Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL
Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL
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DOI：
10.1093/nar/gki164
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Takaku, H
中科院分区：
文献类型：
--
作者：
Habu, Y;Miyano-Kurosaki, N;Takaku, H
tRNA 3 '-加工核糖核酸内切酶（tRNase Z或3' tRNase; EC 3.1.26.11）是从前体tRNA去除3'尾部的必需酶。长形式（tRNase ZL）可以在体外在由适当的小向导RNA（sgRNA）指导的位点切割靶RNA。在这里，我们研究了这种sgRNA/tRNase ZL策略是否可以应用于艾滋病的基因治疗。我们使用瞬时表达试验测试了四种sgRNA表达质粒抑制COS细胞中HIV-1基因表达的能力。这三种sgRNA指导抑制培养的COS细胞中的HIV-1基因表达。对HIV-1 mRNA水平的分析表明，sgRNA指导tRNase ZL介导靶RNA的降解。sgRNA主要定位于细胞核中的观察结果表明，当在该位置与sgRNA复合时，tRNase ZL切割HIV-1 mRNA。我们还检查了表达sgRNA的两种逆转录病毒载体在HIV-1感染的Jurkat T细胞中抑制HIV-1表达的能力。sgRNA-SL 4在感染细胞中几乎完全抑制HIV-1表达长达18天。这些结果表明，sgRNA/tRNase ZL方法在下调HIV-1基因表达中是有效的。
The tRNA 3'-processing endoribonuclease (tRNase Z or 3' tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3' trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression.