Target sequence-specific inhibition of HIV-1 replication by ribozymes directed to tat RNA.
The structural motif formed between a hammerhead ribozyme and its substrate consists of three RNA double helices in which the sequence 5' to the XUY is termed helix I and the sequence 3' to the XUY helix III. Two hammerhead ribozymes targeted to the tat gene of HIV-1SF2 were designed to study target specificity and the potential effect of helix I mismatch on ribozyme efficacy both in vitro and in vivo. The first ribozyme (Rz1) targeted to the 5' splicing region of the tat gene was designed to cleave GUC*A. In HIV-1IIIB the A is changed to a G. The second ribozyme (Rz2) was targeted to the translational initiation region of the tat gene which is highly conserved among a variety of HIV-1 isolates, including both HIV-1SF2 and HIV-1IIIB. In vitro cleavage studies demonstrated that Rz1 efficiency cleaved HIV-1SF2 substrate RNA, but not HIV-1IIIB, presumably due to the base change from A to G. In contrast, Rz2 cleaved HIV-1SF2 or HIV-1IIIB substrate with equal efficiency. Both ribozymes were cloned into the 3' untranslated region of the neomycin gene (neo) within the pSV2neo vector and transfected into the SupT1 human CD4+ T cell line. Following selection, stable transfectants were challenged with either HIV-1SF2 or HIV-1IIIB virus. While Rz1-expressing cells were significantly protected from HIV-1SF2 infection, they exhibited no protection when infected with HIV-1IIIB virus. In contrast, Rz2 was effective in inhibiting the replication of both HIV-1SF2 and HIV-1IIIB in SupT1 cells. Expression of both ribozymes in these cells was demonstrated by Northern analysis. RT-PCR sequencing analysis confirmed the respective HIV-1 target sequence integrity. These data demonstrate the importance of the first base pair distal to the XUY within helix I of the hammerhead structure for both in vitro and in vivo ribozyme activities and imply that the effectiveness of the anti-HIV-1 ribozymes against appropriate target sequences is due to their catalytic activities rather than any antisense effect.
Sun
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LQ
R.W. Johnson Pharmaceutical Research Institute Sydney, NSW, Australia.
Wang
L
L
Gerlach
W L
WL
Symonds
G
G
D001483:Base Sequence; D002460:Cell Line; D018387:Codon, Initiator / Q000235:genetics; D015756:Genes, tat / Q000235:genetics*; D014644:Genetic Variation / Q000235:genetics; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D012313:RNA / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D050378:T-Lymphocytes, Regulatory / Q000821:virology; D014779:Virus Replication
D018387:Codon, Initiator; D009692:Nucleic Acid Heteroduplexes; D016337:RNA, Catalytic; D012367:RNA, Viral; C039919:RNA, recombinant; D012313:RNA; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase