The work in this thesis draws on a prior bioinformatics survey, in which it was shown that the existence of <i>cis-</i>acting RNA sequences within the coding regions of influenza A could be deduced from regions of low codon variability. To identify and characterise sequences with a putative role in packaging, synonymous mutations were introduced into the terminal coding sequences of segments 7 and 5 of influenza A/Puerto Rico/8/34 virus, targeting both conserved codons and non-conserved codons. Mutating conserved codons in segment 7 of the virus caused significant growth defects, reducing the output of plaque-forming virus by 10- to 1000-fold. The mutations were shown to affect various <i>cis-</i>acting functions, including, in all cases, disruption of genome packaging. This manifested differently in MDCK cells, where the mutations reduced the total amount of virus produced, and in embryonated chicken eggs, where virus was released in quantities similar to wild-type but with reduced incorporation of all eight genomic segments. In the latter case, it was shown that the defective mutations caused virions to be released with incomplete genomes. In contrast, the introduction of similar mutations into segment 5 had much less effect on virus growth, with most mutant viruses replicating normally and only two showing replication defects. Nevertheless, the most defective virus showed evidence of poor genome packaging. Serial passage of defective segment 7 mutants produced pseudorevertants that regained wild-type growth properties despite retaining their original mutations; preliminary work to characterise the genetic interactions in these viruses is presented. In summary, this thesis characterises a series of influenza A mutations which provide information on various <i>cis-</i>acting RNA functions, particularly genome packaging.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:604852 |
| Date | January 2009 |
| Creators | Hutchinson, E. C. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
Page generated in 0.0019 seconds