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.
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-ribozymes 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.
Chen
C J
CJ
Laboratory of Viral and Molecular Pathogenesis, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
Banerjea
A C
AC
Harmison
G G
GG
Haglund
K
K
Schubert
M
M
D001483:Base Sequence; D003001:Cloning, Molecular; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D015752:Genes, env / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D016337:RNA, Catalytic / Q000378:metabolism* / Q000494:pharmacology; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics*
D016337:RNA, Catalytic; D012367:RNA, Viral