FLUPOL : Host-specific Variants of the Influenza Virus Replication Machinery FLUPOL : Host-specific Variants of the Influenza Virus Replication Machinery
Starting date : 1 January 2007
Duration : 36 months
Contract no : LSH-044263
EC contribution : 1 973 450 €
Instrument : FP6 STREP
Coordinator : Stephen Cusack
Summary. We aim to understand how the influenza virus replication machinery adapts during interspecies transmission and use this knowledge to provide new tools to combat potentially pandemic influenza outbreaks.
Problem : Currently circulating H5N1 avian influenza viruses are lethal to man and could cause a devastating pandemic if they became transmissible between humans. It is therefore crucial to understand the mechanisms whereby influenza virus adapts from avian to human hosts. The most well-understood factors in inter-species transmission are certain characteristics of the surface glycoprotein, haemagglutinin. However several recent studies have highlighted the importance for transmissibility of mutations in the proteins of the viral replicative machinery, in particular the polymerase which transcribes and replicates the viral RNA. We propose a comprehensive study of the molecular structure and function of the influenza virus polymerase with the aim of understanding how it adapts during inter-species transmission.
Aim : We will focus on determination of the atomic structure of polymerase domains as well as the complete trimeric complex by state of the art methods such as X-ray crystallography, nuclear magnetic resonance and cryo-electron microscopy. This will provide the detailed framework required to understand polymerase function and the effect of specific point mutations in inter-species adaptation. We will also undertake biochemical, cellular and animal functional studies of the replication machinery and identification of host cell factors interacting with the polymerase using advanced functional genomics methods. In parallel, candidate mutations that may be important for inter-species transmission and virulence will be identified by bioinformatics analysis of influenza genome sequences, updated with sequences of new H5N1 isolates, as well as from studies of laboratory strains adapted from one host to another [e.g. avian to mouse].
Expected results : We will systematically identify independently folded and soluble domains of polymerase subunits and nucleoprotein and determine their atomic structures. We will elucidate the structure of the polymerase-viral RNA-nucleoprotein complex by cryo-electron microscopy. We will provide a structural interpretation of species specific mutations. Using yeast two-hybrid method and in vivo tagging of complexes we will identify host factors required for transcription and/or replication of the influenza genome. We will use in vitro and cell-based in vivo characterisation [e.g. polymerase activity, replication efficiency] to assess the effects of mutations in polymerase and NP associated with inter-species transmission. All these studies, combined with a systematic bioinformatic analysis of viral sequences and mathematical modelling will potentially contribute to elaboration of a comprehensive model of evolution of lead to elaboration of new strategies for development of anti-viral compounds targeting polymerase or polymerase-host cell factor interactions.
Potential applications : The influenza virus polymerase complex is an excellent target for new anti-viral drugs, since it is essential for viral replication and contains several functional active sites likely to be significantly different from those found in host cell proteins. However the lack of a detailed structure based understanding of polymerase function, in particular the structure of the target active sites, has hindered progress in this direction. Our structural and functional studies on polymerase aim to rectify this situation, by providing atomic resolution detail of the mechanism of action of this complex machine, including interactions with host cell partner proteins. Based on out biochemical and structural results we aim to develop new high-throughput assays to screen for anti-viral compounds. Other applications include new molecular biology tools such as specific monoclonal which could be used for diagnostic purposes.
Partners :
1. Prof. Rob Ruigrok, Unit of Virus Host Cell Interactions [UVHCI], UMR 5233 UJF-EMBL-CNRS
2. Dr. Vincent Lotteau, INSERM Unit 503, Lyon, France
3. Dr. Juan Ortin, National Biotechnology Centre [CNB], CSIC, Madrid, Spain
4. Prof. Hans-Dieter Klenk, Institute of Virology, University of Marburg, Germany
5. Dr. Alan Hay, National Institute for Medical Research, London, UK
First results: Using a novel library-based screening technique called ESPRIT (Expression of Soluble Proteins by Random Incremental Truncation) we identified an independently folded C-terminal domain from PB2 (residues 678-759) and determined its solution structure by NMR. Using GFP fusions, we show that both the domain and 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 5 demonstrates how the last twenty 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.
Reference: Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit. Tarendeau F, Boudet J, Guilligay D, Mas PJ, Bougault CM, Boulo S, Baudin F, Ruigrok RW, Daigle N, Ellenberg J, Cusack S, Simorre JP, Hart DJ. Nat Struct Mol Biol. 2007, 14(3):229-33.
Figure 1.
Top left: Schematic diagram of the influenza virus virion showing the eight segments of the negative strand RNA genome and the location of the ten virally encoded proteins.
Top right : Schematic diagram of the trimeric influenza virus polymerase comprising subunits PS, PB1 and PB2, showing the so far identified functional sites in each subunit.
Bottom : Schematic diagram of the PB2 polymerase subunit which comprises 759 amino acids showing interaction regions with PB1 and nucleoprotein NP, positions of residues implicated in cap-binding, positions of residues implicated in nuclear localisation (NLS), positions of point mutations observed upon adaptation of avian strains to mammals (across the top) and finally the position of the C-terminal domain (residues 678-759, denoted DPDE) identified in this work.
Figure 2.
Top : NMR structure of the isolated DPDE domain showing positions the two parts of the bipartite NLS sequence (NLS1, NLS2) and positions of point mutations observed upon adaptation of avian strains to mammals (residues 701, 702 and 714)
Bottom : Alignment of C-terminal DPDE domains form influenzas A, B and C showing conservation of structural and functional features.
Figure 3.
Crystal structure of the DPDE domain bound to human importin alpha 5, the nuclear import receptor which transports PB2 into the nucleus. The bipartite NLS is unfolded and binds in the groove of the extended importin molecule.