Photochemical crosslinking in oligonucleotide-protein complexes between a bromine-substituted 2-5A analog and 2-5A-dependent RNase by ultraviolet lamp or laser

Nolan-Sorden, Nancy L.; Lesiak, Krystyna; Bayard, Bernard; Torrence, Paul F.; Silverman, Robert H.

doi:10.1016/0003-2697(90)90684-2

Cited by 44 publications

(34 citation statements)

References 23 publications

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“…These cells were observed to contain basal levels of RNase L by a specific, radiolabeled 2-5A crosslinking assay (ref. 39 and data not shown). Because we observed similar antiviral effects when the oligonucleotides were added beginning either 4 h before infection or 2 h after infection, a treatment protocol was developed in which cells were treated only after infection (data not shown).…”

Section: Resultsmentioning

confidence: 76%

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

Cirino

Xiao

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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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, ...

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Section: Resultsmentioning

confidence: 76%

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

Cirino

Xiao

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…2-5A Binding Assay for RNase L-A 32 P-labeled and bromine-substituted 2-5A analog, p(A2Јp) 2 (br 8 A2Јp) 2 A3Ј[ 32 P]pCp, was cross-linked to RNase L present in cytoplasmic extracts of cells under ultraviolet light (37). The cell extracts (200 g protein) were incubated with the probe (0.02 Ci; 750 Ci/mmol) on ice for 60 min and then under 308 nm of light on ice for 60 min.…”

Section: Methodsmentioning

confidence: 99%

An Apoptotic Signaling Pathway in the Interferon Antiviral Response Mediated by RNase L and c-Jun NH2-terminal Kinase

Xiang

Sabapathy

et al. 2004

Journal of Biological Chemistry

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138

127

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Cellular stress responses induced during viral infections are critical to the health and survival of organisms. In higher vertebrates, interferons (IFNs) mediate the innate antiviral response in part through the action of RNase L, a uniquely regulated enzyme. RNase L is activated by 5 -phosphorylated, 2 -5 oligoadenylates (2-5A) produced from IFN-inducible and double stranded RNA-dependent synthetases. We show that viral activation of the c-Jun NH 2 -terminal kinases (JNK) family of MAP kinases and viral induction of apoptosis are both deficient in mouse cells lacking RNase L. Also, JNK phosphorylation in response to 2-5A was greatly reduced in RNase L ؊/؊ mouse cells. In addition, 2-5A treatment of the human ovarian carcinoma cell line, Hey1b, resulted in specific ribosomal RNA cleavage products coinciding with JNK activation. Furthermore, suppression of JNK activity with the chemical inhibitor, SP600125, prevented apoptosis induced by 2-5A. In contrast, inhibition of alternative MAP kinases, p38 and ERK, failed to prevent 2-5A-mediated apoptosis. Short interfering RNA to JNK1/JNK2 mRNAs resulted in JNK ablation while also suppressing 2-5A-mediated apoptosis. Moreover, Jnk1 ؊/؊ Jnk2 ؊/؊ cells were highly resistant to the apoptotic effects of IFN and 2-5A. These findings suggest that JNK and RNase L function in an integrated signaling pathway during the IFN response that leads to elimination of virus-infected cells through apoptosis.

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“…Assay of truncated and endogenous, wild-type RNase L using a bromine substituted 32 P-labeled 2-5A analogue Truncated and wild-type RNase L were specifically radiolabeled by covalent crosslinking to a 32 P-labeled and bromine-substituted 2-5A probe, p(A2'p) 2 (br 8 A2'p) 2 A3'-[ 32 P]Cp (Nolan-Sorden et al, 1990). Cells were washed in phosphate buffered saline, harvested by scraping, and used to prepare cell extracts as described previously (Nolan-Sorden et al, 1990).…”

Section: Construction Of Stably Transfected Cell Linesmentioning

confidence: 99%

“…Cells were washed in phosphate buffered saline, harvested by scraping, and used to prepare cell extracts as described previously (Nolan-Sorden et al, 1990). About 4610 5 counts/min.…”

Section: Construction Of Stably Transfected Cell Linesmentioning

confidence: 99%

The role of 2′-5′ oligoadenylate-activated ribonuclease L in apoptosis

et al. 1998

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Apoptosis of viral infected cells appears to be one defense strategy to limit viral infection. Interferon can also confer viral resistance by the induction of the 2-5A system comprised of 2'-5' oligoadenylate synthetase (OAS), and RNase L. Since rRNA is degraded upon activation of RNase L and during apoptosis and since both of these processes serve antiviral functions, we examined the role RNase L may play in cell death. Inhibition of RNase L activity, by transfection with a dominant negative mutant, blocked staurosporine-induced apoptosis of NIH3T3 cells and SV40-transformed BALB/c cells. In addition, K562 cell lines expressing inactive RNase L were more resistant to apoptosis induced by decreased glutathione levels. Hydrogen peroxide-induced death of NIH3T3 cells did not occur by apoptosis and was not dependent upon active RNAse L. Apoptosis regulatory proteins of the Bcl-2 family did not exhibit altered expression levels in the absence of RNase L activity. RNase L is required for certain pathways of cell death and may help mediate viral-induced apoptosis.

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Photochemical crosslinking in oligonucleotide-protein complexes between a bromine-substituted 2-5A analog and 2-5A-dependent RNase by ultraviolet lamp or laser

Cited by 44 publications

References 23 publications

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

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

An Apoptotic Signaling Pathway in the Interferon Antiviral Response Mediated by RNase L and c-Jun NH2-terminal Kinase

The role of 2′-5′ oligoadenylate-activated ribonuclease L in apoptosis

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