Nucleic Acids Research, 1995, Vol. 23, No. 15 2909-2913
© 1995
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Target sequence-specific inhibition of Hiv- replication by ribozymes directed to tatRNA
The R.W. Johnson Pharmaceutical Research Institute Sydney, Johnson and Johnson Research Pty Limited GPO Box 3331, Sydney NSW 2001, Australia
*To Whom correspondence should be addressed
Received April 21, 1995. Accepted June 30, 1995.
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-1 SF2 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-11MB 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-1 SF2 and HIV-1 IIIB. In vitro cleavage studies demonstrated that Rz1 efficiently cleaved HIV-1 SF2 substrate RNA, but not HIV-1 IIIB, presumably due to the base change from A to G. In contrast, Rz2 cleaved HIV-1 SF2 or HIV-1 IIIB 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 SupTl human CD4+ T cell line. Following selection, stable transfectants were challenged with either HIV-1 SF2 or HIV-1 IIIB virus. While Rz1-expressing cells were significantly protected from HIV-1 SF2 infection, they exhibited no protection when infected with HIV-1 IIIB virus. In contrast, Rz2 was effective in inhibiting the replication of both HIV-1 SF2 and HIV-1 IIIB 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
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