Inhibition of HIV-1 gene expression by novel macrophage-tropic DNA enzymes targeted to cleave HIV-1 TAT/Rev RNA.
Many regions of the HIV-1 genome have been targeted in earlier studies by RNA-cleaving DNA enzymes possessing the 10-23 catalytic motif, and efficient inhibition of HIV-1 gene expression was reported. All these studies employed charged synthetic lipids to introduce the catalytic DNA into the mammalian cells, which severely limits its practical application and usefulness in vivo. Taking advantage of the ability of G residues to interact directly with the scavenger receptors on the macrophages, we synthesized a DNA enzyme 5970 that contained 10 G residues at the 3' end. With the aim of improving the intracellular stability of the DNA enzyme 5970, we added two short stretches of stem-loop structures that were 12 bases long on either side of the DNA enzyme 5970. DNA enzyme 5970 without the poly-G tracts cleaved the synthetic RNA of HIV-1 TAT/Rev, two important regulatory proteins of HIV, very efficiently in a sequence-specific manner. Addition of 10 G residues at the 3' end of the DNA enzyme affected the cleavage efficiency only marginally whereas the same DNA enzyme with stem-loop structures on either end was significantly less efficient. The DNA enzyme with the poly-G tract at its 3' end was taken up specifically by a human macrophage-specific cell line directly in the absence of Lipofectin and was also able to inhibit HIV-1 gene expression in a transient-expression system as well as when challenged with the virus. The potential applications of these novel macrophage-tropic DNA enzymes are discussed.
Unwalla
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Laboratory of Virology, National Institute of Immunology, JNU Campus, Aruna Asaf Ali Marg, New Delhi-110067, India.
Banerjea
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D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D021881:DNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D015694:Gene Products, rev / Q000235:genetics; D015696:Gene Products, tat / Q000235:genetics; D015757:Genes, rev / Q000502:physiology*; D015756:Genes, tat / Q000502:physiology*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology* / Q000821:virology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D021881:DNA, Catalytic; D015694:Gene Products, rev; D015696:Gene Products, tat; D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus