Detection of HIV-1 nef miRNAs and inhibition of Nef expression by nef RNAs. (A) Schematic representation of HIV-1 nef in its genome and synthetic DNA probes (#) used in this study. (B) Total RNAs were extracted from MT-4 T cells persistently infected with HIV-1 IIIB, separated on a 15% polyacrylamide-7 M urea gel, and subjected to northern blot analysis. The approximate sizes of the three classes of HIV-1 transcripts and small RNAs are indicated on the right. The loading control was rRNA stained with ethidium bromide. Relative expression (%) of nef small RNAs to the three classes of HIV-1 transcripts is at the bottom of figure. (C) Schematic representation of effector plasmids (E) H1 promoter-driven shRNA expression plasmids. Reporter (R) Nef-EGFP expression plasmid (pYM2.2) is also shown. (D) Inhibition by sinefs in pH1 plamids of Nef-EGFP expression. Either sinef, siluc or siegfp in each plasmid was transfected into Jurkat T cells in the presence of either pYM2.2 or control pEGFP-N1. At 36 h after transfection EGFP-positive cells were counted by flow cytometry. Data represent the relative activity of EGFP-positive cells where the percentage of positive cells in the sample transfected with pYM2.2 or pEGFP-N1 plus pcDNA3.1 or si(-) in pH1 plasmid was scored as 100%. Data are averages of three independent experiments + SD. Bars, SD. (E) Immunoblot analysis showing inhibition of YM2.2 expression by different nef shRNAs. Jurakat T cells were transfected with pYM2.2 and pH1/sinefs or siluc plasmid, cellular lysates were prepared 48 h after transfection, and immunoblotted with rabbit serum against Nef (upper panel) and anti-β-actin antibody (lower panel). The β-actin expression shows equal loading of all samples.