Sequence specific cleavage of the HIV-1 coreceptor CCR5 gene by a hammer-head ribozyme and a DNA-enzyme: inhibition of the coreceptor function by DNA-enzyme.
The chemokine receptor CCR5 is used as a major coreceptor for fusion and entry by non-syncytia inducing macrophage tropic isolates of HIV-1, which is mainly involved in transmission. Individuals who are homozygous for the delta32 allele of CCR5 are usually resistant to HIV-1 infection and continue to lead a normal healthy life. Thus this gene is dispensable and is, therefore, an attractive target in the host cell for interfering specifically with the virus-host interaction. With the aim to develop a specific antiviral approach at the molecular level, we have synthesized a hammer-head ribozyme and a DNA-enzyme. Both ribozyme and DNA-enzyme cleaved the CCR5 RNA in a sequence specific manner. This cleavage was protein independent but Mg2+ dependent. The extent of cleavage increased with increasing concentration of magnesium chloride. DNA-enzyme was more effective in cleaving a full length (1376 bases) in vitro generated transcript of CCR5 gene. In this communication, we show that the DNA-enzyme when introduced into a mammalian cell, results in decreased CD4-CCR5-gp160 mediated fusion of cell membranes. Potential applications of these trans acting molecules are discussed.
Laboratory of Virology, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
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D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D019713:Receptors, CCR5 / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity; D014158:Transcription, Genetic
D016337:RNA, Catalytic; D019713:Receptors, CCR5; D011994:Recombinant Proteins; D004247:DNA; C025580:endodeoxyribonuclease XBAI; D015252:Deoxyribonucleases, Type II Site-Specific