Inhibition of human respiratory syncytial virus infectivity by a dendrimeric heparan sulfate-binding peptide - PubMed (original) (raw)
Inhibition of human respiratory syncytial virus infectivity by a dendrimeric heparan sulfate-binding peptide
Manuela Donalisio et al. Antimicrob Agents Chemother. 2012 Oct.
Abstract
Respiratory 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 (IC(50)s) 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.
Figures
Fig 1
Screening of a peptide minilibrary against RSV. Hep-2 cells were preincubated with compounds (33 μg/ml) for 45 min at 4°C before the addition of virus for 3 h at room temperature. Cells were then washed to remove unbound virus and overlaid with 1.2% methylcellulose medium. Three days postinfection, cells were fixed and subjected to RSV-specific immunostaining. Plaques were counted, and the percent infection was determined with respect to untreated control wells. The results are means and standard deviations for triplicates. *, P < 0.001.
Fig 2
Effect of SB105-A10 on virus infectivity. RSV (104) was pretreated with 33 μg/ml of SB105-A10 (7.02 μM) or heparin (2.43 μM) for 2 h at 4°C or 37°C. The mixture was then titrated on Hep-2 cells at high dilutions at which the concentration of compound was not active. The titers, expressed as PFU/ml, are means and standard deviations for triplicates.
Fig 3
Investigation of SB105-A10 antiviral mechanism in Hep-2 cells. (A) Preattachment assay. Hep-2 cells were pretreated with increasing concentrations of SB105-A10 or heparin for 1 h at 4°C, washed, and infected. Three days postinfection, the cells were fixed and subjected to RSV-specific immunostaining, the plaques were counted, and the percent infection was calculated by comparing treated and untreated cells. (B) In the attachment assay, RSV was added to cells for 2 h at 4°C for preincubation in the absence or presence of SB105-A10 or heparin. Cells were shifted to 37°C, and at 72 h postinfection they were subjected to RSV-specific immunostaining, the plaques were counted, and the percent infection was calculated by comparing treated to untreated cells. The results are means and standard deviations for triplicates. (C) In the binding assay, the virus bound to cells was detected by ELISA immediately after the removal of the virus inoculum. (D) Interaction between RSV and SB105-A10 using the ELISA method. Plates were coated with SB105-A10, SB047, or 1% BSA for 16 h at 4°C. After blocking with 1% BSA, the plates were incubated with the virus for 2 h at 37°C. The virus bound to plates was detected by enzyme-linked immunosorbent assay.
Fig 4
Binding of SB105-A10–PEG–biotin to HSPGs expressed at the surfaces of A549 and Hep2 epithelial cells. Monolayers of A549 and Hep2 epithelial cells were incubated at 4°C for 2 h with increasing concentrations of (A) or 220 nM (B) SB105-A10–PEG–biotin peptide alone or in the presence of heparin (100 μg/ml, i.e., 7.35 μM) and washed with PBS containing 2 M NaCl. Alternatively, cell monolayers were pretreated with sodium chlorate (40 mM) or heparinase III (15 mU/ml) before incubation with SB105-A10–PEG–biotin. In a second set of experiments, monolayers of A549 and Hep2 epithelial cells were incubated at 37°C for different periods (from 30 min to 48 h) with 1.1 μM SB105-A10–PEG–biotin peptide (C). The amount of biotinylated SB105-A10 bound to cells was subsequently measured. Each point is the mean and SEM for three or four determinations performed in duplicate. *, P < 0.05, and **, P < 0.01, with respect to untreated controls. (D) Representative microphotographs of A549 epithelial cell monolayers incubated at 37°C for 48 h in the absence or presence of SB105-A10 PEG-biotin (1.1 μM). Similar results were obtained with Hep2 epithelial cells (data not shown).
Fig 5
Inhibition of RSV-induced syncytium formation by SB105-A10. Hep-2 cells (A) and A549 cells (B) were infected with RSV in the absence of SB105-A10. (C and D) The inoculum was removed at 3 h postinfection, and cells were left untreated (a) or incubated in the presence of the following concentrations of SB105-A10 in 1.2% methylcellulose medium: (b) 100 μg/ml (21.3 μM), (c) 66 μg/ml (14.05 μM), (d) 33 μg/ml (7.02 μM), and (e) 3.6 μg/ml (0.76 μM). Formation of syncytia was assessed 72 h after infection, as described in Materials and Methods. The histograms show the plaque area and the plaque count as a function of SB105-A10 concentration. The images in panels C and D show representative syncytia in Hep-2 and A549 cells, respectively. The results are means and standard deviations for triplicates. *, P < 0.05.
Fig 6
Reduction of RSV-infected cells in EpiAirway tissue by SB105-A10. Immunohistochemistry of control tissue (A), RSV-infected tissue (66,000 PFU) (B), and RSV-infected tissue treated with 10 μg/ml of SB105-A10 (2.13 μM) at 3 days postinfection (C) using a monoclonal antibody to RSV (brown signal). The pictures shown are representative of many analyzed sections, ranging from 5 to 12 per condition.
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