Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and Rev-Response Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses.
Keywords: Ago2, Argonaute-2; EGFP, enhanced green fluorescent protein; EMSA, electrophoresis mobility shift assay; FL, firefly luciferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HIV, human immunodeficiency virus; HIV-1; IP, immunoprecipitation; NC, nucleocapsid; PAGE, polyacrylamide gel electrophoresis; RISC, RNA-Induced Silencing Complex; RL, Renilla luciferase; RNA interference; RNA silencing suppressor; RNAi, RNA interference; RRE, Rev Response Element; RSS, RNA silencing suppressor; RT, reverse transcription; Rev-Response Element RNA; TAR RNA Binding Protein (TRBP); TAR, trans-activation responsive element; TRBP, TAR RNA Binding Protein; Trans-Activation Response Element; UTR, untranslated region; VA, virus-associated; WT, wild-type; adenovirus; ds, double-stranded; lentiviral vectors; miRNA, micro RNA; pre-miRNA, precursor miRNA; siRNA, small interfering RNA.