Interaction of the Influenza A Virus Polymerase PB2 C-terminal Region with Importin α Isoforms Provides Insights into Host Adaptation and Polymerase Assembly

Stephane Boivin,Darren J Hart

doi:10.1074/jbc.m110.182964

Abstract

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.

Highlights

(FINOVI) and European Union FLUPOL Contract SP5B-CT-2007-044263. □S The on-line version of this article contains supplemental Figs
The association with importin ␣ isoforms is necessary for nuclear transport, and it seems possible that host adaptation could improve binding of PB2 to the importins of the new host animal
The single PB2 adaptive mutation D701N was observed to enhance 4-fold the binding of importin ␣1 (Imp-␣1) in mammalian cells but not avian cells [38], suggesting that improved binding to specific importins could result from single adaptive mutations, as suggested by structural studies of the PB2 nuclear localization signal (NLS) domain–Imp-␣5 complex [27]

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—A codon-optimized human H3N2 pb synthetic gene encoding amino acids 538 – 759 from A/Victoria/3/1975 [41] and a typical avian H5N1 isolate exemplified by A/duck/Shantou/4610/2003 were synthesized to facilitate expression in Escherichia coli (Geneart, Regensburg, Germany). All constructs were subcloned into a pET9a-derived vector with N-terminal hexahistidine tag and TEV protease cleavage sequence (MGHHHHHHDYDIPTTENLYFQG). For expression of the MBP-NLSbipartite construct, a DNA cassette encoding the PB2 NLS with two linking serines (SSKRKRDSSILTDSQTATKRIRMAIN; NLS regions underlined) was fused directly to the 3Ј end of the malE gene via the SacI/BamHI sites of pMAL-c2g (New England Biolabs); this cassette was expressed from a pET9a vector with an N-terminal TEV-cleavable hexahistidine tag. Purification of proteins was achieved using Ni2ϩ-affinity chromatography and 20 mM Tris-HCl, pH 7.5, containing 200 mM NaCl and 0.5 M imidazole. After dialysis against 20 mM HEPES, pH 7.5, with 200 mM NaCl, the cleaved protein was purified through an additional Ni2ϩ-affinity chromatography step to remove unwanted material. Purified C-terminal PB2 recombinant proteins and variants were covalently coupled to sensor chips CM5 using standard amine-coupling pro-

Hu Imp

RESULTS

DISCUSSION