Sir William Dunn School of Pathology

 

Dr Ervin Fodor

Reader in Virology

 

Sir William Dunn School of Pathology

University of Oxford

South Parks Road

Oxford  OX1 3RE

United Kingdom

 

Email: ervin.fodor@path.ox.ac.uk

Phone: +44 (0)1865 275580

Fax:     +44 (0)1865 275556

 

 

Group Membership:

 

Katja Bier – D.Phil Student

Gabriel Borvák – Research Assistant

Tatiana Fislová – Postdoctoral Research Assistant

Katherine Graef – D.Phil Student

Edward Hutchinson – Postdoctoral Research Assistant

Nicole Robb – D.Phil Student

Jane Sharps – Research Assistant

Frank Vreede – Postdoctoral Research Assistant

 

Research Studentship to read for a DPhil from October 2010

 

 

Research Topic

 

Molecular and cell biology of influenza viruses

 

 

Aims

 

The general aim of our research is to elucidate the molecular interactions between influenza virus and the host cell during the influenza virus life cycle with the aim of understanding the molecular basis of host range restriction and pathogenicity of influenza viruses. There is a special emphasis on the viral RNA-dependent RNA polymerase complex and its interaction with host factors and the cellular responses to viral infection.

 

 

Specific interests

 

  • Nuclear import and assembly of the influenza virus RNA polymerase complex – identification of participating host chaperons and import factors
  • Localization of viral transcription and replication sites in the cell nucleus – functional implications of the association of the viral RNA polymerase with the cellular RNA polymerase II transcriptional machinery
  • Molecular mechanisms of the assembly and nuclear export of viral mRNPs
  • Functional implications of the mitochondrial association of the PB2 subunit of the influenza virus RNA polymerase
  • The role of the RNA polymerase in determining the host range and pathogenicity of influenza viruses
  • Structural studies of the influenza virus RNA polymerase

 

 

Interactions between the viral and host transcriptional machineries

 

Although it is known that transcription and replication of the influenza virus RNA genome by the viral RNA-dependent RNA polymerase are performed in the nucleus of infected cells, little information is available on the exact subnuclear location of these processes. We have found that the viral RNA polymerase associates with the host transcriptional machinery by interacting with the C-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II) (Engelhardt et al, 2005). We hypothesized that this interaction is required for viral mRNA synthesis since viral transcription is dependent on cellular functions, i.e. capping and splicing - activities associated with Pol II transcription. In addition, the viral RNA polymerase, by binding to the CTD of the large subunit of Pol II and cleaving the nascent host transcript, might interfere with Pol II function that could contribute to the virus-induced host shut-off phenomenon (reviewed in Engelhardt and Fodor, 2006).

 

 

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Studies are currently under way to determine whether the interaction between the viral RNA polymerase and the CTD of Pol II is direct or whether it is mediated by a cellular factor(s). We are also interested in determining the functional implications of this interaction for the virus life cycle as well as for the functionality of the Pol II transcriptional machinery (Chan et al, 2006; Vreede et al, 2009).

 

 

Transport and assembly of the viral RNA polymerase complex

 

Another major area of interest is the elucidation of the nuclear import and assembly pathways of the influenza virus RNA polymerase complex (Fodor and Smith, 2004; Deng et al, 2005). We have proposed that the assembly of the RNA polymerase complex is sequential: a PB1-PA dimer and PB2 monomer are transported into the nucleus separately and then are assembled in the nucleus. In collaboration with Prof GG Brownlee and Dr A Akoulitchev (Sir William Dunn School of Pathology) we have identified the host import factor, Ran Binding Protein 5 (RanBP5), as the nuclear import factor responsible for the nuclear import of the PB1-PA polymerase dimer (Deng et al, 2006).

 

 

 

 

Studies are currently under way to identify the nuclear import factors involved in the nuclear import of the PB2 subunit of the viral RNA polymerase. In addition, we are extending these studies to RNA polymerases derived from avian influenza viruses as recent evidence suggests that nuclear import of the influenza virus RNA polymerase might be an important determinant of the host range specificity of influenza viruses.

 

 

Mitochondrial localization of the PB2 subunit of the influenza virus RNA polymerase complex

 

The PB2 subunit of the influenza virus RNA polymerase complex associates with the mitochondria in infected cells (Carr et al, 2006). We have mapped the mitochondrial targeting signal (MTS) of PB2 and prepared recombinant influenza viruses expressing PB2 proteins with knocked out MTS. Cells infected with these viruses showed increased mitochondrial damage suggesting that PB2 might act at the mitochondria during viral infection preventing their damage. In collaboration with Dr A García-Sastre (Mount Sinai School of Medicine, New York) we showed that these viruses were attenuated in a mouse model. The functional significance of the mitochondrial localization is further investigated in collaboration with Dr K Subbarao (NIAID, NIH, Bethesda).

 

 

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Structural studies of the influenza virus RNA-dependent RNA polymerase

 

There is no structural information available on the trimeric RNA polymerase complex of influenza viruses. Recently, we have identified a C-terminal region of the PB1 polymerase subunit that is involved in the regulation of viral transcription (Kerry et al, 2007). In addition, together with Prof GG Brownlee (Sir William Dunn School of Pathology), Prof D Stuart, and Dr J Grimes (Division of Structural Biology, The Wellcome Trust Centre for Human Genetics) we are involved in a collaborative effort to establish expression and purification protocols for the influenza RNA polymerase and initiate studies directed at solving the 3D structure of the RNA polymerase, at high resolution, by X-ray crystallography.

