Figure 5

Expression of a cytosolic full-length CPSF6. (A) The wild type CPSF6 (NCBI Reference Sequence: NP_008938.2) protein with a C-terminal FLAG epitope is depicted on top. The numbers of the amino acid residues at the boundaries of the different domains are indicated (RRM: RNA recognition motive, Pro-rich: Proline-rich domain, RS: Arginine/Serine repeats). The HIV-1 capsid binding region is shown (residues 277-285). The nuclear export signal of the protein kinase inhibitor α (NES-PKIα). The amino acid sequence of NES-PKIα is NELALKLAGLDI. The NES-PKIα was fused to the N-terminus of CPSF6. (B) Cf2Th cells stably transduced with the different CPSF6 variants were analyzed for expression by Western blotting using anti-FLAG antibodies. As a loading control, cell lysates were Western blotted against β-actin. (C) Intracellular distribution of the different CPSF6 variants stably expressed in Cf2Th was studied by immunofluorescence microscopy, as described in Methods. The different CPSF6 variants were stained using anti-FLAG antibodies (red). The cellular nuclei were stained by using DAPI (blue). Image quantification is shown in Additional file 3. (D) The ability of the different CPSF6 variants to bind in vitro assembled HIV-1 CA-NC complexes was measured. 293T cells were transfected with plasmids expressing the indicated CPSF6 variants. Thirty-six hours after transfection, cells were lysed. The lysates were incubated at room temperature for 1hour with in vitro assembled HIV-1 CA-NC complexes. The mixtures were applied to a 70% sucrose cushion and centrifuged. INPUT represents the lysates analyzed by Western blotting before being applied to the 70% sucrose cushion. The input mixtures were Western blotted using anti-FLAG antibodies. The pellet from the 70% sucrose cushion (BOUND) was analyzed by Western blotting using anti-FLAG and anti-p24.