Characterization of ORFV119
|dc.contributor.author||Harfoot, Rhodri Thomas|
|dc.identifier.citation||Harfoot, R. T. (2015). Characterization of ORFV119 (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/5706||en|
|dc.description.abstract||A large number of viruses are known to produce proteins that interact with pRB. For the most part these are small DNA viruses, such as Adenovirus and Human papilloma virus, which require that the cell be in a replicative state itself before they can replicate themselves. For this purpose, these viruses produce proteins (E1A and E7 respectively) that bind to pRB and disrupt its interaction with E2F family members, thereby pushing the cells into the synthesis phase of the cell cycle, and enabling the virus to utilize the resources that the cell generates for replication. At this point, the invading virus co-opts the host-cell replication machinery to replicate the viral genome and produce the protein coat. Poxviruses complete their replication cycle in the cytoplasm rather than the nucleus, and encode much of their own replication machinery, and as such were, until recently not thought to co-opt host cellular replicative mechanisms. Moreover, until recently, poxviruses were also not thought to alter the cell cycle extensively. Based on these concepts, it seemed unlikely that poxviruses would encode a putative pRB binding protein. This thesis aimed to identify and characterize a novel pRB binding protein from orf virus (ORFV), known as ORFV119. Bioinformatics analyses predicted that, the ORFV119 protein contained two putative functional motifs, a pRB binding motif (LXCXE) in the C terminus and a mitochondrial targeting motif in the N terminus. These motifs both matched closely those found in other proteins known to contain the respective motifs. It was found that ORFV119 was completely conserved within isolates orf virus, and homologues were present in other species within the Parapoxvirus genus, but largely absent outside this clade, apart from in Molluscipoxvirus. ORFV119 was determined to be an early gene by detection of the protein 8 hours post infection using an antiserum against ORFV119 developed during this thesis. The protein was detected as a punctate staining in the cytoplasm, that co-localized with mitochondria. In the absence of the putative mitochondrial targeting motif, the staining was dispersed throughout the cell with occasionally enhanced signal in the nucleus. A construct expressing only the predicted pRB binding motif fused to GFP showed a similar pattern to that seen for constructs where the mitochondrial targeting motif had been removed. A construct which had had the LXCXE domain removed showed staining similar to full-length protein. Co-immunoprecipitation showed that full-length ORFV119 was capable of binding to, and co-precipitating pRB, and that this was dependent on the presence of the LXCXE motif, indicating that ORFV119 does bind to pRB through a canonical mechanism. Luciferase assays for E2F responsive promoter activity found that ORFV is capable of a mild activation of E2F responsive promoters. Full length ORFV119 was also able to stimulate E2F responsive promoter activity. The same assays performed using ORFV119 truncations showed that upon removal of the LXCXE motif, ORFV119 was still capable of activating E2F responsive promoters, but removal of the mitochondrial targeting motif removed this ability. It is postulated that this is due to the structure of ORFV119 and its respective truncation mutants, where ORFV119 and the delta-LXCXE mutants both had structures similar to pRB, whereas the delta-Mito did not, thus it could be that ORFV119 is mimicking pRB and competing with pRB for E2F. A Molluscum contagiosum virus (MOCV) protein with a similar range of properties has been identified (Mohr et al., 2008). Phylogenetic comparison of the ORFV and MOCV proteins indicates low sequence similarity and identity, despite overall similarities in protein size and positions of the motifs. This indicates that it is unlikely that the two proteins are related, but rather a common solution to a common problem. In summary, this is the first report of a novel pRB binding and mitochondrial targeting protein from orf virus, which may enable the virus to create a cellular environment conducive for viral replication.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.subject||retinoblastoma associated protein|
|dc.title||Characterization of ORFV119|
|thesis.degree.discipline||Microbiology and Immunology|
|thesis.degree.name||Doctor of Philosophy|
|thesis.degree.grantor||University of Otago|
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