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

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.Respiratory syncytial virus (RSV), a nonsegmented, negativestrand RNA virus in the pneumovirus genus of Paramyxoviridae, is a major cause of lower respiratory disease, resulting in 90,000 hospitalizations and 4500 deaths per year in infants and young children in the United States (1). Of growing concern are outbreaks of RSV within institutionalized elderly (2) and immunodeficient (1) adults. There is only one approved anti-RSV compound, ribavirin, which reduces virus shedding in infected children, but has no effect on mortality or duration of hospitalization (3). In the search for alternative antiviral strategies, we have investigated the potential of novel chimeric antisense molecules, 2-5A-antisense, in the control of RSV infections.Zamecnik and Stephenson were the first to report an antiviral effect of antisense oligonucleotides by showing that replication of the retrovirus Rous sarcoma virus could be inhibited by the addition of oligonucleotides complementary in sequence to reiterated terminal sequences of the viral 35S RNA (4). Subsequently, antisense oligonucleotides have been used against a wide range of different types of viruses, including the negative-strand RNA viruses such as rabies virus (5) and influenza virus (6), positive-strand RNA viruses such as dengue virus (7), DNA viruses including hepatitis B (8) and herpes simplex virus type 1 (9), and the retrovirus HIV-1 (10, 11). Despite encouraging results, significant challenges remain in the development of antisense oligonucleotides as antiviral agents. For instance, ...

Targeted therapy of human malignant glioma in a mouse model by 2-5A antisense directed against telomerase RNA

“…Only the glioma cells demonstrate telomeraseactivity 2-5A antisense therapy for malignant gliomas S Kondo et al inverted linkage to inhibit 3' and 5' exonuclease activities Xiao et al, 1994Xiao et al, , 1996Cirino et al, 1997).…”

“…2-5A antisense refers to a class of oligonucleotide therapeutic agents that cause the catalytic breakdown of target RNA molecules Maran et al, 1994;Maitra et al, 1995;Cirino et al, 1997). These drugs contain a 2',5'-linked tetraadenylate (2-5A) covalently attached through linkers to antisense oligonucleotides.…”

“…RNase L is a ubiquitous protein that has no detectable ribonuclease activity until it comes into contact with 2-5A. Attachment of 2-5A to antisense oligonucleotides greatly enhances the degradation rates of target RNA molecules in intact, human cells (Maran et al, 1994;Cirino et al, 1997). In essence, the 2-5A component of the oligonucleotide activates RNase L while the antisense segment of the chimera directs RNase L to the target RNA molecule.…”

“…For example, unfacilitated uptake of 2-5A antisense into human HeLa cells resulted in the ablation of a cellular mRNA for protein kinase PKR (Maran et al, 1994). Recently, the recruitment of RNase L by 2-5A antisense to a respiratory syncytial virus (RSV) mRNA resulted in a potent inhibition of RSV replication in human tracheal epithelial cells (Cirino et al, 1997). Antisense molecules`charged' by the addition of a functional, tetrameric 2-5A were e ective against RSV replication whereas antisense molecules containing a nonfunctional, dimeric 2-5A moiety were ine ective (Cirino et al, 1997).…”

“…Recently, the recruitment of RNase L by 2-5A antisense to a respiratory syncytial virus (RSV) mRNA resulted in a potent inhibition of RSV replication in human tracheal epithelial cells (Cirino et al, 1997). Antisense molecules`charged' by the addition of a functional, tetrameric 2-5A were e ective against RSV replication whereas antisense molecules containing a nonfunctional, dimeric 2-5A moiety were ine ective (Cirino et al, 1997). Because RNase L is present in nearly all mammalian cells, it is clear that the application of this technology is a powerful method for preventing gene expression.…”