Abstract
BackgroundRNA 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.ResultsAn 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.ConclusionsThe 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.
Highlights
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
Most miRNAs are processed from longer primary miRNA transcripts, which are transcribed from genome sequence by RNA polymerase II promoter [4,5]
This study demonstrated that the flanking pri-miRNA sequence can be replaced and optimized with artificial sequence to construct the polycistronic transcript that expresses three anti-HIV small interfering RNAs (siRNAs) simultaneously and efficiently inhibits HIV-1 replication
Summary
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. 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. Many strategies have been proposed to inhibit HIV-1 replication in cell culture and animal models, including siRNA or short hairpin RNA (shRNA) vectorbased or pri-miRNA vector-based approaches [23,24,25]. These vector-based approaches have demonstrated longterm inhibition of HIV replication. A combination of multiple antiviral inhibitors to overcome escape has been proposed
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