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.
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 (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.
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 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.
Coordinator:
Dr Stephen Cusack
European
Molecular Biology Laboratory Grenoble Outstation
6
Rue Jules Horowitz
38042 Grenoble
France
Tel: +33 47 62 07 238
cusack@embl.fr
Partners:
