Figure 1

TNPO3 depletion decreases the level of HIV-1 2-LTR circles and has no effect on autointegration. (A) TNPO3 protein in TZM-bl cells transduced with lentiviral vectors expressing shRNAs targeting TNPO3 or control (Ctrl). Cell lysate was probed in western blots with anti-TNPO3 antibody (upper panel) or anti-β-actin antibody (lower panel). (B) TZM-bl control (Ctrl) cells and TNPO3 KD cells were challenged with HIV-1-GFP reporter vectors bearing either WT or A105T CA mutation. 72 hrs after transduction, the percent GFP⁺ cells was determined by flow cytometry as an indication of infectivity. (C) Quantification of late RT products 24 hrs post-infection with WT or A105T CA mutant viruses, on control (Ctrl) or TNPO3 KD cells. (D) Schematic representation of qPCR for 2-LTR circles using primers flanking the circle junction. Arrows represent primers. Dashed line represents the PCR product. (E) Quantification of 2-LTR circles (as indicated in B) 24 hrs post-infection with WT or A105T CA mutant viruses, on control (Ctrl) or TNPO3 KD cells. The PCR products were detected using Sybr green. (F) The PCR products from (E) were cloned and sequenced. The ratio between 2-LTR circles and autointegration events is plotted. (G) The analysis from (E) was repeated in the presence of raltegravir (RAL) 10 μM. (H) Level of autointegration events by qPCR when WT or A105T CA mutant viruses infect control (Ctrl) or TNPO3 KD cells.