Isidoro Martínez

Antigenic structure, evolution and immunobiology of human respiratory syncytial virus attachment (G) protein

IP: 54.70.40.11 On: Fri, 07 Dec 2018 06:42:32 Journal of General Virology (1997), 78, 2411–2418. Printed in Great Britain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Binding of human respiratory syncytial virus to cells: implication of sulfated cell surface proteoglycans

Binding of human respiratory syncytial virus (HRSV) to cultured cells was measured by flow cytometry. Using this assay and influenza virus as a control virus with a well-characterized receptor, a systematic search of cell surface molecules that might be implicated in HRSV binding was carried out. Treatment of cells with different enzymes or with other reagents suggested that heparin-like glycosaminoglycans (GAGs) were involved in attachment of HRSV, but not influenza virus, to host cells. This was further confirmed by a lack of binding of HRSV to CHO-K1 mutant cell lines deficient in glycosylation or GAGs biosynthesis and by an inhibition of binding after preincubation of virus with heparin and other GAGs. The degree of sulfation, more than the polysaccharide backbone of GAGs, seems to be critical for virus binding.

A model for the generation of multiple A to G transitions in the human respiratory syncytial virus genome: predicted RNA secondary structures as substrates for adenosine deaminases that act on RNA

Human respiratory syncytial virus (HRSV) escape mutants selected with antibodies specific for the attachment (G) protein contain diverse genetic alterations, including point mutations, premature stop codons, frame shift changes and A to G hypermutations. The latter changes have only been found in mutants selected with antibodies directed against the conserved central region of the G protein. This gene segment fulfils substrate requirements for adenosine deaminases that act on RNA (ADARs): i.e. it is an A+U rich region of 137 residues, and 98 or 106 of them--for A/Mon/3/88 or Long HRSV strains, respectively--are predicted to form intramolecular base pairs leading to a stable RNA secondary structure. In addition, when sequences of the G gene from natural isolates are compared in terms of pairwise substitutions, A to G+G to A changes are preferentially observed in regions where stable intramolecular dsRNA secondary structures are predicted to occur. In this study, a model is proposed in which, in addition to nucleotide misincorporations, reiterative A to G changes in HRSV are generated by ADAR activity operating in short segments (100-200 ribonucleotide residues) of the HRSV genome with high tendency for intramolecular base pairing.

Vesicular Stomatitis Virus Glycoprotein Is a Determinant of Pathogenesis in Swine, a Natural Host

ABSTRACT There are two major serotypes of vesicular stomatitis virus (VSV), Indiana (VSIV) and New Jersey (VSNJV). We recovered recombinant VSIVs from engineered cDNAs that contained either (i) one copy of the VSIV G gene (VSIV-GI); (ii) two copies of the G gene, one from each serotype (VSIV-GNJGI); or (iii) a single copy of the GNJ gene instead of the GI gene (VSIV-GNJ). The recombinant viruses expressed the appropriate glycoproteins, incorporated them into virions, and were neutralized by antibodies specific for VSIV (VSIV-GI), VSNJV (VSIV-GNJ), or both (VSIV-GNJGI), according to the glycoprotein(s) they expressed. All recombinant viruses grew to similar titers in cell culture. In mice, VSIV-GNJ and VSIV-GNJGI were attenuated. However, in swine, a natural host for VSV, the GNJ glycoprotein-containing viruses caused more severe lesions and replicated to higher titers than the parental virus, VSIV-GI. These observations implicate the glycoprotein as a determinant of VSV virulence in a natural host and emphasize the differences in VSV pathogenesis between mice and swine.

