Binding stoichiometry and structural model of the HIV-1 Rev/importin beta complex

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein importin beta (Imp beta), but how Rev associates with Imp beta is poorly understood. Here, we report biochemical, mutational, and biophysical studies of the Imp beta/Rev complex. We show that Imp beta binds two Rev monomers through independent binding sites, in contrast to the 1:1 binding stoichiometry observed for most Imp beta cargos. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix alpha 2 within Rev's arginine-rich motif (ARM) as a primary Imp beta-binding epitope. Cross-linking mass spectrometry and compensatory mutagenesis data combined with molecular docking simulations suggest a structural model in which one Rev monomer binds to the C-terminal half of Imp beta with Rev helix alpha 2 roughly parallel to the HEAT-repeat superhelical axis, whereas the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Imp beta and highlight an atypical binding behavior that distinguishes Rev from canonical cellular Imp beta cargos.