 

 

Regulation of signalling pathways in innate immune response during influenza virus infection

 

The group participates in the EU 6th Framework Programme “FLUINNATE” entitled “Innate immunity in influenza virus infection of mammalian airways”. The FLUINNATE objectives focus on the identification of influenza A virus genes and gene products which contribute to virulence/pathogenicity in experimental animal and tissue culture models. As co-participants in this programme, we are focusing on the role of the influenza virus RNA polymerase in determining virulence/pathogenicity.

 

 

Publications

  • Vreede F, Cahn AY, Sharps J, Fodor E (2009) Mechanisms and functional implications of the degradation of host RNA polymerase II in influenza virus infected cells. Virology (in press).
  • Loucaides EM, von Kirchbach JC, Foeglein A, Sharps J, Fodor E, Digard P (2009) Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies. Virology (in press).
  • Robb NC, Smith M, Vreede FT, Fodor E (2009) NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome. J Gen Virol 90: 1398-1407.
  • Yuan P, Bartlam M, Lou Z, Chen S, Zhou J, He X, Lv Z, Ge R, Li X, Deng T, Fodor E, Rao Z, Liu Y (2009) Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site. Nature 458: 909-913.
  • Chase G, Deng T, Fodor E, Leung BW, Mayer D, Schwemmle M, and Brownlee G (2008) Hsp90 inhibitors reduce influenza virus replication in cell culture. Virology 377: 431-439.
  • Kerry PS, Willsher N, and Fodor E (2008) A cluster of conserved basic amino acids near the C-terminus of the PB1 subunit of the influenza virus RNA polymerase is involved in the regulation of viral transcription. Virology 373: 202-210.
  • Kreysa EM, Kirchbach JC, Fodor E, and Digard P (2008) Intranuclear dynamics of the influenza A virus RNA polymerase as revealed by live cell imaging studies, p. 82-84. In Katz JM (ed), Options for the Control of Influenza VI. International Medical Press, Atlanta.
  • Graef K, Carr S, Subbarao K, and Fodor E (2008) Mitochondrial localization of the influenza A virus PB2 protein, p. 87-89. In Katz JM (ed), Options for the Control of Influenza VI. International Medical Press, Atlanta.
  • Fodor E, Engelhardt OG, Smith M, Sharps J, Brownlee GG, Deng T (2008) Nuclear import and assembly of the influenza virus RNA polymerase complex, 90-92. In Katz JM (ed), Options for the Control of Influenza VI. International Medical Press, Atlanta.
  • Deng T, Engelhardt OG, Thomas B, Akoulitchev AV, Brownlee GG, and Fodor E (2006) Role of Ran Binding Protein 5 (RanBP5) in nuclear import and assembly of the influenza virus RNA polymerase complex. J Virol 80: 11911-11919.
  • Engelhardt OG, and Fodor E (2006) Functional association between viral and cellular transcription during influenza virus infection. Rev Med Virol 16: 329-345.
  • Chan AY, Vreede FT, Smith M, Engelhardt OG, and Fodor E (2006) Influenza virus inhibits RNA polymerase II elongation. Virology 351: 210-217.
  • Carr SM, Carnero E, García-Sastre A, Brownlee GG, and Fodor E (2006) Characterization of a mitochondrial targeting signal in the PB2 protein of influenza viruses. Virology 344: 492-508.
  • Engelhardt OG, Smith M, and Fodor E (2005) Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II. J Virol 79: 5812-5818.
  • Deng T, Sharps J, Fodor E, and Brownlee GG (2005) In vitro assembly of PB2 with a PB1-PA dimer supports a new model of assembly of influenza A virus polymerase subunits into a functional trimeric complex. J Virol 79: 8669-8674.
  • Fodor E., and Smith M (2004) The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the influenza A virus RNA polymerase complex. J Virol 78: 9144-9153.
  • Neumann G, Brownlee GG, Fodor E, and Kawaoka Y (2004) Orthomyxovirus replication, transcription, and polyadenylation, p. 121-143. In Kawaoka Y (ed), Biology of negative strand RNA viruses: the power of reverse genetics (Current Topics in Microbiology and Immunology, 283). Springer-Verlag, Berlin.
  • Fodor E, Fechter P, Crow M, Deng T, Mingay L, Sharps J, and Brownlee GG (2004) Mutational analysis of the PA and PB2 subunits of the influenza RNA polymerase complex leads to new insights into function, p. 25-28. In Kawaoka Y (ed), Options for the Control of Influenza V. Elsevier Science, Amsterdam.
  • Fechter P, Mingay L, Sharps J, Chambers A, Fodor E, and Brownlee GG (2003) Two aromatic residues in the PB2 subunit of influenza A RNA polymerase are crucial for cap binding. J Biol Chem 278: 20381-20388.
  • Catchpole AP, Mingay LJ, Fodor E, and Brownlee GG (2003) Alternative base pairs attenuate influenza A virus when introduced into the duplex region of the conserved viral RNA promoter of either the NS or the PA gene. J Gen Virol 84: 507-515.
  • Subbarao K, Chen H, Swayne D, Mingay L, Fodor E, Brownlee GG, Xu X, Lu X, Katz J, Cox N, and Matsuoka Y (2003) Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics. Virology 305: 192-200.
  • Fodor E, Mingay LJ, Crow M, Deng T, and Brownlee GG (2003) A single amino acid mutation in the PA subunit of the influenza RNA polymerase promotes the generation of defective interfering RNAs. J Virol 77: 5017-5020.

 

Research Funding

The group is supported by grants from the Medical Research Council, the Wellcome Trust and the European Commission.