Targeting RNA decay with 2′,5′ oligoadenylate-antisense in respiratory syncytial virus-infected cells

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Abstract
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.