Targeting RNase L to Human Immunodeficiency Virus RNA with 2-5A-Antisense

• Published in: Antiviral chemistry & chemotherapy Vol. 9; no. 3; pp. 225 - 231
• Main Authors: Player, Maitra, RK, Silverman, RH, Torrence, PF
• Format: Journal Article
• Language: English
• Published: London, England SAGE Publications 01-05-1998; International Medical Press; Sage Publications Ltd
• Subjects: Adenine Nucleotides - genetics; Adenine Nucleotides - pharmacology; AIDS/HIV; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antisense RNA; Antiviral agents; Antiviral Agents - chemical synthesis; Antiviral Agents - pharmacology; Biological and medical sciences; Cell Line; Chimeras; Endoribonucleases - metabolism; Gag protein; HIV; HIV - genetics; Human immunodeficiency virus; Humans; Medical sciences; Nucleic Acid Conformation; Oligoribonucleotides - chemical synthesis; Oligoribonucleotides - genetics; Oligoribonucleotides - pharmacology; Pharmacology. Drug treatments; Plasmids; Protein structure; Recombinant Proteins - genetics; Ribonuclease L; RNA viruses; RNA, Antisense - genetics; RNA, Messenger - genetics; RNA, Viral - metabolism; RNA-Binding Proteins - genetics; Secondary structure; Tat protein; Transcription; Viral Proteins - genetics; 2–5A; RNase L; RNA secondary structure; human immunodeficiency virus; AIDS; RNA degradation; oligonucleotides; Targeting; RNA; Enzyme; Retroviridae; Esterases; Ribonuclease; Antisense oligonucleotide; Lentivirus; In vitro; Biological activity; Virus; Characterization; Biological fixation; Preparation; Hydrolases; Antiviral; Cleavage; Human immunodeficiency virus; Gene therapy; Hybrid gene
• ISSN: 2040-2066; 0956-3202; 2040-2066
• DOI: 10.1177/095632029800900303

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, approximately10-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.