Targeting RNA decay with 2',5' oligoadenylate-antisense in respiratory syncytial virus-infected cells

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection. Infection is critically dependent on the RSV fusion (F) protein, which mediates fusion between the viral envelope and airway epithelial cells. The F protein is also expressed on infected cells and is responsible for fusion of infected cells with adjacent cells, resulting in the formation of multinucleate syncytia. The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that is constitutively highly expressed by type I alveolar epithelial cells. Here, we report that RAGE protected HEK cells from RSV-induced cell death and reduced viral titres in vitro. RAGE appeared to interact directly with the F protein, but, rather than inhibiting RSV entry into host cells, virus replication and budding, membrane-expressed RAGE or soluble RAGE blocked F-protein-mediated syncytium formation and sloughing. These data indicate that RAGE may contribute to protecting the lower airways from RSV by inhibiting the formation of syncytia, viral spread, epithelial damage and airway obstruction.

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Inhibitory Effect of Ferulic Acid and Isoferulic Acid on the Production of Macrophage Inflammatory Protein-2 in Response to Respiratory Syncytial Virus Infection in RAW264.7 Cells

Mediators of Inflammation ◽

10.1080/09629359990513 ◽

1999 ◽

Vol 8 (3) ◽

pp. 173-175 ◽

Cited By ~ 68

Author(s):

S. Sakai ◽

H. Kawamata ◽

T. Kogure ◽

N. Mantani ◽

K. Terasawa ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Ferulic Acid ◽

Dependent Manner ◽

Inflammatory Drug ◽

Inflammatory Protein ◽

Isoferulic Acid ◽

Infected Cells ◽

Anti Inflammatory ◽

Syncytial Virus ◽

Macrophage Inflammatory Protein

We investigated the effect of ferulic acid (FA) and isoferulic acid (IFA), which are the main active components of the rhizoma ofCimicifuga heracleifolia(CH), an anti-inflammatory drug used frequently in Japanese traditional medicine, on the production of macrophage inflammatory protein-2 (MIR-2) in a murine macrophage cell line, RAW264.7, in response to respiratory syncytial virus (RSV) infection. Following the exposure of cells to RSV for 20 h, the MIP-2 level in condition medium was increased to about 20 ng/ml, although this level in mock-infected cells was negligible. In the presence of either FA or IFA, RSV-infected cells reduced MIP-2 production in a dose-dependent manner. These data suggest that FA and IFA might be responsible, at least in part, for the anti-inflammatory drug effect of CH extract through the inhibition of MIP-2 production.

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Respiratory Syncytial Virus Infection Sensitizes Cells to Apoptosis Mediated by Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

Journal of Virology ◽

10.1128/jvi.77.17.9156-9172.2003 ◽

2003 ◽

Vol 77 (17) ◽

pp. 9156-9172 ◽

Cited By ~ 87

Author(s):

Alexander Kotelkin ◽

Elena A. Prikhod'ko ◽

Jeffrey I. Cohen ◽

Peter L. Collins ◽

Alexander Bukreyev

Keyword(s):

Respiratory Syncytial Virus ◽

Tumor Necrosis Factor ◽

Transcriptional Activation ◽

Tumor Necrosis ◽

Death Receptor ◽

Rnase Protection ◽

Rsv Infection ◽

Infected Cells ◽

Syncytial Virus ◽

Necrosis Factor

ABSTRACT Respiratory syncytial virus (RSV) is an important cause of respiratory tract disease worldwide, especially in the pediatric population. For viruses in general, apoptotic death of infected cells is a mechanism for reducing virus replication. Apoptosis can also be an important factor in augmenting antigen presentation and the host immune response. We examined apoptosis in response to RSV infection of primary small airway cells, primary tracheal-bronchial cells, and A549 and HEp-2 cell lines. The primary cells and the A549 cell line gave generally similar responses, indicating their appropriateness as models in contrast to HEp-2 cells. With the use of RNase protection assays with probes representing 33 common apoptosis factors, we found strong transcriptional activation of both pro- and antiapoptotic factors in response to RSV infection, which were further studied at the protein level and by functional assays. In particular, RSV infection strongly up-regulated the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its functional receptors death receptor 4 (DR4) and DR5. Furthermore, RSV-infected cells became highly sensitive to apoptosis induced by exogenous TRAIL. These findings suggest that RSV-infected cells in vivo are susceptible to killing through the TRAIL pathway by immune cells such as natural killer and CD4+ cells that bear membrane-bound TRAIL. RSV infection also induced several proapoptotic factors of the Bcl-2 family and caspases 3, 6, 7, 8, 9, and 10, representing both the death receptor- and mitochondrion-dependent apoptotic pathways. RSV also mediated the strong induction of antiapoptotic factors of the Bcl-2 family, especially Mcl-1, which might account for the delayed induction of apoptosis in RSV-infected cells in the absence of exogenous induction of the TRAIL pathway.

