Fig. 4. TASOR and CNOT1 cooperate to repress HIV-1 expression in latently HIV-1-infected Jurkat T cells.

a Schematic of the LTR-EGFP provirus used to analyze HIV-1 LTR-driven EGFP expression in infected Jurkat T cells. Primers used to quantify LTR-driven transcripts are mapped in orange. b Generation of latently HIV-1-infected Jurkat cells based on the EGFP expression by flow cytometry. The sorted cells were then transduced with the doxycycline-inducible miR CNOT1 or Luc vectors. Without doxycycline treatment, cells remained EGFP negative by flow cytometry, whereas LTR-driven RNAs (US) were detected by RT-qPCR in the latently infected cells. RNA was normalized on the 18s rRNA (n = 2). c Dox-inducible miR-CNOT1, but not miR-Luc, decreases CNOT1 expression while the delivery of VLPs containing HIV-2 Vpx WT induces TASOR depletion in contrast to Vpx R42A in these latently infected Jurkat cells. The latently HIV-1-infected Jurkat miR Luc and miR CNOT1 cells were treated or not for 72 h with doxycycline 1 µg/mL, with VLPs containing HA-Vpx WT or R42A or mock were delivered for 24 h and with TNF (1 ng/mL) 16 h prior to protein analysis. The co-depletion of CNOT1 and TASOR synergistically increases HIV-1 LTR at the protein level with the proportion of cells that became EGFP positive indicated in green and the median of GFP expression in the whole cell population in italic (d), at the RNA level (e), but not MORC2 RNA (f) or TNF RNA (g) in these latently HIV-1-infected T cell lines. The latently HIV-1-infected Jurkat miR Luc and miR CNOT1 cells were treated or not for 72 h with doxycycline 1 µg/mL and VLPs containing HA-Vpx WT or R42A or mock were delivered for 24 h and TNF (1 ng/mL) 16 h prior to flow cytometric analysis and RT-qPCR analyses (for d–g, n = 3 independent experiments, mean and SEM are represented; two-sided unpaired t test was applied: p values are indicated in the graph). Source data are provided as a Source data file.