NUP214 and TNPO1 are required for full anti-viral activity of MX2 in U87-MG cells.

U87-MG CD4+ CXCR4+ cells were transduced with EasiLV vectors expressing FLAG-tagged MX2 or Luciferase (control). After 48 h, transduced cells were transfected twice, 24 h apart, with 20 nM of specific siRNAs (a non-targeting siRNA was included as a control, CTRL). Expression of MX2 or Luciferase was then induced by treatment of cells with doxycycline (0.5 μg/ml) for ~72 h prior to challenge with a HIV-1 based lentiviral vector expressing GFP (HIV-1/GFP). Transduction efficiency was assessed 48 h post challenge by flow-cytometry. (A) NUP358, NUP214, NUP98, hRIP, PNRC1, KLHL6, NUP62, NUP153, TNPO1 or TNPO3 were depleted independently or in pairs and MX2 anti-viral activity was analyzed. A dotted line indicates the fold inhibition of HIV-1 obtained in cells treated with CTRL siRNA. Conditions where NUP214 or TNPO1 where depleted are highlighted in red and blue, respectively, while depletion of both is indicated in purple. (B-C) Effect of siRNA-mediated depletion of NUP214 and/or TNPO1 on the anti-HIV-1 activity of MX2. In C, the same data as in B are represented as MX2-mediated fold inhibition by dividing %GFP+ luciferase expressing cells by %GFP+ MX2 expressing cells (n = 3; mean ± standard deviation (SD); *p-value (D) The effect of NUP214 and/or TNPO1 depletion on MLV infectivity in the presence of MX2 was studied by challenging siRNA-transfected cells with an MLV based vector expressing GFP. (E) Immunoblot analysis of MX2 expressing cells from the experiment described in B-D, indicating levels of FLAG-tagged MX2 and endogenous NUP214 (detected using mab414) and TNPO1 after siRNA treatment. Tubulin is included as a loading control.