Open AccessTargeting RNA decay with 2′,5′ oligoadenylate-antisense in respiratory syncytial virus-infected cells
Author(s) -
Nick M. Cirino,
Guiying Li,
Wei Xiao,
Paul F. Torrence,
Robert H. Silverman
Publication year - 1997
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.94.5.1937
Subject(s) - rnase h , antisense rna , rna , biology , rnase p , microbiology and biotechnology , reverse transcriptase , messenger rna , sense (electronics) , viral replication , nuclease protection assay , oligonucleotide , antisense therapy , virology , virus , rna dependent rna polymerase , gene , chemistry , biochemistry , locked nucleic acid
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.