNAs in monocytes lacking DICER indicates that some miRNAs can be generated by proteins other than DICER.