scholarly journals Distinct gene subsets are induced at different time points after human respiratory syncytial virus infection of A549 cells

2007 ◽  
Vol 88 (2) ◽  
pp. 570-581 ◽  
Author(s):  
Isidoro Martínez ◽  
Luis Lombardía ◽  
Blanca García-Barreno ◽  
Orlando Domínguez ◽  
José A. Melero
Keyword(s):  
Respiratory Syncytial Virus ◽  
Time Course ◽  
A549 Cells ◽  
Cellular Protein ◽  
Human Respiratory Syncytial Virus ◽  
Virus Infectivity ◽  
Dependent Manner ◽  
Down Regulation ◽  
Cytoskeleton Organization ◽  
Syncytial Virus

cDNA microarray technology was applied to time course analysis of differentially expressed genes in A549 cells following human respiratory syncytial virus (HRSV) infection. Both up- and down-regulation of cellular genes were observed in a time-dependent manner. However, gene up-regulation prevailed over gene down-regulation. Virus infectivity was required as UV-inactivated virus failed to up-regulate/down-regulate those genes. At early times post-infection (0–6 h p.i.) 85 genes were up-regulated. Some of those genes were involved in cell growth/proliferation, cellular protein metabolism and cytoskeleton organization. Among the most strongly up-regulated genes at that time were the urokinase plasminogen activator (PLAU) and its receptor (PLAUR), a pleiotropic system involved in many biological processes, including chemotaxis and inflammation. Functionally related genes encoding the α- and β-chains of several integrins were also up-regulated within the first 12 h of infection. Genes up-regulated between 6 and 12 h p.i. included interferon-stimulated genes (ISGs), genes related to oxidative stress and genes of the non-canonical NF-κB pathway. At later times, genes involved in the immune response became predominant among the up-regulated genes, most of them being ISGs. Different up-regulation kinetics of cytokine and cytokine-signalling-related genes were also observed. These results highlight the dynamic interplay between the virus and the host cell and provide a general picture of changes in cellular gene expression along the HRSV replicative cycle.

scholarly journals Identification of Linear Heparin-Binding Peptides Derived from Human Respiratory Syncytial Virus Fusion Glycoprotein That Inhibit Infectivity

2006 ◽  
Vol 81 (1) ◽  
pp. 261-271 ◽  
Author(s):  
Roberta L. Crim ◽  
Susette A. Audet ◽  
Steven A. Feldman ◽  
Howard S. Mostowski ◽  
Judy A. Beeler
Keyword(s):  
Respiratory Syncytial Virus ◽  
Cell Surface ◽  
A549 Cells ◽  
Vero Cells ◽  
Human Respiratory Syncytial Virus ◽  
Virus Infectivity ◽  
Heparin Binding ◽  
The Third ◽  
Fusion Glycoprotein ◽  
Syncytial Virus

ABSTRACT It has been shown previously that the fusion glycoprotein of human respiratory syncytial virus (RSV-F) interacts with cellular heparan sulfate. Synthetic overlapping peptides derived from the F-protein sequence of RSV subtype A (strain A2) were tested for their ability to bind heparin using heparin-agarose affinity chromatography (HAAC). This evaluation identified 15 peptides representing eight linear heparin-binding domains (HBDs) located within F1 and F2 and spanning the protease cleavage activation site. All peptides bound to Vero and A549 cells, and binding was inhibited by soluble heparins and diminished by either enzymatic treatment to remove cell surface glycosaminoglycans or by treatment with sodium chlorate to decrease cellular sulfation. RSV-F HBD peptides were less likely to bind to glycosaminoglycan-deficient CHO-745 cells than parental CHO-K1 cells that express these molecules. Three RSV-F HBD peptides (F16, F26, and F55) inhibited virus infectivity; two of these peptides (F16 and F55) inhibited binding of virus to Vero cells, while the third (F26) did not. These studies provided evidence that two of the linear HBDs mapped by peptides F16 and F55 may mediate one of the first steps in the attachment of virus to cells while the third, F26, inhibited infectivity at a postattachment step, suggesting that interactions with cell surface glycosaminoglycans may play a role in infectivity of some RSV strains.

scholarly journals The Baculovirus GP64 Protein Mediates Highly Stable Infectivity of a Human Respiratory Syncytial Virus Lacking Its Homologous Transmembrane Glycoproteins

