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. 2022 Jan 10;13:66. doi: 10.1038/s41467-021-27650-5

Fig. 3. TASOR and CNOT1 cooperate to repress HIV-1 expression in the J-Lat A1 model of HIV-1 latency.

Fig. 3

a TASOR interacts with CNOT1 in the J-Lat A1 T cell line. TASOR or Rabbit IgG immunoprecipitations were performed in J-Lat A1 lysates. The interaction was then assessed by western blot. β-Actin is a negative control. (n = 2 in Jurkat cell lines). b Generation of Dox-inducible miR-RNA CNOT1 J-Lat A1 cells. J-Lat A1 cells, harboring one copy of integrated and latent LTR-tat-IRES-GFP-LTR construct, were transduced with a pINDUCER10 vector containing a doxycycline-inducible miRNA targeting CNOT1 or Luciferase (Mock) transcripts. c Expression of the miRNA targeting CNOT1 reactivates GFP expression from the latent HIV-1 minigenome. J-Lat A1 miR Luc or miR CNOT1 were treated or not with doxycyline 1 µg/mL for 72 h and GFP expression was analyzed by flow cytometry. The proportion of cells that became GFP positive is indicated in green. d Dox-inducible miR-CNOT1, but not miR-Luc, decreases CNOT1 expression while delivery of VLPs containing HIV-2 Vpx WT induces TASOR depletion in contrast to Vpx R42A in the J-Lat A1 cells. J-Lat A1 miR Luc and miR CNOT1 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 prior to protein analysis. e–f Co-silencing of CNOT1 and TASOR synergistically increases LTR-driven GFP expression at the protein level with the proportion of cells that became GFP positive indicated in green and the median of GFP expression in the whole cell population in italic (e), at the RNA level (f), but not MORC2 RNA (g) or TNF RNA (h) in the J-Lat A1 model. J-Lat A1 miR Luc and miR CNOT1 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 (from d to h, n = 3 for miR Luc; n = 6 for miR CNOT1; each independent replicate, mean and SEM are shown; two-sided unpaired t test was applied). Source data are provided as a Source data file.