Inhibition of retroviral infections and retro-transposition by Daxx.

<p>(A) Wt and Daxx KO MEFs were transduced with increasing doses of HIV-1 vector and the percentage of GFP-expressing cells was determined 48 h later by flow cytometry. Means +/- SD of a typical experiment are shown. Similar results were obtained in three independent experiments. (B) Wt HeLa cells were transfected with an empty (EV) or a Daxx-encoding plasmid. Two days post-transfection, Daxx expression was evaluated by Western-blot analysis (left panel) and the percentage of GFP-expressing cells was determined by flow cytometry 48 h post-transduction with increasing doses of HIV-1 vector (right panel). Graph shows means of triplicate values +/- SD, representative of two independent experiments. (C) PBMCs of healthy adult individuals were activated by anti-CD3/anti-CD28 for 48 h and were subsequently transduced with either a non-coding control (RV_CTR) or a Daxx-expressing (RV_Daxx) MLV-derived retroviral vector. Relative expression of Daxx was estimated by RT-qPCR analysis performed two days post-transduction, and expressed as a ratio to RPL13A mRNA expression. Two days post transduction, activated PBMCs were infected with HIV-1 NL4.3 at a dose corresponding to 0.1 ng of p24 and virus replication was followed at given time points post-infection by measuring the amount of HIV-1 p24 by ELISA on the culture supernatants. Results show the mean of triplicates +/- SD and are representative of two independent experiments. (D) Wt HeLa cells were transfected with an EV or a Daxx-encoding plasmid. Two days later, cells were transduced with VSV-pseudotyped HIV-1 vector for 6 h and early and late reverse transcript were quantified by qPCR. (E) Wt HeLa cells transfected with an EV or a Daxx-encoding plasmid were transduced 48 h later with GFP-expressing VSV-G pseudotyped Mo-MLV or SIVmac vectors. The percentage of GFP-expressing cells was determined by flow cytometry. Graphs show means of triplicate values +/- SD, representative of three independent experiments. (F) Experimental procedure for the detection of retrotransposition. (G) Analysis of the activity of Daxx on IAP and MusD retrotransposons. Retrotranspositions (defined by quantifying G418R clones) are presented as percentages relative to samples containing retroelement alone. Results show the mean of three independent experiments +/- SD.