2004 ◽  
Vol 78 (1) ◽  
pp. 124-135 ◽  
Author(s):  
A. G. P. Oomens ◽  
Gail W. Wertz
Keyword(s):  
Respiratory Syncytial Virus ◽  
Membrane Fusion ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Low Ph ◽  
Human Respiratory Syncytial Virus ◽  
Dependent Manner ◽  
Practical Applications ◽  
Ph Dependent ◽  
Syncytial Virus

ABSTRACT Baculovirus GP64 is a low-pH-dependent membrane fusion protein required for virus entry and cell-to-cell transmission. Recently, GP64 has generated interest for practical applications in mammalian systems. Here we examined the membrane fusion function of GP64 from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressed in mammalian cells, as well as its capacity to functionally complement a mammalian virus, human respiratory syncytial virus (HRSV). Both authentic GP64 and GP64/F, a chimeric protein in which the GP64 cytoplasmic tail domain was replaced with the 12 C-terminal amino acids of the HRSV fusion (F) protein, induced low-pH-dependent cell-cell fusion when expressed transiently in HEp-2 (human) cells. Levels of surface expression and syncytium formation were substantially higher at 33°C than at 37°C. The open reading frames (ORFs) encoding GP64 or GP64/F, along with two marker ORFs encoding green fluorescent protein (GFP) and β-glucuronidase (GUS), were used to replace all three homologous transmembrane glycoprotein ORFs (small hydrophobic SH, attachment G, and F) in a cDNA of HRSV. Infectious viruses were recovered that lacked the HRSV SH, G, and F proteins and expressed instead the GP64 or GP64/F protein and the two marker proteins GFP and GUS. The properties of these viruses, designated RSΔsh,g,f/GP64 or RSΔsh,g,f/GP64/F, respectively, were compared to a previously described HRSV expressing GFP in place of SH but still containing the wild-type HRSV G and F proteins (RSΔsh [A. G. Oomens, A. G. Megaw, and G. W. Wertz, J. Virol., 77:3785-3798, 2003]). By immunoelectron microscopy, the GP64 and GP64/F proteins were shown to incorporate into HRSV-induced filaments at the cell surface. Antibody neutralization, ammonium chloride inhibition, and replication levels in cell culture showed that both GP64 proteins efficiently mediated infectivity of the respective viruses in a temperature-sensitive, low-pH-dependent manner. Furthermore, RSΔsh,g,f/GP64 and RSΔsh,g,f/GP64/F replicated to higher levels and had significantly higher stability of infectivity than HRSVs containing the homologous HRSV G and F proteins. Thus, GP64 and a GP64/HRSV F chimeric protein were functional and efficiently complemented an unrelated human virus in mammalian cells, producing stable, infectious virus stocks. These results demonstrate the potential of GP64 for both practical applications requiring stable pseudotypes in mammalian systems and for studies of viral glycoprotein requirements in assembly and pathogenesis.

scholarly journals Neutralization of Human Respiratory Syncytial Virus Infectivity by Antibodies and Low-Molecular-Weight Compounds Targeted against the Fusion Glycoprotein

2010 ◽  
Vol 84 (16) ◽  
pp. 7970-7982 ◽  
Author(s):  
Margarita Magro ◽  
David Andreu ◽  
Paulino Gómez-Puertas ◽  
José A. Melero ◽  
Concepción Palomo
Keyword(s):  
Molecular Weight ◽  
Respiratory Syncytial Virus ◽  
Vaccine Development ◽  
Human Respiratory Syncytial Virus ◽  
Heptad Repeat ◽  
Low Molecular Weight ◽  
Virus Infectivity ◽  
F Protein ◽  
Low Molecular Weight Compounds ◽  
Syncytial Virus

ABSTRACT Human respiratory syncytial virus (HRSV) fusion (F) protein is an essential component of the virus envelope that mediates fusion of the viral and cell membranes, and, therefore, it is an attractive target for drug and vaccine development. Our aim was to analyze the neutralizing mechanism of anti-F antibodies in comparison with other low-molecular-weight compounds targeted against the F molecule. It was found that neutralization by anti-F antibodies is related to epitope specificity. Thus, neutralizing and nonneutralizing antibodies could bind equally well to virions and remained bound after ultracentrifugation of the virus, but only the former inhibited virus infectivity. Neutralization by antibodies correlated with inhibition of cell-cell fusion in a syncytium formation assay, but not with inhibition of virus binding to cells. In contrast, a peptide (residues 478 to 516 of F protein [F478-516]) derived from the F protein heptad repeat B (HRB) or the organic compound BMS-433771 did not interfere with virus infectivity if incubated with virus before ultracentrifugation or during adsorption of virus to cells at 4°C. These inhibitors must be present during virus entry to effect HRSV neutralization. These results are best interpreted by asserting that neutralizing antibodies bind to the F protein in virions interfering with its activation for fusion. Binding of nonneutralizing antibodies is not enough to block this step. In contrast, the peptide F478-516 or BMS-433771 must bind to F protein intermediates generated during virus-cell membrane fusion, blocking further development of this process.

