The immunomodulatory protein, GIF, encoded by orf virus

Abstract

Inflammatory diseases cause considerable morbidity throughout the world. Examples of inflammatory diseases include rheumatoid arthritis, Crohn’s disease, atherosclerosis and asthma. Current treatments options are insufficient, and novel treatment options must be explored. Immunomodulatory proteins observed in nature could be repurposed as biotherapeutics, as a form of biomimicry. Large double-strand DNA viruses encode a number of virulence proteins which are capable of dampening host immune response. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and Interleukin-2 (IL-2) inhibition factor (GIF) is one such protein encoded by orf virus. Orf virus is a zoonotic virus which infects mostly sheep and goats. GIF is able to bind ovine and bovine GM-CSF and IL-2, but not human variants despite high sequence homology of both cytokines. The expression of virulence proteins including GIF allows orf virus to infect and re-infect individual animals and herds repeatedly. This work reports on the establishment of an expression and purification protocol for the GIF protein, an examination of the structural and binding behaviour for GIF, as well as the exploration of a potential mechanism for GIF interacting with and inhibiting human GM-CSF. Work consisted of establishing an expression and purification protocol of GIF in enough quantity for downstream experiments. Expression trials optimised the amount of DNA used in HEK 293-6E cell transfection, best day of harvest and the addition of supplements to the expression mixture. The purification protocol involved a three-step fast-performance liquid chromatography purification with an additional dialysis step to remove heat-shock protein 70 (HSP70) bound to GIF. Binding and modelling studies of GIF were carried out to help explain its biological properties. Structural binding experiments confirmed that GIF is subject to N-linked glycosylation which is required for secretion from the cell. Enzyme-linked immunosorbent assays (ELISAs) confirmed that GIF did not bind human GM-CSF or IL-2, but was able to bind bovine GM-CSF. A cell proliferation assay with GM-CSF-dependent TF-1 cells revealed that GIF treatment reduced proliferation of cells treated with human GM-CSF compared to the no-GIF control, indicating that GIF may be able to have some form of interaction with human GM-CSF. We studied the interface of the GIF-ovine GM-CSF complex and observed the differences in residues between human and ovine GM-CSF. It was concluded that GIF might be able to form a transitory interaction with human GM-CSF, however this interaction is likely to be weak due to steric hinderance across the binding interface. Further examination of the structural basis and binding behaviour of GIF is suggested for it to be repurposed as a biotherapeutic.