Delaforge, E. | Search bioRxiv

bioRxiv2021

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, biophysical and structural studies of the Imp{beta}/Rev complex. Gel shift, native mass spectrometry and isothermal titration calorimetry data reveal that Imp{beta} binds two Rev monomers through independent binding sites. Small-angle X-ray scattering (SAXS) data suggest that the HEAT repeats of Imp{beta} 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 2 within Revs Arginine-Rich Motif (ARM) as a primary Imp{beta} 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 Imp{beta} with Rev helix 2 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 Imp{beta} and highlight an atypical binding behaviour that distinguishes Rev from canonical cellular Imp{beta} cargos.

biochemistry↗

bioRxiv2020

Molecular Basis of Host-Adaptation Interactions between Influenza Virus Polymerase PB2 Subunit and ANP32A

Avian influenza polymerase undergoes host adaptation in order to efficiently replicate in human cells. Adaptive mutants are localised on the C-terminal (627-NLS) domains of the PB2 subunit. In particular mutation of PB2 residue 627 from E to K in avian polymerase rescues activity in mammalian cells. A host transcription regulator ANP32A, comprising a long C-terminal intrinsically disordered domain (IDD), has also been shown to be responsible for this viral adaptation. Human ANP32A IDD lacks a 33 residue insertion compared to avian ANP32A, a deletion that restricts avian influenza polymerase activity in mammalian cells. We determined conformational descriptions of the highly dynamic complexes between 627E and 627K forms of the 627-NLS domains of PB2 and avian and human ANP32A. The negatively charged intrinsically disordered domain of human ANP32A transiently binds to a basic face of the 627 domain, exploiting multiple binding sites to maximize affinity for 627-NLS. This interaction also implicates residues 590 and 591 that are responsible for human-adaptation of the the 2009 pandemic influenza polymerase. The presence of 627E interrupts the polyvalency of the interaction, an effect that is compensated by extending the interaction surface and exploiting an avian-unique motif in the unfolded domain that interacts with the 627-NLS linker. In both cases the interaction favours the open, dislocated form of the 627-NLS domains. Importantly the two binding modes exploited by human- and avian-adapted PB2 are strongly abrogated in the cross interaction between avian polymerase and human ANP32A, suggesting that this molecular specificity may be related to species adaptation. The observed binding mode is maintained in the context of heterotrimeric influenza polymerase, placing ANP32A in the immediate vicinity of known host-adaptive PB2 mutants. This study provides a molecular framework for understanding the species-specific restriction of influenza polymerase by ANP32A and will inform the identification of new targets for influenza inhibition.

biophysics↗