Host-specific variants of the Influenza Virus Replication Machinery
We aim to understand how the influenza virus replication machinery adapts during interspecies transmission and to use this knowledge to provide new tools to combat potentially pandemic influenza outbreaks.
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 best 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.[+] Read More
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).
We will systematically identify independently folded and soluble domains of polymerase subunits and nucleoprotein (NP) 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.
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 our 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 antibodies which could be used for diagnostic purposes.