The RNA polymerase of influenza A virus is a heterotrimerk- complex of PS1, PS2 and PA subunits, that is required for transcription and replication of the viral genome. The polymerase complex binds to viral RNA (vRNA) and complementary RNA (cRNA) via conserved promoter regions at the 5' and 3' ends of each segment. Regions within PSl that bind to both promoters have been identified by others; however, promoter binding by PS2 and PA has also been reported. In this thesis a differential requirement of the PA subunit for binding to the vRNA and cRNA promoters is demonstrated - specifically, PA is more important for binding to the cRNA than to the vRNA promoter. Furthermore, five point mutations were identified in the L163-I178 region of PA, which resulted in an inhibition of polymerase activity when provided with a cRNA compared to vRNA promoter. Cross-linking studies suggested that this inhibition was due to a reduction in promoter binding of the mutant polymerases to the cRNA promoter. This suggested that the L163-I178 region of PA is directly or indirectly involved in cRNA promoter binding and suggested a novel function for PA in modulating promoter binding. Characterisation and comparison of the in vitro activity of mutant polymerase complexes . required that similar amounts of polymerase were used. In this thesis a quantitative method for polymerase normalisation by silver stained SDS-PAGE was developed. Mutant PA subunits L163A, D164A, R168A, I171A, K172A, R174A, L175A and I178A were observed to have reduced trimeric polymerase formation because these mutants were targeted to the proteasome. Furthermore, an inhibition of their nuclear accumulation suggested that these mutants may be misfolded and that this region of PA may be required for intra-molecular interactions. Newly synthesised trimeric polymerase requires import of the three subunits into the nucleus and assembly of the trimeric complex. It has been suggested that a [PS1, PA] dimer is transported into the nucleus where assembly with the PS2 monomer occurs. In this thesis, preliminary data has suggested binding of the monomeric PA subunit to cellular karyopherin 02 and 03. Furthermore, it was suggested that binding of karyopherin 02 and 03 to PA was not via a previously characterised bipartite nuclear localisation signal (NLS). Interestingly, however, functionality of the bipartite NLS in the nuclear targeting of monomeric PA was confirmed, suggesting that another, as yet unidentified, nuclear import factor (other than karyopherin 02 and 03) may bind to this NLS.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:491365 |
| Date | January 2007 |
| Creators | Maier, Helena J. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
Page generated in 0.0013 seconds