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.

Analysis of the proteins of capripoxvirus (KS-1) revealed a 32kd protein that is one of the major structural proteins of the virus and is localised in the virus envelope. Monospecific serum prepared against the 32kd envelope protein neutralised the virus indicating that this protein contains neutralising epitopes. Lymphocyte proliferation studies, using the 32kd protein and peripheral blood mononuclear cells from capripoxvirus (KS-i) vaccinated sheep, showed that this protein strongly induced cellmediated immune responses. The 32kd protein is capripoxvirus specific and induced antibodies in early stages of capripoxvirus infections. Immunoblot analysis of antibody responses against this protein has provided a basis for the differential diagnosis of capripoxvirus and orf virus infections. The 32kd protein bound to the surface of cultured lamb testis cells. The binding of the 32kd protein was completely inhibited by prior incubation of cells with purified capripoxvirus (KS-1) but not by bovine serum albumin. Trypsin treatment of capripoxvirus (KS-1) degraded the majority of the 32kd protein with a minimal effect on a few other virus proteins. Trypsin removed an external 10kd fragment from the 32kd protein, leaving a 22kd fragment associated with the virus. In addition, the trypsin treatment reduced the virus infectivity by at least ten fold, suggesting that the cell surface binding domain of the 32kd protein is located within the external 10kd fragment. The monospecific serum to the 32kd protein had no effect of the infectivity titre of the trypsin treated virus further supporting the concept that the external 10kd fragment of the 32kd protein is involved in binding of the virus particle to the cell surface. A degenerate oligonucleotide probe, based on an internal amino acid sequence obtained from V8 protease cleavage products of the 32kd protein, was used to identify the gene encoding the 32kd protein. The gene encoding the 32kd protein was identified within the 2.8kb HindI1l Q1 fragment of the capripoxvirus (KS-1) genome. The nucleotide sequence analysis of the Hindu Q1 fragment revealed five open reading frames (Q11L, Q12R, Q13L, Q14R and Q15L), one of these open reading frames, Q13L, is capable of encoding a 30.6kd protein and contains the complete internal amino acid sequence obtained from the V8 protease cleavage products of the 32kd protein, indicating that the Q13L encodes the 32kd envelope protein of capripoxvirus (KS-1). The deduced amino acid sequence of the Q13L shows a 34.1% identity and 61.3% similarity with that of H3L open reading frame of vaccinia virus.

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.

The anaphase promoting complex (APC/C) is an ubiquitin ligase that is an essential regulator of multiple steps in the cell cycle. The complex consists of at least 12 subunits with a catalytic core formed by a scaffold protein, APC2, and a RING-H2 protein, APC11. The Parapoxvirus, Orf virus (OV), encodes a RING-H2 protein, B5L, with clear sequence similarities to APC11. The disruption of APC/C function leads to pre-mature entry into S phase and a delayed M phase exit and, potentially, apoptosis. This investigation explored the functional significance of the similarity between B5L and APC11 and specifically sought to determine if B5L manipulates cell cycle regulation by targeting APC/C function. Co-immunoprecipitation experiments from lysates of cells expressing a range of constructs revealed an interaction between B5L and APC2 in the same manner as seen with APC11. Furthermore, B5L was found to associate with endogenous APC/C. However, although APC11 promoted the formation of polyubiquitin chains in substrate-independent in vitro assays, B5L was inactive in this assay. Bioinformatics comparisons of APC11 and other known RING ubiquitin ligases with B5L and its poxviral homologues revealed some subtle differences. In particular a domain of APC11 (amino acids 61-74), that is essential for its ubiquitin ligase activity is not conserved in B5L or its homologues. When this APC11 domain was incorporated in place of the corresponding region of B5L (amino acids 59-67), the mutated B5L acquired ubiquitin ligase activity. On the other hand, APC11 protein in which the domain was replaced with that of B5L lost ubiquitin ligase activity. Stable cell lines expressing B5L showed an increased number of cells in G2/M phase (30�4%) compared with cell lines expressing APC11 (11�2%, n=3, p<0.05, ANOVA, Tukey�s), consistent with impaired APC/C function. APC/C substrates such as cyclin A, cyclin B and the thymidine kinase were stablized in B5L-expressing cells compared with control cells. Furthermore, transient hyper-expression of B5L induced apoptosis in 25�2% (n=3, p<0.05) of the cell population compared with only 6�1% apoptotic cells when APC11 was hyper-expressed. Analysis of the DNA content of OV-infected cells revealed enhanced DNA synthesis compared with cells infected with a B5L knockout OV. These observations indicate that B5L is a non-functional mimic of APC11. It associates with APC/C, but lacks ubiquitin ligase activity, and hence disrupts APC/C function. These abilities may enable OV to induce a cellular environment that enhances viral replication.

