AbstractThe aim of this study was to characterise the function of the Ham1-like protein encoded by cassava brown streak virus (CBSV) and ugandan cassava brown streak virus (UCBSV), which cause CBSD. It has been proposed that this protein functions as a nucleotide triphosphate pyrophosphatase, due to its homology to cellular Inosine triphosphate pyrophosphatase (ITPase) proteins. It might therefore act to reduce the viral mutation rate during infection to preserve viral stability, which would be an unusual role for an RNA virus protein. To test this hypothesis, recombinant Ham1 proteins of CBSV and UCBSV were over-expressed in E. coli and purified by nickel-affinity chromatography, and their ITPase activity tested in a pyrophosphatase assay against a range of nucleotide substrates. It was demonstrated that both viral Ham1 proteins were active and preferred the mutagen-causing noncanonical nucleotides deoxy inosine triphosphate and xanthosine triphosphate. Therefore, Ham1 may act as ITPase proteins to protect the viral genome from mutation during infection.
To help understand why viruses benefit from encoding a Ham1-like protein, the role of host plant ITPase proteins was studied. To assess whether viruses require the presence of an ITPase protein for stable infections, the viral titres of the RNA virus cucumber mosaic virus (CMV) were studied in mutant Arabidopsis with abolished ITPase proteins. Systemic leaf CMV titres were found to be much lower in mutant plants versus WT Arabidopsis, suggesting ITPase proteins are somehow involved in virus stability and infectivity. However, the expression of WT Arabidopsis ITPase was found to not change in response to CMV infection. This provides the first proof that CBSV and UCBSV are indeed functional ITPase proteins, and that ITPase activity is important during viral propagation in plantae.
|Date of Award||23 Jan 2019|
|Supervisor||Andy M Bailey (Supervisor) & Gary D Foster (Supervisor)|