Inhibition of respiratory syncytial virus replication by vector-derived small interfering RNAs against NS1 protein

Respiratory syncytial virus (RSV) is an important virus associated with bronchiolitis and asthma. The available antiviral treatments are partially effective and nonspecific. Small interfering RNAs (siRNA) provide a novel approach to inhibit specific viral gene expression. The possibility to inhibit virus replication by a plasmid expressing siRNAs capable of silencing the NS1 gene of RSV was examined in this study. A549 cells were transfected with either siRSV-NS1 or siHPV 18 E7 (control), then infected twenty-four hours later with rgRSV (recombinant RSV with EGFP gene inserted) at an MOI of 1. The numbers of rgRSV virus infected cells were quantified by fluorescence microscopy or by flow cytometry one day post-infection. Also, viral protein expression in cultured cells was detected by western blotting. The percentage of cells expressing GFP decreased in siRSV-NS1 treated cells suggesting that rgRSV replication was silenced in a dose-dependent and sequence-specific manner. Expression of NS1 could not be detected in siRSV-NS1 treated cells. SiRSV-NS1 also significantly decreased the rgRSV virus titer compared to control, as determined in culture supernatants from infected A549 cells by the cell-based plaque assay. The cytotoxicity of si-RSV-NS1 plasmid on A549 cells was tested by the MTT assay. There was no difference (P>0.05) in viability between siRSV-NS1-treated cells and controls at the highest dose of plasmid (4 μg) used. These results demonstrate that siRSV-NS1 is capable of significantly decreasing RSV replication in human epithelial cells and provide a basis for the development of siRSV-NS1 in potential prophylaxis and therapy against RSV infection of humans.