Figure 2

Binding of endogenously expressed CPSF6 to in vitro assembled HIV-1 CA-NC complexes. (A) Cellular extracts from human 293T cells were incubated with wild type or mutant (N74D) in vitro assembled HIV-1 CA-NC complexes for 1 h. Samples were subsequently applied onto 70% sucrose cushion and centrifuged, as described in Methods. A small fraction of each lysate was collected before centrifugation and analyzed by Western Blotting using anti-CPSF6 antibodies (INPUT). Pelleted fractions (BOUND) were analyzed for the presence of CPSF6 and HIV-1capsid (p24) by Western blotting using anti-CPSF6 and anti-p24 antibodies, respectively. (B, C) The ability of endogenously expressed CPSF6 from human 293T cells to bind in vitro assembled HIV-1 CA-NC complexes was measured in the presence of the small molecule PF74 (PF-3450074). (D) The ability of the restriction factor TRIMCyp to bind in vitro assembled HIV-1 CA-NC in the presence of PF74. (E) Similarly, we measured the ability of endogenously expressed CPSF6 from TNPO3 K.D. and shRNA control HeLa cells to bind in vitro assembled HIV-1 CA-NC complexes. (F) HeLa cells stably expressing the nuclearly localized CPSF6 and the cytoplasmicly localized NES-CPSF6 were lysed in capsid binding buffer (10 mM Tris pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT) or whole cell extract buffer (50 mM Tris pH 8, 2 mM MgCl₂, 280 mM NaCl, 0.5% NP-40,10% Glycerol). Extracts were analyzed by Western blotting using antibodies against FLAG. As a control, we blotted extracts using antibodies against the nuclear marker β-laminin. Similar results were obtained in three independent experiments and a representative experiment is shown.