In the adaptation of avian viruses to mammalian hosts, mutations in the viral polymerase, notably in the PB2 subunit, play an important role. A PB2 C-terminal domain rich in putative host adaptation residues has been shown to bind importin α nuclear import receptors. Adaptation has been proposed to involve binding of PB2 to importins of the new host. To date PB2-importin complexes have been characterized semiquantitatively with no precise measurement of binding parameters. To investigate the effects of adaptive mutations on importin interaction and selectivity, surface plasmon resonance was used to compare the binding rate constants and affinities of avian H5N1 and human H3N2 PB2 C-terminal variants with importin isoforms human α 1, 3, 5 and 7, and avian α 1. Using purified proteins eliminates host environment effects and permits measurement of intrinsic affinities and rates of complex formation and dissociation. Two effects were observed: first, adaptive mutations D701N, R702K, and S714R in the nuclear localization signal domain increased 2–4-fold the association rates with avian and human importins; second, measurement of different structural forms of the PB2 C terminus demonstrated that the upstream 627 domain reduced binding affinity, consistent with a steric clash predicted from crystal structures. From these kinetic data, structural analyses, and the data of others, a model is proposed in which an increase in charged surface residues during host adaptation increases the association rate of PB2 to cytoplasmic importins and where the C-terminal 627-nuclear localization signal domain may reorganize upon importin binding, consistent with a role in active polymerase assembly.
Characterization of a nuclear localization signal in the C-terminus of the adeno-associated virus Rep68/78 proteins
