Secondary RNA Structure and its Role in RNA Interference to Silence the Respiratory Syncytial Virus Fusion Protein Gene

Abstract

RNA interference (RNAi) is a post-transcriptional, gene silencing mechanism which uses small interfering RNA molecules (siRNA) for gene silencing. Respiratory Syncytial Virus (RSV) is an important respiratory pathogen of medical significance that causes high mortality in infants. The fusion (F) protein of RSV is a good target for therapeutic purposes as it is primarily responsible for penetration of the virus into host cells and subsequent syncytium formation during infection. In the present study, four siRNAs were designed and used individually as well as a mixture, to silence the RSV F gene. The relationship between siRNA design, target RNA structure, and their thermodynamics was also investigated. Silencing of F gene was observed using indirect immunofluorescence, western blot, reverse transcription PCR, and progeny viral titers. Our results show F gene silencing by all the four siRNAs individually and collectively. RT-PCR analysis revealed a decrease in mRNA level which corresponded to decreased F protein expression. siRNAs also inhibited RSV progeny as shown by viral titer estimation on infected HEp-2 cells. The present study demonstrates the silencing of the F gene using siRNA. Thermodynamic characteristics of the target RSV mRNA and siRNA seem to play an important role in siRNA gene silencing efficiency.

Gold nanorods inhibit respiratory syncytial virus by stimulating the innate immune response

“…32 Briefly, following treatment with GNR and GNR-RSV, cells were incubated for another 48 h. After 48 h, cells were fixed with 10% trichloroacetic acid and washed successively with 70%, 90% and 100% ethanol. Cells were then blocked with 3% dry milk powder and incubated for 1 h with 1:500 goat anti-RSV antibody (Millipore, Temecula, CA, USA) followed by anti-goat antibody (FITC-conjugated IgG H+L, 1:1000).…”

“…32 HEp-2 cells were infected with RSV and treated with GNRs (1.25 and 2.5 μg/mL). Lungs were collected in DMEM and processed to determine the RSV titers using gentleMACS dissociator (Miltenyi Biotec Inc., Auburn, CA, USA) following the manufacturer’s procedures.…”

Atomic force microscopic investigation of respiratory syncytial virus infection in HEp‐2 cells

“…RSV Long strain was obtained from the ATCC (Manassas, VA, U.S.A.) and stocks were prepared as described previously (Sun et al , 2008; Vig et al , 2009). …”

Recent Advances in Diagnosis, Prevention, and Treatment of Human Respiratory Syncytial Virus

“…This approach was also effective when targeting the RSV-F gene [193]. Similar work on HEp-2 cell lines was replicated using four siRNA, designed to silence RSV F gene, which showed inhibitory action against RSV at various concentrations [194]. Silencing different RSV genes too had an inhibitory action on the RSV, a plasmid encoding siRNA which was complexed with chitosan targeting RSV-NS1 gene decreased RSV infection in BALB/c mice and Fischer 344 rats and also reduced the associated inflammation [195,196].…”