Site-Specific Cleavage of the Transactivation Response Site of Human Immunodeficiency Virus RNA with a Tat-Based Chemical Nuclease

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 89; no. 8; pp. 3526 - 3530
• Main Authors: Jayasena, Sumedha D., Johnston, Brian H.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 15-04-1992; National Acad Sciences; National Academy of Sciences
• Subjects: Acquired immune deficiency syndrome; AIDS; AIDS/HIV; Amino Acid Sequence; Arginine; Base Sequence; Binding Sites; Biological and medical sciences; Copper - pharmacology; Fundamental and applied biological sciences. Psychology; Gels; Gene Products, tat - genetics; Gene Products, tat - metabolism; Genetics; HIV; HIV 1; HIV 2; HIV-1 - genetics; HIV-1 - metabolism; HIV-2 - genetics; HIV-2 - metabolism; Humans; Lysine; Medical research; Microbiology; Molecular Sequence Data; Nucleic Acid Conformation; Nucleotides; Peptides - chemical synthesis; Peptides - metabolism; Phenanthrolines - pharmacology; Ribonucleases - metabolism; Ribonucleic acid; RNA; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Viral - genetics; RNA, Viral - metabolism; Sequencing; tat Gene Products, Human Immunodeficiency Virus; Transactivation; Transcriptional Activation; Transfer RNA; Virology; Peptides; Transcription; HIV-1 virus; Enzyme; Cleavage site; Retroviridae; Binding site; Ribonuclease; Virus; Synthetic product; Enzymatic activity; Lentivirinae; Trans acting regulatory factor; Regulatory sequence; Human immunodeficiency virus
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.8.3526

tat, an essential transactivator of gene transcription in the human immunodeficiency virus (HIV), is believed to activate viral gene expression by binding to the transactivation response (TAR) site located at the 5' end of all viral mRNAs. The TAR element forms a stem-loop structure containing a 3-nucleotide bulge that is the site for tat binding and is required for transactivation. Here we report the synthesis of a site-specific chemical ribonuclease based on the TAR binding domain of the HIV type 1 (HIV-1) tat. A peptide consisting of this 24-amino acid domain plus an additional C-terminal cysteine residue was chemically synthesized and covalently linked to 1,10-phenanthroline at the cysteine residue. The modified peptide binds to TAR sequences of both HIV-1 and HIV-2 and, in the presence of cupric ions and a reducing agent, cleaves these RNAs at specific sites. Cleavage sites on TAR sequences are consistent with peptide binding to the 3-nucleotide bulge, and the relative displacement of cleavage sites on the two strands suggests peptide binding to the major groove of the RNA. These results and existing evidence of the rapid cellular uptake of tat-derived peptides suggest that chemical nucleases based on tat may be useful for inactivating HIV mRNA in vivo.