Fig. (3).

Per-1 suppresses HIV-1 transcription in post-activated resting CD4+ T-cells. (A) Schematic representation of the experimental design. CD4+ T-cells were stimulated with CD3/CD28 activator magnetic beads and IL-2 for 48 h and transduced with shRNA against Per-1 or control lentivirus in the presence of puromycin selection. CD4+ T-cells were cultured with gradual dilutions of IL-2 to transform them into the resting state (i.e., CD69, CD25, and cell proliferation were measured by flow cytometry as shown in Fig. S3). The resting cells were further treated with VLP-Vpx for 6 h and spinoculated with HIV-1_NL4.3.Luc. At 48 h after infection, the cells were lysed to measure the luciferase reporter levels (B). Genomic DNA and total RNA were extracted for qPCR to assess HIV-1 integration by Alu-PCR (C) and viral Gag (D) and Per-1 (E) transcripts normalized to GAPDH. (F) Resting CD4+ T-cells were electroporated with siRNAs against Per-1 or control along with pNL4.3.Luc or CMV-Luc vectors and cultured for 2 days before lysis and then measured for the luciferase reporter levels, which were normalized between the pNL4.3.Luc and CMV-Luc samples. Total RNA was extracted for qPCR to assess Per-1 transcripts normalized to GAPDH. (G) Resting CD4+ T-cells were electroporated with siRNAs against Per-1 along with LTR- or CMV-Luc vectors and then cultured for 2 days. The cells were lysed to measure the luciferase reporter levels, which were normalized between the LTR- and CMV-Luc samples. Total RNA was extracted for qPCR to assess Per-1 transcripts normalized to GAPDH. RLU, relative luminescence units. *P < 0.05, **P < 0.01 (Student's t-test). NS, not significant. Data are presented as mean ± S.E.M. from three independent experiments.