Inhibition of nef expression in human T cells by nef siRNAs. (A) Schematic representation of shRNA-expressing STYLE vector (E) and HIV-1 SF2 nef gene expressing pPFV/nef vector (R). The helper plasmid, pFFenv expresses FFV envelope protein under the control of CMV promoter. (B) Detection of expression of nef mRNA and integration of vectors. STYLE, SKY3.0, PFV/nef, PFV or mock was used to infect Jurkat T cells and the infected cells were cultured for 2 weeks. After 2 weeks, gag and nef mRNA expression was measured by RT-PCR. Genomic DNA of LTR, gag and nef of STYLE or PFV/nef were also detected by PCR. β-actin was used as a control. (C) Inhibition of Nef-EGFP expression by nef siRNA-expressing STYLE in Jurkat T cells. The pYM2.2 was transfected into each of the STYLE or mock-infected Jurkat T cells and EGFP-expressing cells were counted by flow cytometry at 48 h after transfection. Data represent the relative activity of EGFP-positive cells, where the percentage of positive cells in the sample transfected with pYM2.2 upon the STYLE-si(-) infected cells was scored as 100%. (D) Inhibition of HIV-1 transcription and replication by nef STYLE-367. HIV-1 IIIB persistently infected MT-4 T cells were transfected with the pLTRSF2 reporter and β-gal expressing control pCMVβ plasmids at 72 h after infection with STYLE. At 48 h post-transfection, Luc activity was measured and normalized as Luc values (Luc/β-gal). Absolute levels of Luc activity in the samples of pLTRSF2 plus SRYLE-si(-) were 16,311 + 1,253 or 783 + 87 light units for STYLE-367/miR-N367 transfectants. Data represent the relative Luc activities where the percentage of positive cells in the samples infected with the STYLE-si(-) was scored as 100%. After 48 h, p24 antigen was also measured in the cell culture supernatant of STYLE-infected Jurkat T cells. Data are averages of three independent experiments + SD. Bars, SD. (E) Inhibition of nef expression by nef siRNA in Jurkat T cells. Cells were infected with PFV/nef 48 h after infection with the STYLE and then subjected to semi-quantitative RT-PCR analysis. Data represent the relative expression of mRNA, where the percentage of positive cells in the sample of mock-infected cells (E: Mock) relative to the PFV/nef (R: PFV/nef) infected cells was scored as 100%. Data averages were derived from three independent experiments + SD. Bars, SD.