Functional analysis of Parapoxvirus-encoded vascular endothelial growth factors

The poxviruses are an extensive family of large DNA viruses, including the causative agent for smallpox, variola virus. The success of poxviruses as pathogens is in part due to their expression of an array of gene products that redirect host responses to infection and contribute to virus virulence. An example of this is a novel member of the vascular endothelial growth factor (VEGF) family, VEGF-E, encoded exclusively by Parapoxviruses (PPV). VEGFs are essential regulators of blood vessel formation (angiogenesis), and act via the VEGF receptors (VEGFR)-1, -2 and -3. VEGF-E has a unique receptor-recognition profile of interacting selectively with VEGFR-2, the main receptor mediating angiogenesis. Orf virus (ORFV), the type species of the Parapoxvirus genus, was the first virus shown to encode a VEGF. ORFV VEGF appears to be responsible for the extensive vascularisation and marked epidermal proliferation characteristic of the viral lesions. Other members of the PPVs encode VEGF homologs, and the first aim of this study was to compare receptor-recognition profiles across the PPV VEGF group with direct and competitive receptor-binding assays. Analysis of the purified proteins revealed that all PPV VEGFs are functional homologs of the VEGF family that conserve selectivity for VEGFR-2, despite their unusual degree of amino acid sequence variation. This sequence divergence and receptor specificity led to the second aim of this study; to investigate the role of a Thr/Pro-rich C-terminus, containing putative O-glycosylation that is highly conserved across the PPV VEGFs but not present in mammalian VEGFs. Receptor-binding assays revealed that the O-glycosylated C-terminus of the PPV VEGFs prevents interaction with VEGFR-1 and optimises interaction with VEGFR-2. The third aim was to investigate the biological effects of the variant receptor-binding profiles exhibited by the VEGFs, in a mouse model. Cellular assays showed that VEGFR-2-specific ORFVNZ2VEGF induced keratinocyte proliferation and migration, and endothelial cell proliferation in vitro, as well as epidermal thickening, endothelial cell proliferation and blood vessel development in vivo; reminiscent of the extensive epidermal proliferation and vascularisation observed in PPV lesions. The lack of VEGFR-1 recognition meant that ORFVNZ2VEGF did not stimulate monocyte migration in vivo, or increase dermal monocytes in vivo. This would impair the adaptive immune response and represents an important mechanism for immune evasion by the virus. The limited host range and lack of systemic spread of ORFV makes it an attractive tool for vaccine antigen delivery to sheep. Recombinant ORFV expressing relevant antigens and attenuated via deletion of the VEGF-E gene would lack the characteristic proliferative lesion but could still stimulate a protective immune response. The different receptor-binding profiles displayed by VEGF-E variants constructed and examined in this thesis suggest that receptor-recognition may be further fine-tuned for optimal therapeutic angiogenesis. This provides exciting new options to treat several human diseases with significant morbidity and mortality, such as revascularisation and healing of diabetic ulcers, and promoting post-ischemia neurovascular remodeling after stroke