Two expression systems were utilised in an attempt to produce suitable quantities of the influenza polymerase proteins for protein purification. Using the methylotrophic yeast <I>Pichia pastoris, </I>both PB1 and PB2 were expressed at around 0.1-0.4 mg/l. However, it did not prove possible to purify the polypeptides which appeared to be aggregated. The P proteins and NP were also expressed in mammalian cells using recombinant Semliki Forest viruses. However, soluble protein did not accumulate at levels sufficient for large scale purification studies. A transient "reverse genetics" assay whereby the expression of a synthetic model viral RNA segment is driven by influenza P proteins expressed by recombinant vaccinia viruses and NP from a T7 driven plasmid, was used for the analysis of mutant influenza proteins in the processes of viral transcription and replication. Recombinant vaccinia viruses expressing mutant forms of PB1 were generated and their ability to support viral replication determined. One mutant which was shown not to bind PB2 retained reduced but signifciant replication activity. In contrast, mutants that were unable to bind PA were defective in viral RNA synthesis. Of the mutants that were defective in viral transcription, one acted as a <I>trans</I>-dominant inhibitor of wild type protein function. Mutants that were unable to bind RNA were unable to support viral transcription, whilst those with reduced RNA binding capacity were similarly impaired in transcription. The mutants that were unable to bind RNA were also shown to be transdominant inhibitors of replication, indicating that they were able to compete with wild type NP for essential components of the transcription machinery. Mutations in the N-terminal region of NP previously described as the karyopherin α binding site were also analysed for their ability to support viral transcription. Neither mutation of a residue described as essential for karyopherin α binding nor deletion of the karyopherin α binding site affected viral RNA synthesis, though these mutants localised in the cell differently to wild type NP.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:596669 |
| Date | January 1998 |
| Creators | Bishop, K. J. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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