Respiratory Syncytial Virus Inhibits Ciliagenesis in Differentiated Normal Human Bronchial Epithelial Cells: Effectiveness of N-Acetylcysteine

Persistent respiratory syncytial virus (RSV) infections have been associated with the exacerbation of chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). This virus infects the respiratory epithelium, leading to chronic inflammation, and induces the release of mucins and the loss of cilia activity, two factors that determine mucus clearance and the increase in sputum volume. These alterations involve reactive oxygen species-dependent mechanisms. The antioxidant N-acetylcysteine (NAC) has proven useful in the management of COPD, reducing symptoms, exacerbations, and accelerated lung function decline. NAC inhibits RSV infection and mucin release in human A549 cells. The main objective of this study was to analyze the effects of NAC in modulating ciliary activity, ciliagenesis, and metaplasia in primary normal human bronchial epithelial cell (NHBEC) cultures infected with RSV. Our results indicated that RSV induced ultrastructural abnormalities in axonemal basal bodies and decreased the expression of β-tubulin as well as two genes involved in ciliagenesis, FOXJ1 and DNAI2. These alterations led to a decrease in ciliary activity. Furthermore, RSV induced metaplastic changes to the epithelium and increased the number of goblet cells and the expression of MUC5AC and GOB5. NAC restored the normal functions of the epithelium, inhibiting ICAM1 expression, subsequent RSV infection through mechanisms involving nuclear receptor factor 2, and the expression of heme oxygenase 1, which correlated with the restoration of the antioxidant capacity, the intracellular H2O2 levels and glutathione content of NHBECs. The results presented in this study support the therapeutic use of NAC for the management of chronic respiratory diseases, including COPD.

ISG15 Is Upregulated in Respiratory Syncytial Virus Infection and Reduces Virus Growth through Protein ISGylation

ABSTRACT Human respiratory syncytial virus (RSV), for which neither a vaccine nor an effective therapeutic treatment is currently available, is the leading cause of severe lower respiratory tract infections in children. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is highly increased during viral infections and has been reported to have an antiviral or a proviral activity, depending on the virus. Previous studies from our laboratory demonstrated strong ISG15 upregulation during RSV infection in vitro. In this study, an in-depth analysis of the role of ISG15 in RSV infection is presented. ISG15 overexpression and small interfering RNA (siRNA)-silencing experiments, along with ISG15 knockout (ISG15−/−) cells, revealed an anti-RSV effect of the molecule. Conjugation inhibition assays demonstrated that ISG15 exerts its antiviral activity via protein ISGylation. This antiviral activity requires high levels of ISG15 to be present in the cells before RSV infection. Finally, ISG15 is also upregulated in human respiratory pseudostratified epithelia and in nasopharyngeal washes from infants infected with RSV, pointing to a possible antiviral role of the molecule in vivo. These results advance our understanding of the innate immune response elicited by RSV and open new possibilities to control infections by the virus. IMPORTANCE At present, no vaccine or effective treatment for human respiratory syncytial virus (RSV) is available. This study shows that interferon-stimulated gene 15 (ISG15) lowers RSV growth through protein ISGylation. In addition, ISG15 accumulation highly correlates with the RSV load in nasopharyngeal washes from children, indicating that ISG15 may also have an antiviral role in vivo. These results improve our understanding of the innate immune response to RSV and identify ISG15 as a potential target for virus control.

Biological Differences between Vesicular Stomatitis Virus Indiana and New Jersey Serotype Glycoproteins: Identification of Amino Acid Residues Modulating pH-Dependent Infectivity

