Liu Ying Poi, Haasnoot Joost, ter Brake Olivier, Berkhout Ben, Konstantinova Pavlina
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
Nucleic Acids Res. 2008 May;36(9):2811-24. doi: 10.1093/nar/gkn109. Epub 2008 Mar 16.
RNA interference (RNAi) is a powerful approach to inhibit human immunodeficiency virus type 1 (HIV-1) replication. However, HIV-1 can escape from RNAi-mediated antiviral therapy by selection of mutations in the targeted sequence. To prevent viral escape, multiple small interfering RNAs (siRNAs) against conserved viral sequences should be combined. Ideally, these RNA inhibitors should be expressed simultaneously from a single transgene transcript. In this study, we tested a multiplex microRNA (miRNA) expression strategy by inserting multiple effective anti-HIV siRNA sequences in the miRNA polycistron mir-17-92. Individual anti-HIV miRNAs that resemble the natural miRNA structures were optimized by varying the siRNA position in the hairpin stem to obtain maximal effectiveness against luciferase reporters and HIV-1. We show that an antiviral miRNA construct can have a greater intrinsic inhibitory activity than a conventional short hairpin (shRNA) construct. When combined in a polycistron setting, the silencing activity of an individual miRNA is strongly boosted. We demonstrate that HIV-1 replication can be efficiently inhibited by simultaneous expression of four antiviral siRNAs from the polycistronic miRNA transcript. These combined results indicate that a multiplex miRNA strategy may be a promising therapeutic approach to attack escape-prone viral pathogens.
RNA干扰（RNAi）是一种抑制人类免疫缺陷病毒1型（HIV-1）复制的有效方法。然而，HIV-1可通过在靶向序列中选择突变来逃避RNAi介导的抗病毒治疗。为防止病毒逃逸，应将针对保守病毒序列的多个小干扰RNA（siRNA）联合使用。理想情况下，这些RNA抑制剂应从单个转基因转录本中同时表达。在本研究中，我们通过在miRNA多顺反子mir-17-92中插入多个有效的抗HIV siRNA序列，测试了一种多重微小RNA（miRNA）表达策略。通过改变发夹茎中siRNA的位置，优化了类似于天然miRNA结构的单个抗HIV miRNA，以获得对荧光素酶报告基因和HIV-1的最大抑制效果。我们表明，抗病毒miRNA构建体比传统短发夹（shRNA）构建体具有更强的内在抑制活性。当在多顺反子环境中联合使用时，单个miRNA的沉默活性会大大增强。我们证明，从多顺反子miRNA转录本中同时表达四种抗病毒siRNA可有效抑制HIV-1复制。这些综合结果表明，多重miRNA策略可能是一种有前途的治疗方法，可用于对抗易逃逸的病毒病原体。