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Caveolin-1 is incorporated into mature respiratory syncytial virus particles during virus assembly on the surface of virus-infected cells

Journal of General Virology ◽

10.1099/0022-1317-83-3-611 ◽

2002 ◽

Vol 83 (3) ◽

pp. 611-621 ◽

Cited By ~ 56

Author(s):

Gaie Brown ◽

James Aitken ◽

Helen W. McL. Rixon ◽

Richard J. Sugrue

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Respiratory Syncytial Virus ◽

Confocal Microscopy ◽

Cell Surface ◽

Filamentous Form ◽

Caveolin 1 ◽

Immuno Electron Microscopy ◽

Infected Cells ◽

Syncytial Virus

We have employed immunofluorescence microscopy and transmission electron microscopy to examine the assembly and maturation of respiratory syncytial virus (RSV) in the Vero cell line C1008. RSV matures at the apical cell surface in a filamentous form that extends from the plasma membrane. We observed that inclusion bodies containing viral ribonucleoprotein (RNP) cores predominantly appeared immediately below the plasma membrane, from where RSV filaments form during maturation at the cell surface. A comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the pattern of caveolin-1 (cav-1) fluorescence staining. Analysis by immuno-electron microscopy showed that RSV filaments formed in close proximity to cav-1 clusters at the cell surface membrane. In addition, immuno-electron microscopy showed that cav-1 was closely associated with early budding RSV. Further analysis by confocal microscopy showed that cav-1 was subsequently incorporated into the envelope of RSV filaments maturing on the host cell membrane, but was not associated with other virus structures such as the viral RNPs. Although cav-1 was incorporated into the mature virus, it was localized in clusters rather than being uniformly distributed along the length of the viral filaments. Furthermore, when RSV particles in the tissue culture medium from infected cells were examined by immuno-negative staining, the presence of cav-1 on the viral envelope was clearly demonstrated. Collectively, these findings show that cav-1 is incorporated into the envelope of mature RSV particles during egress.

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The Respiratory Syncytial Virus M2-1 Protein Forms Tetramers and Interacts with RNA and P in a Competitive Manner

Journal of Virology ◽

10.1128/jvi.00335-09 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6363-6374 ◽

Cited By ~ 52

Author(s):

Thi-Lan Tran ◽

Nathalie Castagné ◽

Virginie Dubosclard ◽

Sylvie Noinville ◽

Emmanuelle Koch ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Amino Acid ◽

Rna Binding ◽

Amino Acid Residues ◽

Polymerase Complex ◽

Bacterial Rna ◽

Infected Cells ◽

Syncytial Virus ◽

Α Helix ◽

Zinc Binding Domain

ABSTRACT The respiratory syncytial virus (RSV) M2-1 protein is an essential cofactor of the viral RNA polymerase complex and functions as a transcriptional processivity and antitermination factor. M2-1, which exists in a phosphorylated or unphosphorylated form in infected cells, is an RNA-binding protein that also interacts with some of the other components of the viral polymerase complex. It contains a CCCH motif, a putative zinc-binding domain that is essential for M2-1 function, at the N terminus. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in Escherichia coli and purified to >95% homogeneity by using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were copurified with bacterial RNA, which could be eliminated by a high-salt wash. Circular dichroism analysis showed that M2-1 is largely α-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses led to the conclusion that M2-1 forms a 5.4S tetramer of 89 kDa and ∼7.6 nm in diameter at micromolar concentrations. By using a series of deletion mutants, the oligomerization domain of M2-1 was mapped to a putative α-helix consisting of amino acid residues 32 to 63. When tested in an RSV minigenome replicon system using a luciferase gene as a reporter, an M2-1 deletion mutant lacking this region showed a significant reduction in RNA transcription compared to wild-type M2-1, indicating that M2-1 oligomerization is essential for the activity of the protein. We also show that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1.

