ABSTRACT
Effective and specific RNA interference (RNAi) elements are essential for the RNAi-based anti-HIV-1 research which has achieved extensive application. vif37 targeted to HIV-1 vi f is a highly effective and conserved RNAi target obtained from the previous study on screening. In this study, we explored the construction of artificial miRNAs to induce RNAi targeted to vif37, which had advantages on inhibition efficiency and flexibility of promoter selection. Three artificial miRNA targeted to vif37 were constructed by walking method using native miR-155 as a backbone and expressed by RNA polymerase II promoter. Then, expression vectors of artificial miRNA were co-transfected with HIV-1 infectious clone pNL4-3 to score its inhibition ability and showed that only miR-vif37 had the significant inhibition efficiency similar to shRNA-vif37. Subsequently, co-transfections with luciferase reporter plasmids into which different target sequences were inserted proved the specificity of miR-vif37 H. The replication of HIV-1 was inhibited in MT-4-miR37 H cells which could express miR-vif37 H stably and were cloned from MT-4 cells transducted with recombinant lentiviral vectors containing the miR-vif37 H expression element. Real-time RT-PCR revealed that miR-vif37 H had much lower expression level than shRNA-vif37. Results also showed that intracellular miR-181 and miR-16 expression levels and stat1 mRNA levels were not effected by the expression of miR-vif37 H in MT-4-miR37 H cells. We conclude miR-vif37 is a specific and highly effective artificial miRNA which will promote the further application of vif37 target.
Subject(s)
Humans , Cell Line , Gene Targeting , Methods , Genetic Vectors , Genetics , Metabolism , HIV Infections , Virology , HIV-1 , Genetics , Physiology , Lentivirus , Genetics , Metabolism , MicroRNAs , Genetics , Metabolism , Nucleic Acid Conformation , RNA Interference , vif Gene Products, Human Immunodeficiency Virus , Chemistry , Genetics , MetabolismABSTRACT
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.