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Title: The characterisation of a GM-CSF and IL-2 inhibitory protein encoded by Orf virus
Author: Deane, David Leslie
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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In this study, the gene encoding the GM-CSF inhibitory factor (GIF) was isolated and mapped to the right terminal quarter of the orf virus genome. The orf virus GIF cDNA was expressed as a secreted protein in Chinese hamster ovarian cells as detected by GM-CSF inhibition ELISA. Recombinant GIF was purified by ovine GM-CSF affinity chromatography and gel filtration. Sequence analysis of the 20 N-terminal amino acids was performed on the purified GIF. The GIF gene encodes a 28 kDa protein that exhibits 32% amino acid sequence similarity to the predicted sequence of the A41L gene product encoded by vaccinia virus. Although the vaccinia virus A41L protein has sequence similarity to the T1 secreted chemokine-binding proteins of leporipoxviruses, its function is not known. GIF did not share any homology with any cytokine receptor molecule identified to date. In contrast to other parapoxvirus immunomodulatory proteins that are products of early viral genes, GIF was found to be the product of an intermediate/late viral gene of orf virus infected cells. GIF formed homodimers and homotetramers in solution and bound ovine GM-GSF with a Kd of 369 pM. In addition GIF bound ovine IL-2 with a Kd of 1.04 nM. Although orf virus infects humans, GIF did not bind human GM-CSF or IL-2. GIF was shown to inhibit the binding of ovine GM-CSF labelled with 125I to its receptor on isolated sheep neutrophils and it inhibits the haematopoietic activity of ovine GM-CSF in a soft agar bone marrow colony assay. GIF also inhibited the binding of ovine IL-2 labelled with 125I to CD4+ T cells and inhibited the stimulatory activity of ovine IL-2 in a T cell proliferation assay. This inhibitory activity was neutralised by a rabbit antiserum raised against purified GIF. GIF was produced in vivo during orf virus reinfection. GIF was detected in skin, localised to the area of orf virus infected cells and in afferent lymph draining the skin site of infection. The presence of GIF, 3-7 days after virus infection was associated with reduced levels of GM-CSF in the lymph plasma and the period of maximum viral replication in the skin.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available