Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit

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The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin α5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.

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Figure 1: Identification and solution NMR structure of PB2 C-terminal domain.
Figure 2: Nuclear import of PB2 C-terminal domain and full-length PB2 subunit is directed by a bipartite NLS.
Figure 3: X-ray structure of the PB2 C-terminal domain complexed with importin α5.

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We thank J. Ortin (Centro Nacional de Biotecnologia, CSIC, Madrid) for the pb2 gene, A. Favier (Institut de Biologie Structurale, Grenoble) for NMR scripts and the EMBL Centre for Molecular and Cellular Imaging for suggestions. Screening for crystals was done by the Partnership for Structural Biology high-throughput crystallization facility. We thank the European Synchrotron Radiation Facility and EMBL Joint Structural Biology group for assistance with the synchrotron beamtime and T. Crepin (EMBL, Grenoble) for help with data collection. Partial funding was provided by the European Commission Framework 5 Integrated Project 'Structural Proteomics in Europe' (SPINE, contract QLG-CT-2002-00988).

Author information

D.J.H. conceived the ESPRIT method. D.J.H., F.T. and P.J.M. implemented ESPRIT. D.G. purified wild-type and mutant DPDE for in vitro binding studies and crystallization and made double-labeled protein for NMR. C.M.B., J.B. and J.-P.S. performed the NMR measurements and structural analysis. S.B. purified importin α5 under the supervision of F.B. and cocrystallized it with DPDE. R.W.H.R. and S.C. initiated the influenza polymerase project, and S.C. determined the crystallographic structure. F.T. and N.D. performed nuclear import assays with instrumentation and methodology established by J.E. S.C. and D.J.H. compiled the text, with contributions from all authors.

Correspondence to Stephen Cusack or Darren J Hart.

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The authors have applied for a patent on ESPRIT technology.

Supplementary information

Supplementary Fig. 1

Assigned 1H-15N HSQC spectrum of the C-terminal domain of PB2 at 10 °C (PDF 101 kb)

Supplementary Fig. 2

Plot of r.m.s. deviation and S2 generalized order parameters against primary sequence (PDF 61 kb)

Supplementary Fig. 3

Interaction of the PB2 C-terminal domain with human importin α5 (PDF 176 kb)

Supplementary Fig. 4

Electron density for the bipartite NLS in the complex of human importin α5 with influenza PB2 DPDE domain (PDF 162 kb)

Supplementary Fig. 5

Interactions of the bipartite NLS with human importin α5 (PDF 142 kb)

Supplementary Table 1

NMR and refinement statistics for PB2 C-terminal domain (PDF 34 kb)

Supplementary Methods (PDF 183 kb)

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Tarendeau, F., Boudet, J., Guilligay, D. et al. Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit. Nat Struct Mol Biol 14, 229–233 (2007) doi:10.1038/nsmb1212

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