scholarly journals Inhibition of Human Respiratory Syncytial Virus Infectivity by a Dendrimeric Heparan Sulfate-Binding Peptide

2012 ◽  
Vol 56 (10) ◽  
pp. 5278-5288 ◽  
Author(s):  
Manuela Donalisio ◽  
Marco Rusnati ◽  
Valeria Cagno ◽  
Andrea Civra ◽  
Antonella Bugatti ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antiviral Activity ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
A549 Cells ◽  
Epithelial Tissue ◽  
Virus Infectivity ◽  
Human Respiratory Tract ◽  
Rsv Infection ◽  
Syncytial Virus

ABSTRACTRespiratory syncytial virus (RSV) interacts with cell surface heparan sulfate proteoglycans (HSPGs) to initiate infection. The interaction of RSV with HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In the present study, a minilibrary of linear, dimeric, and dendrimeric peptides containing clusters of basic amino acids was screened with the aim of identifying peptides able to bind HSPGs and thus block RSV attachment and infectivity. Of the compounds identified, the dendrimer SB105-A10 was the most potent inhibitor of RSV infectivity, with 50% inhibitory concentrations (IC50s) of 0.35 μM and 0.25 μM measured in Hep-2 and A549 cells, respectively. SB105-A10 was found to bind to both cell types via HSPGs, suggesting that its antiviral activity is indeed exerted by competing with RSV for binding to cell surface HSPGs. SB105-A10 prevented RSV infection when added before the viral inoculum, in line with its proposed HSPG-binding mechanism of action; moreover, antiviral activity was also exhibited when SB105-A10 was added postinfection, as it was able to reduce the cell-to-cell spread of the virus. The antiviral potential of SB105-A10 was further assessed using human-derived tracheal/bronchial epithelial cells cultured to form a pseudostratified, highly differentiated model of the epithelial tissue of the human respiratory tract. SB105-A10 strongly reduced RSV infectivity in this model and exhibited no signs of cytotoxicity or proinflammatory effects. Together, these features render SB105-A10 an attractive candidate for further development as a RSV inhibitor to be administered by aerosol delivery.

scholarly journals Quantitative Proteomic Analysis of A549 Cells Infected with Human Respiratory Syncytial Virus

2010 ◽  
Vol 9 (11) ◽  
pp. 2438-2459 ◽  
Author(s):  
Diane C. Munday ◽  
Edward Emmott ◽  
Rebecca Surtees ◽  
Charles-Hugues Lardeau ◽  
Weining Wu ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Proteomic Analysis ◽  
A549 Cells ◽  
Human Respiratory Syncytial Virus ◽  
Quantitative Proteomic Analysis ◽  
Syncytial Virus

Cimicifuga foetida L. Inhibited Human Respiratory Syncytial Virus in HEp-2 and A549 Cell Lines

2012 ◽  
Vol 40 (01) ◽  
pp. 151-162 ◽  
Author(s):  
Kuo Chih Wang ◽  
Jung San Chang ◽  
Lien Chai Chiang ◽  
Chun Ching Lin
Keyword(s):  
Respiratory Syncytial Virus ◽  
Respiratory Tract ◽  
Cell Lines ◽  
A549 Cells ◽  
Plaque Formation ◽  
Human Respiratory Syncytial Virus ◽  
Major Constituent ◽  
Enzyme Linked Immunosorbent Assay ◽  
Viral Attachment ◽  
Syncytial Virus

Human respiratory syncytial virus (HRSV) causes serious pediatric infection of the lower respiratory tract without effective therapeutic modality. Sheng-Ma-Ge-Gen-Tang (SMGGT; Shoma-kakkon-to) has been proven to be effective at inhibiting HRSV-induced plaque formation, and Cimicifuga foetida is the major constituent of SMGGT. We tested the hypothesis that C. foetida effectively inhibited the cytopathic effects of HRSV by a plaque reduction assay in both human upper (HEp2) and lower (A549) respiratory tract cell lines. Its ability to stimulate anti-viral cytokines was evaluated by an enzyme-linked immunosorbent assay (ELISA). C. foetida dose-dependently inhibited HRSV-induced plaque formation (p < 0.0001) before and after viral inoculation, especially in A549 cells (p < 0.0001). C. foetida dose-dependently inhibited viral attachment (p < 0.0001) and could increase heparins effect on viral attachment. In addition, C. foetida time-dependently and dose-dependently (p < 0.0001) inhibited HRSV internalization. C. foetida could stimulate epithelial cells to secrete IFN-β to counteract viral infection. However, C. foetida did not stimulate TNF-α secretion. Therefore, C. foetida could be useful in managing HRSV infection. This is the first evidence to support that C. foetida possesses antiviral activity.

