Targeting RNase L to human immunodeficiency virus RNA with 2-5A-antisense.
In an attempt to develop a lead for the application of 2-5A-antisense to the targeted destruction of human immunodeficiency virus (HIV) RNA, specific target sequences within the HIV mRNAs were identified by analysis of the theoretical secondary structure. 2-5A-antisense chimeras were chosen against a total of 11 different sequences: three in the gag mRNA, three in the rev mRNA and five in the tat mRNA. 2-5A-antisense chimera synthesis was accomplished using solid-phase phosphoramidite chemistry. These chimeras were evaluated for their activity in a cell-free assay system using purified recombinant human RNase L to effect cleavage of 32P-labelled RNA transcripts of plasmids derived from HIV NL4-3. This screening revealed that of the three 2-5A-antisense chimeras targeted against gag mRNA, only one had significant HIV RNA cleavage activity, approximately 10-fold-reduced compared to the parent 2-5A tetramer and comparable to that reported for the prototypical 2-5A-anti-PKR chimera, targeted against PKR mRNA. The cleavage activity of this chimera was specific, since a scrambled antisense domain chimera and a chimera without the key 5'-monophosphate moiety were both inactive. The 10 other 2-5A-antisense chimeras against tat and rev had significantly less activity. These results imply that HIV gag RNA, like PKR RNA and a model HIV tat-oligoA-vif RNA, can be cleaved using the 2-5A-antisense approach. The results further imply that not all regions of a potential RNA target are accessible to the 2-5A-antisense approach.
Player
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MR
Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0805, USA.
Maitra
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Silverman
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Torrence
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PF
D000227:Adenine Nucleotides / Q000235:genetics / Q000494:pharmacology*; D000998:Antiviral Agents / Q000138:chemical synthesis / Q000494:pharmacology; D002460:Cell Line; D004722:Endoribonucleases / Q000378:metabolism*; D006678:HIV / Q000235:genetics*; D006801:Humans; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000235:genetics / Q000494:pharmacology*; D016372:RNA, Antisense / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
D000227:Adenine Nucleotides; D000998:Antiviral Agents; D009843:Oligoribonucleotides; D016372:RNA, Antisense; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C023505:2',5'-oligoadenylate; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease