Binding stoichiometry and structural model of the HIV-1 Rev/Importin β complex

Abstract

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 β (Impβ), but how Rev associates with Impβ is poorly understood. Here we report biochemical, biophysical and structural studies of the Impβ/Rev complex. Gel shift, native mass spectrometry and isothermal titration calorimetry data reveal that Impβ binds two Rev monomers through independent binding sites. Small-angle X-ray scattering (SAXS) data suggest that the HEAT repeats of Impβ retain an extended conformation upon binding Rev, which according to NMR data is primarily recognized through its helical hairpin domain. Peptide scanning data and charge-reversal mutations identify the N-terminal tip of Rev helix α2 within Rev’s Arginine-Rich Motif (ARM) as a primary Impβ binding epitope. Crosslinking 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 Impβ with Rev helix α2 roughly parallel to the HEAT-repeat superhelical axis while the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by Impβ and highlight an atypical binding behaviour that distinguishes Rev from canonical cellular Impβ cargos.