scholarly journals Infectivity of a Human Respiratory Syncytial Virus Lacking the SH, G, and F Proteins Is Efficiently Mediated by the Vesicular Stomatitis Virus G Protein

2003 ◽  
Vol 77 (6) ◽  
pp. 3785-3798 ◽  
Author(s):  
A. G. P. Oomens ◽  
A. G. Megaw ◽  
G. W. Wertz
Keyword(s):  
Respiratory Syncytial Virus ◽  
G Protein ◽  
Fluorescent Protein ◽  
Syncytium Formation ◽  
Transmembrane Domains ◽  
Human Respiratory Syncytial Virus ◽  
Virus Infectivity ◽  
Reading Frame ◽  
Vesicular Stomatitis ◽  
Syncytial Virus

ABSTRACT To examine the requirements of the human respiratory syncytial virus (HRSV) SH (small hydrophobic), G (attachment), and F (fusion) proteins for virus infectivity and morphology, we used the prototype A2 strain of HRSV to generate a series of cDNAs from which (i) the SH open reading frame (ORF), (ii) the SH and G ORFs, or (iii) the SH, G, and F ORFs were deleted. Each deleted ORF was replaced as follows: the SH ORF was replaced with that of green fluorescent protein; the G ORF was replaced with that of Gvsv, a chimeric glycoprotein consisting of the vesicular stomatitis Indiana virus (VSIV) G protein ecto- and transmembrane domains coupled to the HRSV F cytoplasmic tail; and the F ORF was replaced with that of marker protein β-glucuronidase. The number of genes and the intergenic junctions in the constructs were kept as found in A2 virus in order to maintain authentic levels of transcription. Infectious viruses were recovered from all three engineered cDNAs and designated RSΔsh, RSΔsh,g/Gvsv, and RSΔsh,g,f/Gvsv, respectively. Low-pH-induced syncytium formation was observed in cells infected with viruses RSΔSH,G/Gvsv and RSΔSH,G,F/Gvsv, indicating that Gvsv was expressed and functional. Neutralization of infectivity by anti-VSIV G antibodies and inhibition of entry by ammonium chloride showed that RSΔSH,G,F/Gvsv infectivity was mediated by Gvsv and that an acidification step was required for entry into the host cell, similar to VSIV virions. All three engineered viruses displayed growth kinetics and virus yields similar to a wild-type A2 virus, both in Vero and HEp-2 cells. Abundant virus-induced filaments were observed at the surface of cells infected with each of the three engineered viruses or with virus A2, indicating that neither the SH and G proteins nor the F protein ecto- and transmembrane domains were required for the formation of these structures. This is the first report of the recovery of an infectious HRSV lacking a fusion protein of the Paramyxoviridae family and of manipulation of the HRSV entry pathway via incorporation of a nonparamyxoviral transmembrane glycoprotein.

scholarly journals Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Miaoge Xue ◽  
Boxuan Simen Zhao ◽  
Zijie Zhang ◽  
Mijia Lu ◽  
Olivia Harder ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rational Design ◽  
A549 Cells ◽  
Human Respiratory Syncytial Virus ◽  
Gene Transcript ◽  
Airway Epithelial ◽  
Live Attenuated Vaccine ◽  
Epithelial Cultures ◽  
Cotton Rats ◽  
Syncytial Virus

Abstract N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m6A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m6A methyltransferases decreases RSV replication and gene expression whereas knockdown of m6A demethylases has the opposite effect. The G gene transcript contains the most m6A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m6A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m6A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m6A methylation upregulates RSV replication and pathogenesis and identify viral m6A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.

