Zhang Tao, Cheng Tong, Zhang Ya-Li, Wei Li-Hua, Cai Yi-Jun, Zhang Jun, Zhu Hua, Han Jia-Huai, Xia Ning-Shao
National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen 361005, China.
Bing Du Xue Bao. 2008 Jun;24(2):88-95.
Discovery of the RNA interference (RNAi) pathway has led to exciting new strategies for developing HIV treatment. This study was to find out the highly effective and conserved siRNA target sequences for improving RNAi-based therapy against the HIV-1. We constructed 30 shRNA expression plasmids for expressing different siRNAs targeted to HIV-1 vif and co-transfected them with the pNL4-3 to score for its ability to inhibit the expression of p24 protein of HIV-1. Then, the highly effective siRNAs targeting sequences were selected to align with 625 HIV-1 sequences in database including all HIV-1 subtypes to ana lyze their conserved character. In addition, vif37 the highly effective and most conserved target sequence was confirmed of its sequence-specific inhibition by independent reporter assays. MT-4 cell transduced with lentiviral shRNA-vif37 vector could inhibit HIV-1(NL4.3) replication in vitro. Moreover, MT-4-vif37 cloned from transduced MT-4 cell could stably express shRNA-vif37 and inhibit virus replication more efficiently when challenged with high titer virus. These results showed that RNAi has great potential as an antiviral gene therapy approach and supports the efforts to develop treatment for HIV-1-infected individuals.
RNA干扰（RNAi）途径的发现为开发艾滋病病毒治疗方法带来了令人兴奋的新策略。本研究旨在寻找高效且保守的小干扰RNA（siRNA）靶序列，以改进基于RNAi的抗HIV-1疗法。我们构建了30个短发夹RNA（shRNA）表达质粒，用于表达靶向HIV-1病毒感染因子（vif）的不同siRNA，并将它们与pNL4-3共转染，以评估其抑制HIV-1 p24蛋白表达的能力。然后，选择高效siRNA靶向序列与数据库中包括所有HIV-1亚型的625条HIV-1序列进行比对，分析其保守性。此外，通过独立的报告基因检测证实了高效且最保守的靶序列vif37具有序列特异性抑制作用。用慢病毒shRNA-vif37载体转导的MT-4细胞能够在体外抑制HIV-1（NL4.3）复制。此外，从转导的MT-4细胞克隆的MT-4-vif37能够稳定表达shRNA-vif37，并且在受到高滴度病毒攻击时更有效地抑制病毒复制。这些结果表明，RNAi作为一种抗病毒基因治疗方法具有巨大潜力，并支持为感染HIV-1的个体开发治疗方法的努力。
