Multiple components of the nuclear pore complex interact with the amino-terminus of MX2 to facilitate HIV-1 restriction
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.