Abstract
Influenza virus mRNAs are synthesized by the trimeric viral polymerase using short capped primers obtained by a 'cap-snatching' mechanism. The polymerase PB2 subunit binds the 5′ cap of host pre-mRNAs, which are cleaved after 10–13 nucleotides by the PB1 subunit. Using a library-screening method, we identified an independently folded domain of PB2 that has specific cap binding activity. The X-ray structure of the domain with bound cap analog m7GTP at 2.3-Å resolution reveals a previously unknown fold and a mode of ligand binding that is similar to, but distinct from, other cap binding proteins. Binding and functional studies with point mutants confirm that the identified site is essential for cap binding in vitro and cap-dependent transcription in vivo by the trimeric polymerase complex. These findings clarify the nature of the cap binding site in PB2 and will allow efficient structure-based design of new anti-influenza compounds inhibiting viral transcription.
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Acknowledgements
We thank P. Mas for help with ESPRIT screening; H. Belrhali, A. McCarthy and M. Walsh (MRC-France) for help in data collection and structure determination; Y. Fernández (CSIC) for help in the polymerase assays; and B. Dublet, L. Signor and E. Forest (IBS) for MS measurements. We acknowledge the ESRF, EMBL and MRC-France for access to synchrotron facilities, the Partnership for Structural Biology for an integrated structural biology environment and the EU for funding of the FLUPOL contract (SP5B-CT-2007-044263). Work at CNB was also supported by the Spanish Ministry of Education and Science (Ministerio de Educación y Ciencia) (BFU2004-491) and the Research Programme VIRHOST-CM from Comunidad de Madrid (S-SAL/0185/2006). Thanks to Catherine for inspiration.
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D.J.H. and F.T. devised and implemented the extension of ESPRIT to internal domains and identified soluble central fragments of PB2. D.G. and F.T. purified and biochemically characterized soluble central fragments of PB2. D.G. identified and crystallized the minimal cap binding domain, made and crystallized selenomethionated protein and made and assayed cap binding of domain mutants. S.C. performed the crystal structure analysis with T.C. P.R.-I. established conditions for binding of polymerase to cap-analog resins. P.R.-I. and R.C. contributed equally to the generation of and functional characterization of mutants in the context of recombinant polymerase and RNPs. J.O. designed and supervised the mutant phenotype analyses. S.C. compiled the text with contributions from J.O. and D.H. P.S. and J.L. performed and analysed the SPR measurements. J.O., R.W.H.R. and S.C. are principal investigators of the EU FP6 FLUPOL project.
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D.J.H. and F.T. have applied for a patent on ESPRIT technology. S.C., D.G., D.J.H. and F.T. have applied for a patent on the use of recombinantly expressed constructs of the PB2 cap binding domain and the three-dimensional structure for anti-influenza drug design.
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Guilligay, D., Tarendeau, F., Resa-Infante, P. et al. The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat Struct Mol Biol 15, 500–506 (2008). https://doi.org/10.1038/nsmb.1421
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DOI: https://doi.org/10.1038/nsmb.1421
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