Anti-Viral Activity of Water Extract of Paeonia lactiflora Pallas Against Human Respiratory Syncytial Virus in Human Respiratory Tract Cell Lines

2013 ◽  
Vol 41 (03) ◽  
pp. 585-599 ◽  
Author(s):  
Tzeng-Jih Lin ◽  
Kuo-Chih Wang ◽  
Chun-Ching Lin ◽  
Lien-Chai Chiang ◽  
Jung-San Chang
Keyword(s):  
Respiratory Syncytial Virus ◽  
Respiratory Tract ◽  
Cell Line ◽  
Carcinoma Cell ◽  
A549 Cells ◽  
Water Extract ◽  
Human Respiratory Syncytial Virus ◽  
Paeonia Lactiflora ◽  
Viral Attachment ◽  
Syncytial Virus

Paeonia lactiflora Pallas (P. lactiflora, Ranunculaceae) is a common ingredient of Sheng-Ma-Ge-Gen-Tang (SMGGT; Shoma-kakkon-to) and Ge-Gen-Tang (GGT; kakkon-to). SMGGT and GGT are different prescriptions of traditional Chinese medicine with different ingredients designed for airway symptoms. Both SMGGT and GGT have anti-viral activity against human respiratory syncytial virus (HRSV). Therefore, P. lactiflora was hypothesized to be the effective ingredient of both SMGGT and GGT against HRSV. However, P. lactiflora does not have any proven antiviral activity. This study used both human upper (Human larynx epidermoid carcinoma cell line, HEp-2) and lower (human lung carcinoma cell line, A549) respiratory tract cells to test the hypothesis that a hot water extract of P. lactiflora could effectively inhibit plaque formation induced by HRSV infection. The ability of P. lactiflora to stimulate anti-viral cytokines was evaluated by enzyme-linked immunosorbent assay (ELISA). The results showed that P. lactiflora was time-dependently and dose-dependently effective against HRSV in HEp-2 and A549 cells, particularly supplemented before viral inoculation (p < 0.0001). 10 μg/ml P. lactiflora had a comparable anti-HRSV activity with 10 μg/ml ribavirin, a broad-spectrum antiviral agent. P. lactiflora was dose-dependently effective against viral attachment (p < 0.0001), with a better effect on A549 cells (p < 0.0001). P. lactiflora was time-dependently (p < 0.0001) and dose-dependently (p < 0.0001) effective against viral penetration. Moreover, P. lactiflora stimulated IFN-β secretion without any effect on TNF-α secretion. Therefore, P. lactiflora could be beneficial at preventing HRSV infection by inhibiting viral attachment, internalization, and stimulating IFN secretion.

Antiviral Effect of Cimicifugin from Cimicifuga foetida Against Human Respiratory Syncytial Virus

2012 ◽  
Vol 40 (05) ◽  
pp. 1033-1045 ◽  
Author(s):  
Kuo-Chih Wang ◽  
Jung-San Chang ◽  
Liang-Tzung Lin ◽  
Lien-Chai Chiang ◽  
Chun-Ching Lin
Keyword(s):  
Respiratory Syncytial Virus ◽  
Respiratory Tract ◽  
A549 Cells ◽  
Antiviral Agent ◽  
Human Respiratory Syncytial Virus ◽  
Major Constituent ◽  
Enzyme Linked Immunosorbent Assay ◽  
Viral Pathogen ◽  
Viral Attachment ◽  
Syncytial Virus

Human respiratory syncytial virus (RSV) causes serious infection of the lower respiratory tract in children and an effective antiviral therapy against the viral pathogen remains unavailable. We previously demonstrated that the oriental medicinal plant, Cimicifuga foetida L. (C. foetida), possessed inhibitory activity against RSV. Since cimicifugin is a major constituent of C. foetida, we sought to examine in this study its anti-RSV effect on both the human upper (HEp-2) and lower (A549) respiratory tract cell lines. Results revealed that cimicifugin dose-dependently inhibited RSV-induced plaque formation in both HEp-2 and A549 cells (p < 0.0001), with a superior effect in the latter cell type (p < 0.0001). The antiviral activity of cimicifugin was time-dependent (p < 0.0001) and was most effective when cells were treated with the compound before viral inoculation. Additional experiments demonstrated that cimicifugin could inhibit viral attachment (p < 0.0001) and viral internalization (p < 0.0001). Furthermore, the drug could potentiate heparin's effect against attachment of RSV, particularly in A549 cells. Enzyme-linked immunosorbent assay (ELISA) analysis of antiviral cytokines induction revealed that cimicifugin could also stimulate epithelial cells to secrete IFN-β to counteract viral infection. Taken together, these results indicate that cimicifugin is an efficient antiviral agent against RSV infection. We suggest that cimicifugin might be useful for the management of RSV pathogenesis.

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