Fig 2. NOP2 suppresses HIV-1 transcription.

(A) TZM-bl cells stably expressing FLAG-tagged HIV-1 Tat protein in the retroviral vector pQXCIP (pQCXIP-Tat) were transiently transduced with the indicated shRNA (shNT or shNOP2) in pAPM vector, and NOP2 knockdown was analyzed by reverse transcription coupled qPCR (RT-qPCR). (B) The RLU of cells in (A) was measured, and normalized to that of shNT. (C) HEK293 cells were stably transduced with the indicated vectors (pLEX-FLAG or pLEX-NOP2), and protein expression of V5-NOP2 was analyzed by immunoblotting. (D) The cells in (C) were transiently co-transfected with pcDNA-Tat, HIV-LTR-Luciferase, and pTK-Renilla vectors, followed by the dual-luciferase reporter assay. The RLU (luciferase/renilla) was measured, and normalized to that of pLEX-FLAG. (E) Jurkat cells were stably transduced with the indicated shRNA (shNT, shNOP2, or shNSUN2) in pAPM vector, and NOP2 or NSUN2 knockdown was analyzed by RT-qPCR. (F) The cells in (E) were infected with HIV-1 IIIB viruses, followed by the RNA extraction and RT-qPCR to measure HIV-1 initiated or elongated (proximal [Pro], intermediate [Int], and distal [Dis]) transcripts. The level of each HIV-1 transcript was normalized to that of shNT. Results were based on n = 3 experiments and presented as mean ± S.D., * p < 0.05; ** p < 0.01; *** p < 0.001, ANOVA.