Secondary RNA structure and its role in RNA interference to silence the respiratory syncytial virus fusion protein gene.
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.
Vig
Komal
K
Center for Nanobiotechnology Research, Alabama State University, Montgomery, AL 36101, USA.
Lewis
Nuruddeen
N
Moore
Eddie G
EG
Pillai
Shreekumar
S
Dennis
Vida A
VA
Singh
Shree R
SR
D045744:Cell Line, Tumor; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012136:Respiratory Syncytial Viruses / Q000235:genetics*; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014760:Viral Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D010948:Viral Plaque Assay
C526850:F protein, human respiratory syncytial virus; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014760:Viral Fusion Proteins