National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Research Center for Medical Molecular Virology of Fujian Province, School of Life Science, Xiamen University, Xiamen, 361005, People's Republic of China.
Virol J. 2012 Jun 18;9:118. doi: 10.1186/1743-422X-9-118.
RNA interference (RNAi) has been used as a promising approach to inhibit human immunodeficiency virus type 1 (HIV-1) replication for both in vitro and in vivo animal models. However, HIV-1 escape mutants after RNAi treatment have been reported. Expressing multiple small interfering RNAs (siRNAs) against conserved viral sequences can serve as a genetic barrier for viral escape, and optimization of the efficiency of this process was the aim of this study.
An artificial polycistronic transcript driven by a CMV promoter was designed to inhibit HIV-1 replication. The artificial polycistronic transcript contained two pre-miR-30a backbones and one pre-miR-155 backbone, which are linked by a sequence derived from antisense RNA sequence targeting the HIV-1 env gene. Our results demonstrated that this artificial polycistronic transcript simultaneously expresses three anti-HIV siRNAs and efficiently inhibits HIV-1 replication. In addition, the biosafety of MT-4 cells expressing this polycistronic miRNA transcript was evaluated, and no apparent impacts on cell proliferation rate, interferon response, and interruption of native miRNA processing were observed.
The strategy described here to generate an artificial polycistronic transcript to inhibit viral replication provided an opportunity to select and optimize many factors to yield highly efficient constructs expressing multiple siRNAs against viral infection.
RNA 干扰 (RNAi) 已被用作抑制人类免疫缺陷病毒 1 型 (HIV-1) 复制的一种有前途的方法，无论是在体外还是在体内动物模型中。然而，据报道，在 RNAi 治疗后 HIV-1 会出现逃逸突变。针对保守病毒序列表达多种小干扰 RNA (siRNA) 可以作为病毒逃逸的遗传障碍，本研究旨在优化这一过程的效率。
设计了一种由 CMV 启动子驱动的人工多顺反子转录本，以抑制 HIV-1 复制。人工多顺反子转录本包含两个 pre-miR-30a 骨架和一个 pre-miR-155 骨架，由靶向 HIV-1 env 基因的反义 RNA 序列的序列连接。我们的结果表明，这种人工多顺反子转录本同时表达三种抗 HIV siRNA，有效抑制 HIV-1 复制。此外，评估了表达这种多顺反子 miRNA 转录本的 MT-4 细胞的生物安全性，未观察到对细胞增殖率、干扰素反应和内源性 miRNA 处理的中断有明显影响。
本研究中描述的生成人工多顺反子转录本以抑制病毒复制的策略为选择和优化产生高效表达多种针对病毒感染的 siRNA 的构建体提供了机会。
