Negative strand RNA viruses : a structural study
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This thesis details the study of proteins derived from four negative strand RNA viruses: Simian Virus 5 (SV5), Bunyamwera virus (BUN), Rabies virus and Newcastle Disease virus (NDV). Each virus is an important human and/or animal pathogen with the capacity to cause outbreaks of epidemic proportions. Biochemical and biological characterisation of viral proteins has been fundamental in determining the pathogenic potential of their associated viruses. Elucidation of a polypeptide's tertiary structure can be pivotal in the full interpretation of this data by defining structure-function relationships intrinsic to protein activity. However, to date the number of solved virus protein structures remains relatively small. The predominant focus of this work was interpretation of biophysical evidence gathered during attempts to elucidate the tertiary structures of the phosphoprotein (SV5 P), nucleoprotein (BUN N) and rabies glycoprotein (CVS G). Following successful expression and purification of SV5 P, attempts to crystallise the protein failed. Further characterisation revealed the N-terminal domain to be predominantly disordered. Several strategies were developed to induce globularity by co-expression with an in vivo binding partner, but concluded without success. Finally, truncated regions of SV5 P were expressed, purified and placed into crystallisation screens. BUN N crystals were successfully obtained following optimisation of an established purification procedure. Pursuit of better quality crystals was hindered by precipitation of BUN N at low concentrations. The aggregation of the protein precluded all attempts to increase soluble protein yield. To produce glycosylated CVS G, recombinant P. pastoris clones were constructed and tested for expression. Initial success in small-scale expression trials was not repeatable in volumes suitable for producing significant protein yields. Finally, this thesis furthers previous studies on the catalytic domain of the hemagglutinin-neuraminidase protein (HN) from NDV. The structures of two HN-inhibitor complexes were solved to 2.5 and 2.8 Å respectively. The inhibitory actions of the substrate analogues are discussed in terms of inhibitor HN interactions.
Thesis, PhD Doctor of Philosopy
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