The anaphase promoting complex (APC/C) is an ubiquitin ligase that is an essential regulator of multiple steps in the cell cycle. The complex consists of at least 12 subunits with a catalytic core formed by a scaffold protein, APC2, and a RING-H2 protein, APC11. The Parapoxvirus, Orf virus (OV), encodes a RING-H2 protein, B5L, with clear sequence similarities to APC11. The disruption of APC/C function leads to pre-mature entry into S phase and a delayed M phase exit and, potentially, apoptosis. This investigation explored the functional significance of the similarity between B5L and APC11 and specifically sought to determine if B5L manipulates cell cycle regulation by targeting APC/C function. Co-immunoprecipitation experiments from lysates of cells expressing a range of constructs revealed an interaction between B5L and APC2 in the same manner as seen with APC11. Furthermore, B5L was found to associate with endogenous APC/C. However, although APC11 promoted the formation of polyubiquitin chains in substrate-independent in vitro assays, B5L was inactive in this assay. Bioinformatics comparisons of APC11 and other known RING ubiquitin ligases with B5L and its poxviral homologues revealed some subtle differences. In particular a domain of APC11 (amino acids 61-74), that is essential for its ubiquitin ligase activity is not conserved in B5L or its homologues. When this APC11 domain was incorporated in place of the corresponding region of B5L (amino acids 59-67), the mutated B5L acquired ubiquitin ligase activity. On the other hand, APC11 protein in which the domain was replaced with that of B5L lost ubiquitin ligase activity. Stable cell lines expressing B5L showed an increased number of cells in G2/M phase (30�4%) compared with cell lines expressing APC11 (11�2%, n=3, p<0.05, ANOVA, Tukey�s), consistent with impaired APC/C function. APC/C substrates such as cyclin A, cyclin B and the thymidine kinase were stablized in B5L-expressing cells compared with control cells. Furthermore, transient hyper-expression of B5L induced apoptosis in 25�2% (n=3, p<0.05) of the cell population compared with only 6�1% apoptotic cells when APC11 was hyper-expressed. Analysis of the DNA content of OV-infected cells revealed enhanced DNA synthesis compared with cells infected with a B5L knockout OV. These observations indicate that B5L is a non-functional mimic of APC11. It associates with APC/C, but lacks ubiquitin ligase activity, and hence disrupts APC/C function. These abilities may enable OV to induce a cellular environment that enhances viral replication.

No description available.

Orf virus (ORFV) has the potential to be developed as a vaccine vector. Its ability to stimulate non-specific as well as specific immune responses in permissive and non-permissive hosts stands it in good stead to be utilised as such a tool. The fusion of immunogenic peptides to vaccinia virus (VACV) structural proteins have been shown to improve their immunogenicity due to presentation of the foreign antigens in a particulate form that can stimulate both B and T cells. The aims of this study were to fuse foreign antigens to ORFV structural proteins to demonstrate proof-of-concept that such surface display could also render the foreign antigens more immunogenic. Little is known about ORFV structure and morphogenesis. When this study commenced, the ORFV genome had recently been sequenced and this revealed a large number of homologues in common with VACV. It was thus assumed that both viruses may share structural similarities and that ORFV also assumes the different morphological forms such as the mature virion (MV) and extracellular virion (EV) that are present in VACV. The MV and EV forms are both infectious, with the EV containing an additional membrane acquired from the trans-Golgi network during viral morphogenesis. Furthermore, specific viral proteins are associated with both the MV and EV membranes. Six ORFV structural proteins ORFV 089, 10 kDa, F1, that are homologues of structural membrane proteins A13, A27 and H3 of VACV MVs, together with ORFV 109, ORFV 110 and B2, that are homologues of structural membrane proteins A33, A34 and F13 of VACV EVs were selected as possible candidates for manipulation. At present, there is some information available only for 10 kDa, F1 and B2. The 10 kDa is required for virus assembly, F1 for mediating cell attachment while B2 has been shown to induce significant antibody responses in sheep. Indeed proteomic analyses predicted similarities in the topologies of all of these proteins with their VACV counterparts. Using this information, preliminary studies were conducted to generate recombinant ORFVs (rORFVs) which had FLAG fused to the terminus of the protein that was exposed on the surface of the virus particle. Three rORFVs 10 kDa, F1L and 110 were successfully generated. Immunogold labelling of FLAG proteins on virus particles isolated from lysed cells showed that FLAG-10 kDa and FLAG-F1 were displayed on the surface of MV particles whereas FLAG-ORFV 110 could not be detected. Western blot analyses of solubilised recombinant ORFV 110-FLAG particles revealed that FLAG-ORFV 110 was abundant and undergoes post-translational modification indicative of endoplasmic reticulum trafficking whereas FLAG-10 kDa and FLAG-F1 did not appear to be subjected to post-translational modifications. Fluorescent microscopy confirmed the prediction that ORFV 110-FLAG localised to the Golgi in virus-infected cells and immunogold labelling of EVs showed that ORFV110-FLAG became exposed on the surface of EV-like particles as a result of egress from the cell, suggesting that the membranes had been acquired from the Golgi. These modifications also appeared to have minimal effect on the infectivity of these rORFVs. The study was extended by replacing the small FLAG peptide with an immunogenic protein (EG95), derived from the oncosphere of the zoonotic parasite Echinococcus granulosus. This protein is known to confer protection in immunised animals. Three rORFVs were generated in which a truncated version of the protein, EG95[Delta]TM, was fused to 10 kDa in the absence (rORFV 699) or presence (rORFV 700) of a linker, and also to F1 (rORFV 701). Western blot analyses of these solubilised particles demonstrated that the fusion proteins appeared to be post-translationally modified while immunogold labelling using anti-EG95 monoclonal antibodies successfully demonstrated the surface labelling on these rORFVs. In order to test the immunogenicity of these rORFVs, prime-boost experiments in sheep were conducted using rORFVs 699, 700 and 701 and a glutathione-S-transferase (GST-EG95) based vaccine. The results showed the production of EG95-specific antibodies. In particular, antibody production by group rORFV 701 compared favourably with a control group that was primed and boosted by GST-EG95 vaccine. This was despite the slightly slower growth rates of rORFVs 700 and 701 and the decreased infectivity of all three rORFVs discovered in in vitro experiments. In conclusion, these studies indicated the feasibility of this strategy to manipulate ORFV structural proteins for use as an agent for vaccine delivery.

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.