Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Mol Ther. 2009 Oct;17(10):1712-23. doi: 10.1038/mt.2009.176. Epub 2009 Aug 11.
RNA interference (RNAi) is a widely used gene suppression tool that holds great promise as a novel antiviral approach. However, for error-prone viruses including human immunodeficiency virus type 1(HIV-1), a combinatorial approach against multiple conserved sequences is required to prevent the emergence of RNAi-resistant escape viruses. Previously, we constructed extended short hairpin RNAs (e-shRNAs) that encode two potent small interfering RNAs (siRNAs) (e2-shRNAs). We showed that a minimal hairpin stem length of 43 base pairs (bp) is needed to obtain two functional siRNAs. In this study, we elaborated on the e2-shRNA design to make e-shRNAs encoding three or four antiviral siRNAs. We demonstrate that siRNA production and the antiviral effect is optimal for e3-shRNA of 66 bp. Further extension of the hairpin stem results in a loss of RNAi activity. The same was observed for long hairpin RNAs (lhRNAs) that target consecutive HIV-1 sequences. Importantly, we show that HIV-1 replication is durably inhibited in T cells stably transduced with a lentiviral vector containing the e3-shRNA expression cassette. These results show that e-shRNAs can be used as a combinatorial RNAi approach to target error-prone viruses.
RNA 干扰 (RNAi) 是一种广泛应用的基因抑制工具，作为一种新型抗病毒方法具有很大的潜力。然而，对于易错的病毒，包括人类免疫缺陷病毒 1 型 (HIV-1)，需要针对多个保守序列的组合方法来防止 RNAi 抗性逃逸病毒的出现。先前，我们构建了扩展短发夹 RNA (e-shRNA)，其编码两个有效的小干扰 RNA (siRNA) (e2-shRNA)。我们表明，获得两个功能 siRNA 需要最小发夹茎长度为 43 个碱基对 (bp)。在这项研究中，我们详细阐述了 e2-shRNA 的设计，以制造编码三个或四个抗病毒 siRNA 的 e-shRNA。我们证明，siRNA 的产生和抗病毒效果对于 66bp 的 e3-shRNA 是最佳的。发夹茎的进一步延伸导致 RNAi 活性丧失。针对连续 HIV-1 序列的长发夹 RNA (lhRNA) 也观察到了同样的情况。重要的是，我们表明，含有 e3-shRNA 表达盒的慢病毒载体稳定转导的 T 细胞中，HIV-1 的复制被持久抑制。这些结果表明，e-shRNA 可作为一种针对易错病毒的组合 RNAi 方法。
