Dr Paul Digard
Research description
Influenza A viruses are the cause of influenza epidemics and pandemics and remain one of the major uncontrolled infectious diseases. Vaccination is rendered less effective by frequent antigenic change in the virus glycoproteins, and antiviral chemotherapy is hindered because of the acute nature of the infection. A better understanding of the virus is therefore of scientific and medical interest. Research in my laboratory focuses on three interlinked areas: (1) Intracellular trafficking of virus components, (2) virion assembly and (3) transcription and replication of the virus genome.
- Virus transcription occurs in the nucleus but progeny virions are assembled at and bud from the apical plasma membrane. This
necessitates bi-directional transport of the genome ribonucleoproteins across the nuclear membrane at different stages of the viral
life-cycle, as well as nuclear export of viral mRNAs. We are currently studying the mechanisms involved in RNP and mRNA transport.
- The process of virion assembly and how all the necessary viral components are targetted to the sites of budding is not well understood. We are interested in how polarised budding is achieved and the processes involved in the assembly of filamentous virus particles, with particular reference to the actin cytoskeleton and lipid raft domains. We also have a strong interest in the mechanism by which a full complement of all eight genome segments is packaged into virus particles.
- Influenza virus is absolutely dependent upon the virus encoded RNA- dependent RNA polymerase for expression and replication of its negatively stranded segmented RNA genome. Also, since the enzyme does not have a counterpart in uninfected cells, it is an obvious target for antiviral chemotherapy. The trimeric polymerase catalyses a wide variety of reactions, including synthesis of polyadenylated mRNAs containing a 5'-cap structure scavenged from host cell mRNA by endonucleolytic cleavage, and replication of the genome segments via a full length plus strand intermediate. We are currently dissecting the polymerase using molecular-genetic and biochemical techniques, primarily to understand how the switch between mRNA transcription and genome replication is regulated. As well as providing insights into the fundamental biological processes of protein-protein and protein-RNA interactions, this information will be used to aid the rational design of novel inhibitors of virus replication.
Our general approach to these topics is to combine viral genetics with biochemical analyses and microscopy. A couple of examples of films where we have applied live-cell imaging techniques to influenza can be seen below.
RNP.mov demonstrates cytoplasmic transport of GFP-tagged RNPs. The film shows images taken over a 5 minute period of a 293T cell transfected with the components necessary to reconstitute viral RNPs tagged with GFP and direct their export from nucleus to the cytoplasm. Cytoplasmic RNPs can be seen exhibiting the rapid, saltatory movement characteristic of microtubule-based transport.
Filaments.mov is a timelapse movie of filamentous virions budding from the surface of infected cells in which 12 hours are compressed into 6 seconds. The spikes that appear from cell edges around half-way through the timecourse are bundles of viral filaments