Targeting RNA decay with 2',5' oligoadenylate-antisense in respiratory syncytial virus-infected cells.
Treatment of human cells with 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) results in the selective cleavage of targeted RNA species by 2-5A-dependent RNase L. Here we show that 2-5A-antisense containing stabilizing modifications at both termini are effective in suppressing the replication of respiratory syncytial virus (RSV) in human tracheal epithelial cells. The affinity of 2-5A-antisense for different regions in the RSV M2 and L mRNAs was predicted from a computer-generated model of the RNA secondary structure. The most potent 2-5A-antisense molecule caused a highly effective, dose-dependent suppression of RSV yields when added to previously infected cells. In contrast, control oligonucleotides, including an inactive dimeric form of 2-5A linked to antisense, 2-5A linked to a randomized sequence of nucleotides, and antisense molecules lacking 2-5A, had minimal effects on virus replication. The specificity of this approach was shown by reverse transcriptase-coupled PCR analysis of RSV M2, P, and N mRNA and of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The RSV M2 mRNA amounts were depleted after treating RSV-infected cells with 2-5A-antisense targeted to this mRNA, whereas the amounts of the other RNA species were unchanged. These studies demonstrate that 2',5' oligoadenylate covalently linked to antisense (2-5A-antisense) can effectively suppress RSV replication by directing the cellular RNase L to selectively degrade an essential viral mRNA.
Cirino
N M
NM
Department of Cancer Biology, Research Institute, The Cleveland Clinic Foundation, OH 44195, USA.
Li
G
G
Xiao
W
W
Torrence
P F
PF
Silverman
R H
RH
D000227:Adenine Nucleotides / Q000235:genetics / Q000494:pharmacology*; D000998:Antiviral Agents / Q000494:pharmacology*; D001665:Binding Sites; D002460:Cell Line; D004587:Electrophoresis, Agar Gel; D004722:Endoribonucleases / Q000378:metabolism; D006801:Humans; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000235:genetics; D009843:Oligoribonucleotides / Q000235:genetics / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D016372:RNA, Antisense / Q000235:genetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D012136:Respiratory Syncytial Viruses / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects
D000227:Adenine Nucleotides; D000998:Antiviral Agents; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D016372:RNA, Antisense; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; C023505:2',5'-oligoadenylate; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease