Multitarget-ribozyme directed to cleave at up to nine highly conserved HIV-1 env RNA regions inhibits HIV-1 replication—potential effectiveness against most presently sequenced HIV-1 isolates

• Published in: Nucleic acids research 1992-09, Vol.20 (17), p.4581-4589
• Main Authors: Chen, Chang-Jie, Banerjea, Akhil C., Harmison, George G., Haglund, Karl, Schubert, Manfred
• Format: Article
• Language: English
• Subjects: AIDS/HIV; Base Sequence; Cloning, Molecular; Gene Expression Regulation, Viral - genetics; Genes, env - genetics; HeLa Cells; HIV-1 - genetics; Human immunodeficiency virus 1; Humans; Molecular Sequence Data; Nucleic Acid Conformation; Plasmids - genetics; RNA, Catalytic - metabolism; RNA, Catalytic - pharmacology; RNA, Viral - genetics; RNA, Viral - metabolism; Simian virus 40; Virus Replication - drug effects; Virus Replication - genetics
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/20.17.4581

Several mono-, di-, tetra-, penta- and nonaribozymes were developed. These multitarget-ribozymes were targeted to cleave HIV-1 env RNA at up to nine different conserved sites. Each multitarget-ribozyme consisted of a chain of up to nine hammerhead motifs, each flanked by a different targeting sequence. The multitarget-ribozymes were functional in vitro and gave rise to multiple, specific partial and/or complete RNA digestion products. Per RNA copy, multitarget ribozymes were more efficient than monoribozymes or ribozymes targeting a subset of the same sites. In contrast to monoribozymes, a 400nt nonaribozyme, targeted to cleave at nine different sites within a 1.3kb HIV-1 env RNA substrate, was active and showed the same specificity of cleavage when it was part of a large 3.3kb transcript. We conclude that multitarget ribozymes retain the specificity of monoribozymes, but they are more efficient per ribozyme RNA copy and they remain active when they are part of a large transcript. A tetra-, penta- or nonaribozyme under control of the SV40 late promoter, the beta-actin gene promoter or the HIV-1 LTR, respectively, were cotransfected with the infectious HIV-1 DNA clone pNL4—3 Into permissive HeLa T4 cells. Each cotransfection resulted in a specific inhibition of HIV-1 replication as determined by syncytia formation and p24 antigen release. In addition, coexpression of the nonaribozyme with an HIV-1 env RNA transcript resulted in the specific dramatic reduction of the env transcript. We conclude that the multitarget-ribozymes are also functional intracellularly. A nucleotide sequence comparison of the target sites indicates that the multitarget-ilbozymes could potentially be effective against all thirty HIV-1 isolates presently sequenced. Their use may help to slow the selection of viral escape mutants and thereby prolong their effectiveness. We anticipate that multitarget-ribozymes will also be more effective in the successful targeting of less variable cellular RNAs.