Fig 1.
Multiple nucleoporins interact with the N-terminal domain of MX2 (N-MX2).
(A) A yeast two-hybrid (Y-2-H) screen was performed using a human leukocyte cDNA library to identify interacting proteins with wild-type or mutant RRR11-13A N-MX2. Interacting partners were assigned a predicted biological score from A-F to assess the confidence of an interaction being specific (with A indicating very high confidence, and F indicating experimentally determined artifacts). (B) Co-immunoprecipitation of candidate interacting proteins with MX2. 293T cells were co-transfected with HA-tagged wild-type or mutant RRR11-13A MX2 and FLAG-tagged candidate interactors from the Y-2-H screen. Cells were lysed and HA-tagged protein immunoprecipitated with anti-HA antibody. Co-transfection of FLAG-tagged candidates with HA-tagged MX1 or GFP served as negative controls. Immunoblots of immunoprecipitated proteins (IP) were probed with anti-FLAG and anti-HA antibodies. As a control for protein expression, samples of lysate prior to immunoprecipitation (IN) were probed with anti-FLAG antibody. All experiments were performed at least three times.
Fig 2.
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 < 0.05; paired t-test to CTRL siRNA). (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.
Fig 3.
Multiple nucleoporins and transport receptors are required for anti-viral activity of MX2 in HeLa cells.
(A) HeLa cells were stably transduced with lentiviral vectors constitutively expressing FLAG-tagged MX2 or CD8 (negative control), and transduced cells selected by treatment with 1 μg/ml puromycin for 72 h. After selection, transduced cells were transfected twice, 24 h apart, with 20 nM siRNA targeting individual nucleoporins or transport receptors. A non-targeting siRNA was included as a control, CTRL. After 48 h, cells were challenged with HIV-1/GFP and transduction efficiency assessed by flow cytometry after a further 48 h post challenge. (B) the same data as in A are represented as fold MX2-mediated inhibition, calculated as in Fig 2 (n = 3; mean ± SD; *p-value < 0.05; paired t-test to CTRL siRNA).
Fig 4.
MX2 interacts with TNPO1 via RRR11-13, and endogenous FG-nucleoporins.
(A) Interaction of MX2 with endogenous FG-nucleoporins. U87-MG CD4+ CXCR4+ cells were treated with siRNA targeting NUP214 or a non-targeting siRNA (CTRL) as described in Fig 2 and incubated for ~48 h prior to culture with or without IFNα (500 U/ml) for a further 24 h. Treated cells were lysed, and mouse monoclonal mab414 used to extract FG-nucleoporins NUP358, NUP214, NUP153 and NUP62. An anti-GFP mouse monoclonal was included as a control (CTRL). Immunoblots were performed on immunoprecipitated material (IP) to detect the presence of associated MX2, and on samples of lysate prior to precipitation (INPUT). (B) TNPO1 interacts with MX2 via RRR11-13. 293T cells were co-transfected with HA-tagged wild-type MX2 or mutant RRR11-13A MX2 and FLAG-tagged TNPO1. Cells were lysed and HA-tagged protein immunoprecipitated with anti-HA antibody. HA-tagged MX1 or GFP were included as negative controls. Immunoblots of immunoprecipitated protein (IP) were probed with anti-FLAG and anti-HA antibodies. As a control for protein expression, samples of lysate prior to immunoprecipitation (IN) were probed with anti-FLAG antibody. (C) TNPO1 interacts with MX2 but not the CTD of NUP214. A similar experiment was performed in 293T cells as described in B, except that a FLAG-tagged construct encoding the 35 kDa CTD of NUP214 was co-expressed with HA-tagged constructs expressing TNPO1, MX1 or MX2. Immunoprecipitation was performed with an anti-HA antibody (D) MX2 interacts with NUP214 CTD and TNPO1 after FLAG immunoprecipitation. In 293T cells, a similar experiment was performed as described in B and C except that HA-tagged MX2 was co-expressed with FLAG-tagged NUP214 CTD, TNPO1 or GFP and immunoprecipitation of cell lysate performed with an anti-FLAG antibody. All experiments were done at least three times.
Fig 5.
Endogenous MX2 requires NUP214 and TNPO1 for a full anti-viral function.
(A) U87-MG cells transduced with a CRISPR-Cas9 control guide RNA were transfected twice with a control siRNA (CTRL) or siRNAs targeting MX2, NUP214 and/or TNPO1, and treated or not with 1000 U/ml of IFNα, prior to infection with an HIV-1/GFP lentiviral vector. On the left, the percentage of infected cells calculated by flow cytometry and the fold inhibition of infection due to IFNα treatment are shown (n = 4; mean ± SD; *p-value < 0.05; (ns) non-significant; paired t-test). On the right is an immunoblot from a representative experiment, showing depletion of the indicated proteins. (B) Same experiment as in A, but using U87-MG cells where the MX2 alleles were disrupted using CRISPR-Cas9 genome editing (n = 4; mean ± SD; *p-value < 0.05; (ns) non-significant; paired t-test). (C) Primary CD4+ T cells were isolated from 4 independent donors, transduced with shRNAs targeting MX2, NUP214, TNPO1 or a control shRNA (CTRL) and treated or not with 3000 U/ml of IFNα. 24 h later, cells were challenged with NL4.3/Nef-IRES-Renilla and luciferase activity determined 48 h later. The mean of three technical replicates are shown for each donor on the left, and the fold inhibition of infection (no IFN/IFN) on the right. (D) Efficiency of MX2, NUP214 and TNPO1 depletion in primary CD4+ T cells following shRNA transduction was quantitated by qPCR and normalized to GAPDH. Data shown represent 4 donors used in (C).
Fig 6.
Depletion of NUP214 and TNPO1 disrupts nuclear envelope accumulation of MX2.
(A) HeLa cells stably expressing MX2 bearing a C-terminal FLAG tag were transfected with siRNA targeting NUP214 and/or TNPO1 or a non-targeting siRNA (CTRL) as described in Fig 3, and then seeded onto glass coverslips. Localization of MX2, and endogenous NUP214 and TNPO1 was visualized by fluorescence confocal microscopy, using anti-FLAG, anti-NUP214, and anti-TNPO1 antibodies respectively. DAPI was used to stain the nuclei. Scale bar represents 20 μm. (B) Predominant cellular localization of MX2 was determined visually (blinded) using a 60x objective and an average of 100 cells, randomly selected (mean ± SD; *p-value < 0.05; paired t-test).
Fig 7.
Nucleoporins interacting with MX2 are located at the cytoplasmic face of the nuclear pore.
Schematic diagram indicating the structure and organization of the nuclear pore complex as described in Hoelz et al [22]. Nucleoporins interacting with MX2 (identified by Y-2-H screening) are shown in red. Nucleoporins required for MX2 activity (defined as those for which specific depletion elicited >50% reduction in MX2 dependent inhibition of HIV-1, in either HeLa or U87-MG cells) are shown in bold. Nucleoporins that interact with MX2 and are required for MX2 inhibition are shown in red and bold.