MULTITARGET-RIBOZYME DIRECTED TO CLEAVE AT UP TO 9 HIGHLY CONSERVED HIV-1 ENV RNA REGIONS INHIBITS HIV-1 REPLICATION POTENTIAL EFFECTIVENESS AGAINST MOST PRESENTLY SEQUENCED HIV-1 ISOLATES
MULTITARGET-RIBOZYME DIRECTED TO CLEAVE AT UP TO 9 HIGHLY CONSERVED HIV-1 ENV RNA REGIONS INHIBITS HIV-1 REPLICATION POTENTIAL EFFECTIVENESS AGAINST MOST PRESENTLY SEQUENCED HIV-1 ISOLATES
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DOI：
10.1093/nar/20.17.4581
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发表时间：
1992-09-11
影响因子：
14.9
通讯作者：
SCHUBERT, M
中科院分区：
文献类型：
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作者：
CHEN, CJ;BANERJEA, AC;SCHUBERT, M
几个单，双，四，五和nonaribozyme开发。这些多靶点核酶靶向切割HIV-1 env RNA在多达9个不同的保守位点。每个多靶点核酶由多达9个锤头状基序的链组成，每个基序的侧翼是不同的靶向序列。多靶核酶在体外是功能性的，并产生多个，特定的部分和/或完整的RNA消化产物。每个RNA拷贝，多靶核酶比单核酶或核酶靶向相同位点的子集更有效。与单核糖酶相比，靶向切割1.3kb HIV-1 env RNA底物内9个不同位点的400 nt nonaribozyme具有活性，并且当它是大的3.3kb转录物的一部分时显示出相同的切割特异性。我们的结论是，多靶核酶保留了单核酶的特异性，但它们更有效的每个核酶RNA拷贝，当它们是一个大的转录本的一部分时，它们仍然是活跃的。分别在SV 40晚期启动子、β-肌动蛋白基因启动子或HIV-1 LTR控制下的四、五或九核糖体酶与感染性HIV-1 DNA克隆pNL 4 - 3共转染到允许的HeLa T4细胞中。通过合胞体形成和p24抗原释放测定，每种共转染导致HIV-1复制的特异性抑制。此外，nonaribozyme与HIV-1 env RNA转录物的共表达导致env转录物的特异性显著减少。我们的结论是，多靶点核酶也是细胞内的功能。靶位点的核苷酸序列比较表明，多靶核酶可能对目前测序的所有30种HIV-1分离株有效。它们的使用可能有助于减缓病毒逃逸突变体的选择，从而延长其有效性。我们预计，多靶点核酶也将更有效地成功靶向变异较小的细胞RNA。
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.