Scripts ( 1 to 40) and elongated transcripts ( 5396 to 5531) (Fig. 1D). The levels of initiated transcript had been comparable in siControl and siNELF-treated cells, indicating that RNAP II was present in the transcriptional start off site, whereas more elongated transcripts were seen in siNELF treated cells, consistent with RNAP II pausing limiting HIV transcription in key T cells. These adjustments in provirus transcription corresponded to about a 7-fold boost in HIV release, as measured by p24 inside the supernatant (Fig. 1E). To achieve insights into how silencing NELF induces HIV transcription inside the cell population, we infected CD4 T cells with a HIV-PLAP reporter virus that expresses PLAP on the surface of HIV-positive cells (20) then transfected these infected cells with siControl or siNELF. PLAP was assessed by flow cytometry. A modest 45 boost in HIV-expressing cells was observed (Fig. 1F), suggesting that the induction of transcription in aspect reflected the activation of infected cells not previously expressing HIV. Activating infected cells with anti-CD3 plus antiCD28 antibodies, which didn’t rescue NELF expression in siRNA-treated CD4 T cells (Fig. 1G), enhanced HIV transcription, monitored by luciferase (Fig. 1H), regardless of whether or not cells have been treated with siControl or siNELF-B. These data indicate that RNAP II pausing is a important checkpoint for basal HIV transcription but is bypassed when conditions favor HIV transcription elongation. As a result, NELF-mediated RNAP II pausing limits provirus transcription in primary CD4 T cells. RNAP II Pausing Is Coupled with Premature Termination in Limiting HIV Transcription–We showed previously that both NELF and Pcf11 restricted HIV transcription in U1 cells (17, 18). We had been interested in exploring no matter whether NELF and Pcf11 act independently or cooperatively to regulate HIV transcription in principal cells. We utilized siRNAs to diminish both Pcf11 and NELF in principal CD4 T cells. RT-PCR and μ Opioid Receptor/MOR Inhibitor Compound immunoblot analyses indicated that expression of Pcf11 and NELF were regularly decreased by 40 ?60 (Figs. two, A ). Attempts to improve the efficiency of these knockdowns promoted cell death, suggesting that these are critical elements. Measuring initiated and elongated HIV transcripts from CD4 T cells infected with HIV-LUC showed that depletion of Pcf11, or both NELF and Pcf11, improved processive transcription compared with siControl-treated cells (Fig. 2D). Moreover, depletingJOURNAL OF BIOLOGICAL CHEMISTRYRESULTS NELF Limits HIV Transcription in Major T Cells–Our earlier research demonstrating that NELF limits HIV transcription utilized latently HIV-infected premonocytic U1 cells, which carry two copies of provirus that harbor Tat mutations (18). It is actually probable that Tat mutations contribute to the lack of RNAP II processivity observed in U1 cells (30). We wanted to establish no matter whether RNAP II pausing had a part in limiting HIVSEPTEMBER 6, 2013 ?VOLUME 288 ?NUMBERRNA Polymerase II Pausing Represses HIV TranscriptionA) B) 1.eight 1.6 1.four 1.2 1.0 0.eight 0.six 0.four 0.two 0 C) Basal Tr 100 80 60 40 20 P 0.D)e NELF-B expression4 3.5 3 2.5 2 1.5 1 0.five P 0.Luciferase unitse HIV transcriptsNELF-Belongatedelongated P 0.ReResiCtrl G)siNELF CD3+ CD28 H) 2000 CD3 + CDE)800 700 600 500 400 300 200 one hundred P 0.F)siControlsiNELFP24 (pg/ml)Luciferase unitsEventsEventsNELF-B1500 1000 S1PR4 Agonist Purity & Documentation 5001116PLAP expressionPLAP expressionFIGURE 1. NELF limits HIV transcription and replication in key CD4 T cells. Human main CD.
