Apoptosis plays important roles in host defences against virus infection. It is therefore not surprising that viruses have developed a vast array of modulators that block this process at different stages within the apoptotic pathways. Intrestingly, Orf virus (ORFV), a member of the Parapoxvirus genus, did not reveal any of the known poxviral inhibitors of apoptosis, but was found to express a unique anti-apoptotic protein, ORFV125. The aim of this PhD project was to determine the subcellular localisation of this protein and to further characterise its anti-apoptotic activity. This included exploring its ability to inhibit early, intermediate and late events of apoptosis and identifying the mechanism by which this viral protein functions to prevent cell death.
Experiments revealed that ORFV125 was localised to the mitochondria through a C-terminal mitochondrial-targeting motif, and this specific location was necessary for the protein�s anti-apoptotic function. Furthermore, the viral protein inhibited UV-induced apoptotic events at and downstream of the mitochondria such as cytochrome c release, caspase activation and DNA fragmentation. However, it was not able to prevent UV-induced activation of the c-Jun-NH₂ kinase (JNK), an event occurring upstream of the mitochondria, consistent with its localisation to this organelle. The ability to prevent apoptosis was comparable with that of the cellular anti-apoptotic protein Bcl-2, which belongs to a family of mitochondrial regulators of apoptosis.
Although standard BLAST analysis failed to detect homology to anti-apoptotic members of the Bcl-2 family, a manual alignment of the primary sequence of ORFV125 with these proteins revealed characteristic residues of Bcl-2 homology (BH) domains within ORFV125. These motifs are conserved within the Bcl-2 proteins and important for their structure and function. In addition, mutating amino acids within the ORFV125 BH domains led to a loss of the anti-apoptotic function of the mutated proteins, indicating the functional importance of these residues for the viral protein. These observations suggest that ORFV125 might be classified as a viral Bcl-2-like protein.
To provide evidence for this hypothesis, it was investigated if ORFV125 acts in a Bcl-2-like manner to inhibit apoptosis. The viral protein was able to entirely block the activation of the pro-apoptotic Bcl-2 family members Bak and Bax, although it did not directly bind to these proteins. Instead, ORFV125 interacted with a subset of the pro-apoptotic BH3-only proteins, which can trigger the activation of Bax and Bak. Furthermore, this study demonstrated that ORFV125 could inhibit apoptosis induced by BH3-only proteins to which the viral protein could bind. On the other hand, ORFV125 was not able to prevent the activity of pro-apoptotic proteins that it failed to interact with. This shows that ORFV125�s mechanism of action is to inhibit the activity of BH3-only proteins by binding and neutralising their function.
Overall, these results provided evidence that ORFV125 is potent anti-apoptotic protein that can prevent UV-induced cell death without the participation of other ORFV proteins. Furthermore, the viral protein shared primary sequence and secondary structure similarities to Bcl-2 family members and acted in a Bcl-2-like manner to inhibit apoptosis.
| Identifer | oai:union.ndltd.org:ADTP/201523 |
| Date | January 2008 |
| Creators | Westphal, Dana, 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 Dana Westphal |
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