ABSTRACT We previously generated recombinant vesicular stomatitis viruses (VSV) based on the Indiana serotype genome which contained either the homologous glycoprotein gene from the Indiana serotype (VSIV-GI) or the heterologous glycoprotein gene from the New Jersey serotype (VSIV-GNJ). The virus expressing the GNJ gene was more pathogenic than the parental VSIV-GI virus in swine, a natural host (26). For the present study, we investigated the biological differences between the GI and GNJ proteins that may be related to the differences in pathogenesis between VSIV-GI and VSIV-GNJ. We show that the capacities of viruses with either the GNJ or GI glycoprotein to infect cultured cells differ depending on the pH. VSIV-GNJ could infect cells at acidic pHs, while the infectivity of VSIV-GI was severely reduced. VSIV-GNJ infection was also more sensitive to inhibition by ammonium chloride, indicating that the GNJ protein had a lower pH threshold for membrane fusion. We applied selective pressure to VSIV-GI by growing it at successively lower pH values and isolated variant viruses in which we identified amino acid changes that conferred low-pH-resistant infectivity. Repeated passage in cell culture at pH 6.8 resulted in the selection of a VSIV-GI variant (VSIV-6.8) that was similar to VSIV-GNJ regarding its pH- and ammonium chloride-dependent infectivity. Sequence analysis of VSIV-6.8 revealed that it had a single amino acid substitution in the amino-terminal region of the glycoprotein (F18L). This alteration was shown to be responsible for the observed phenotype by site-directed mutagenesis of a VSIV-GI full-length cDNA and analysis of the recovered engineered virus. A further adaptation of VSIV-6.8 to pHs 6.6 and 6.4 resulted in additional amino acid substitutions in areas of the glycoprotein that were not previously implicated in attachment or fusion.

Roflumilast Inhibits Respiratory Syncytial Virus Infection in Human Differentiated Bronchial Epithelial Cells

Respiratory syncytial virus (RSV) causes acute exacerbations in COPD and asthma. RSV infects bronchial epithelial cells (HBE) that trigger RSV associated lung pathology. This study explores whether the phosphodiesterase 4 (PDE4) inhibitor Roflumilast N-oxide (RNO), alters RSV infection of well-differentiated HBE (WD-HBE) in vitro. WD-HBE were RSV infected in the presence or absence of RNO (0.1-100 nM). Viral infection (staining of F and G proteins, nucleoprotein RNA level), mRNA of ICAM-1, ciliated cell markers (digital high speed videomicroscopy, β-tubulin immunofluorescence, Foxj1 and Dnai2 mRNA), Goblet cells (PAS), mRNA of MUC5AC and CLCA1, mRNA and protein level of IL-13, IL-6, IL-8, TNFα, formation of H2O2 and the anti-oxidative armamentarium (mRNA of Nrf2, HO-1, GPx; total antioxidant capacity (TAC) were measured at day 10 or 15 post infection. RNO inhibited RSV infection of WD-HBE, prevented the loss of ciliated cells and markers, reduced the increase of MUC5AC and CLCA1 and inhibited the increase of IL-13, IL-6, IL-8, TNFα and ICAM-1. Additionally RNO reversed the reduction of Nrf2, HO-1 and GPx mRNA levels and consequently restored the TAC and reduced the H2O2 formation. RNO inhibits RSV infection of WD-HBE cultures and mitigates the cytopathological changes associated to this virus.

Induction of DNA double-strand breaks and cellular senescence by human respiratory syncytial virus

ABSTRACT Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract infections during infancy and childhood and is associated with significant morbidity and mortality. HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and γH2AFX in cultured cells secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci contained γH2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester (GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs of DNA damage and aging such as γH2AFX and CDKN2A expression in the respiratory epithelia of infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress. The results also suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of HRSV infections.

Reduced innate immune response, apoptosis, and virus release in cells cured of respiratory syncytial virus persistent infection

It has been reported that cell clones isolated at different passages from a culture of HEp-2 cells infected persistently with human respiratory syncytial virus (HRSV) were cured of the virus. Further studies on one of these clones (31C1) are reported here, showing that 31C1 cells can still be infected by HRSV but release low amounts of virus to the culture supernatant, develop smaller and less numerous syncytia than the original HEp-2 cells, and display only a weak innate immune response to the infection. Accordingly, uninfected 31C1 cells, but not clones derived from uninfected HEp-2 cells, express low levels of TLR3 and RIG-I. In addition, 31C1 cells are partly resistant to apoptosis. These results indicate that persistent infection of HEp-2 cells by HRSV has selected cell variants, with changes affecting cell survival, virus growth and the innate immune response that may be valuable for studies of virus-cell interaction.