A lentiviral vector expressing RRE decreases miRNA-mediated RNAi activity in the absence of Rev or GagPol. (A) pHIV7-tdTomato lentiviral vector expressing RRE partially prevents let7-mediated inhibition of RL. (top) Schematic representation of pHIV7-tdTomato vector. Relevant genes and regulatory elements are shown. (bottom) HeLa cells were transfected with 0.25μg of pcDNA3, pHIV7-tdTomato or pNL4–3 as indicated and the assay was carried out as in Fig. 1D. (B) Rev and GagPol restore RNAi suppressed by HIV7-tdTomato lentiviral vector. HeLa cells were cotransfected with 0.4 μg pEGFP or pEGFP-clet7 and with 1μg of pcDNA3, pHIV7-tdTomato, pCMV-Rev, pCHGP expressing GagPol or pHIV7-tdTomato with pCMV-Rev, pCHGP as indicated. The assay was carried out as in Fig. 1C. (C) Expression of TAR and RRE RNAs from HIV7-tdTomato. HeLa cells were mock-transfected or transfected with 1 μg of pcDNA3, pcDNA3-TAR, pcDNA3-RRE or pHIV7-tdTomato as indicated. RNAs were recovered and analyzed by RT-PCR, with primers specific for TAR, RRE and GAPDH.