The nucleopolyhedrovirus of Epiphyas postvittana (EppoMNPV) is being studied as a potential biological control agent for leafroller insects in New Zealand. The aims of this project were for the identification of putative genes that are unique to, variant in or missing from, the EppoMNPV genome and the subsequent analysis of at least one of these genes. The purpose of this was to identify and characterise genes potentially involved in the specific host range and virulence of EppoMNPV. This was achieved in two steps. Three genome regions lacking linearity between EppoMNPV and the closely related OpMNPV were previously identified and the targeted sequencing of these three regions was the first aim of this project. The collation of the entire genome sequence of EppoMNPV and comparison to the genome sequences of 22 other baculoviruses completed the identification of genes that are unique to, variant in or missing from EppoMNPV. The EppoMNPV genome was found to be 118,584 bp in size encoding 136 putative proteins. A total of 29 genes were found to be common to all baculoviruses, while the lepidopteran baculoviruses share a total of 62 genes. The genome of EppoMNPV encodes four putative unique genes, the sequence of which offers no clues as to possible function. EppoMNPV lacks a homologue of the superoxide dismutase gene common to all other lepidopteran baculoviruses The EppoMNPV IE2 homologue was identified as a 311 amino acid protein with a truncation in the N-terminal region. We hypothesised that this truncation would lead to a loss of function, which could contribute to the virulence and/or host range of EppoMNPV For this reason, the characterisation of the EppoMNPV IE2 was taken up as the second part of this project. A comparative study between the AcMNPV and EppoMNPV IE2 proteins identified no differences in function between these two proteins in Sf21 cells. The EppoMNPV IE2 was capable of trans-activating three constitutive promoters and localised to discrete nuclear bodies. Cell cycle arrest was not achieved by either IE2 protein in our cell culture system. The role of four sequence motifs common to all IE2 proteins was studied with the aid of mutational analysis. Mutation of arginine and acidic rich sequences of EppoMNPV IE2 showed only a slight decrease in trans-activation activity while mutation of the RING-finger and coiled-coil motifs reduced trans-activation to less than half that of wild type IE2. Mutation of the coiled-coil motif resulted in reduced amounts of protein localising to discrete nuclear regions. A series of deletion mutants from the N- and C-termini of EppoMNPV IE2 identified that the C-terminal 111 amino acids of EppoMNPV IE2 was sufficient for nuclear targeting. Deletion of the C-terminal 19 amino acids resulted in an IE2 mutant completely defective in both localisation and transactivation. This demonstrates that localisation to discrete nuclear regions is essential for EppoMNPV IE2 to act as a transactivator.
| Identifer | oai:union.ndltd.org:ADTP/217363 |
| Date | January 2005 |
| Creators | Hyink, Otto, n/a |
| Publisher | University of Otago. Department of Microbiology & Immunology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Otto Hyink |
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