Human Nucleoporins Promote HIV-1 Docking at the Nuclear Pore, Nuclear Import and Integration (original) (raw)
Figure 5
Nup358/RanBP2 interacts with HIV-1 CA-NC.
(A) Protein sequence alignment of cyclophilin-homology domain of Nup358/RanBP2 and the human cyclophilin A, showing 66% identity (amino acids conserved are in red in capital letters and the amino acids divergent are in lowercase in black). (B) Domain structure of the wild-type and truncated Nup358-RanBP2 GFP fusion proteins used for interaction with CA-NC complexes. LRR: leucine-rich region. R1–R4: RanBP homology domains (RBH-1-4). Zn fingers: zinc finger domains. IR: internal repeats. Cyp: cyclophilin-homology domain. BPN, BPM, BPC: N-terminal, middle and C-terminal regions of Nup358/RanBP2 (Joseph and Dasso, 2008). Microscopy image showing location of the GFP-RanBP2 constructs in 293T transfected cells. (C) _In vitro_-assembled CA–NC complexes were mixed with 293T lysates containing WT GFP-RanBP2 or GFP-RanBP2-ΔCyp or Trim5αRH-HA and layered onto 70% sucrose before centrifugation. Immediately before mixing, an aliquot of the cell lysate was removed and blotted with α-GFP or α-HA antibodies to determine the steady-state expression levels of the different transgenic proteins (input). After centrifugation, the pellet was resuspended in SDS sample buffer and analyzed by Western blotting with an anti-GFP antibody (to detect RanBP2) or anti-HA antibody (to detect Trim5αRH) or an anti-p24 antibody (to detect CA-NC). As control of the assay we included TRIM5α-HA.