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Differences in fluorescent antibody staining of bovine respiratory syncytial virus-infected cells by ovine and bovine sera

Veterinary Microbiology ◽

10.1016/0378-1135(87)90102-7 ◽

1987 ◽

Vol 13 (1) ◽

pp. 87-91 ◽

Cited By ~ 3

Author(s):

B.M. Adair ◽

J.B. McFerran

Keyword(s):

Respiratory Syncytial Virus ◽

Fluorescent Antibody ◽

Bovine Respiratory Syncytial Virus ◽

Antibody Staining ◽

Infected Cells ◽

Syncytial Virus ◽

Fluorescent Antibody Staining

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Requirements for Human Respiratory Syncytial Virus Glycoproteins in Assembly and Egress from Infected Cells

Advances in Virology ◽

10.1155/2011/343408 ◽

2011 ◽

Vol 2011 ◽

pp. 1-11 ◽

Cited By ~ 16

Author(s):

Melissa Batonick ◽

Gail W. Wertz

Keyword(s):

Plasma Membrane ◽

Respiratory Syncytial Virus ◽

G Proteins ◽

Matrix Protein ◽

Protein Localization ◽

Viral Proteins ◽

Human Respiratory Syncytial Virus ◽

The Other ◽

Infected Cells ◽

Syncytial Virus

Human respiratory syncytial virus (HRSV) is an enveloped RNA virus that assembles and buds from the plasma membrane of infected cells. The ribonucleoprotein complex (RNP) must associate with the viral matrix protein and glycoproteins to form newly infectious particles prior to budding. The viral proteins involved in HRSV assembly and egress are mostly unexplored. We investigated whether the glycoproteins of HRSV were involved in the late stages of viral replication by utilizing recombinant viruses where each individual glycoprotein gene was deleted and replaced with a reporter gene to maintain wild-type levels of gene expression. These engineered viruses allowed us to study the roles of the glycoproteins in assembly and budding in the context of infectious virus. Microscopy data showed that the F glycoprotein was involved in the localization of the glycoproteins with the other viral proteins at the plasma membrane. Biochemical analyses showed that deletion of the F and G proteins affected incorporation of the other viral proteins into budded virions. However, efficient viral release was unaffected by the deletion of any of the glycoproteins individually or in concert. These studies attribute a novel role to the F and G proteins in viral protein localization and assembly.

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The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro

Journal of Virology ◽

10.1128/jvi.76.21.10776-10784.2002 ◽

2002 ◽

Vol 76 (21) ◽

pp. 10776-10784 ◽

Cited By ~ 37

Author(s):

Bin Lu ◽

Chien-Hui Ma ◽

Robert Brazas ◽

Hong Jin

Keyword(s):

Respiratory Syncytial Virus ◽

Virus Replication ◽

Vero Cells ◽

Phosphorylation Sites ◽

N Protein ◽

P Protein ◽

Infected Cells ◽

Syncytial Virus

ABSTRACT The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of 33Pi-labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2- and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.

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Obtaining and Characterization of the Monoclonal Antibodies Against G-Protein of the Respiratory Syncytial Virus

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2020-1-7-14 ◽

2020 ◽

pp. 7-14

Author(s):

N. A. Demidova ◽

R. R. Klimova ◽

A. A. Kushch ◽

E. I. Lesnova ◽

O. V. Masalova ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Respiratory Syncytial Virus ◽

G Protein ◽

Clinical Samples ◽

Enzyme Linked Immunosorbent Assay ◽

Rsv Infection ◽

Hybridoma Technology ◽

Infected Cells ◽

Syncytial Virus

The aim of this study was to obtain hybridomas producing monoclonal antibodies (Mabs) to the G-protein of the respiratory syncytial virus (RSV), and to evaluate their immunological characteristics and virus-neutralizing activity.Material and methods. Mouse Mabs were obtained using hybridoma technology. The properties of Mabs were studied by enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining (IF) of infected cells, as well as by biological neutralization test in vitro (NT). To identify epitopes recognized by the Mabs on G protein ELISA additivity test was used.Results. Hybridization of splenocytes with Sp2/0 myeloma cells and primary screening showed that 75 hybridomas produce antibodies interacting with purified virus, 17 of them also react with the recombinant G-protein in ELISA. In NT 4, hybridomas suppressed in vitro RSV infection by more than 50%. Cloning of these hybridomas revealed 4 monoclones producing the most active Mabs. Mab 1C11 was IgG2a, 3 others (5D4, 5G11 and 6H4) were IgM. Three IgM Mabs actively reacted with both RSV A2 and Long, and with G-protein; Mab 1C11 was less reactive with all antigens tested. All Mabs suppressed RSV infection, while Mab 5D4 supressed it almost completely (98%). IF analysis showed that all Mabs detected RSV G-protein in the cell cytoplasm, the largest number of infected cells was detected using Mab 5D4 (80%). It was shown that the isolated Mabs were directed to two non-overlapping epitopes on the RSV G-protein.Conclusion. The isolated Mabs can be used to detect RSV in clinical samples by ELISA and IF. The isolated Mabs can be used for humanized recombinant antibodies construction and for the treatment of RSV infection in future.

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