Biophysical characterisation of the orf poxviral ankyrin/F-box OV008 protein

Abstract

Orf poxvirus OV008 protein contains multiple ankyrin repeats and an F-box motif, through which it interacts with the Skp1 component of the host Skp1:Cul1:F-box (SCF-1) ubiquitin ligase complex. This thesis demonstrates that a linker region between the OV008 ankyrin repeat domain and the F-box motif produces an oligomerisation of the heterodimeric OV008:Skp1 complex, and that the OV008 protein also demonstrates this oligomeric interaction in an SCF-1 complex of OV008:Skp1:Cul1:Rbx1. The oligomeric nature of the OV008:Skp1 complex was examined using solution based biophysical techniques including small angle X-ray scattering, analytical ultracentrifugation, multi-angle laser light scattering, and electro-spray ionisation mass spectrometry. Truncations to the C-terminal linker of the OV008 protein enabled the identification of this previously uncharacterised region of the protein as the mediator of the self-association. A number of these truncates formed stable OV008-F-box:Skp1 complexes that no longer self-associated. The OV008 linker region is highly conserved within the Parapoxvirus genus, and shows high residue conservation in other poxvirus species. However this region lacks homology to sequences outside of the ankyrin/F-box proteins and is absent in known interaction motifs, and therefore could contain a novel oligomeric domain. The dimerisation of F-box domains is being recognised as a fundamental part of the regulation of the SCF-1 ubiquitin ligases. This poxviral mimicry of cellular F-box behaviour suggests that the incorporation of poxviral ankyrin/F-box proteins into the SCF-1 complex would still produce a dimeric complex that would be redirected to the viral mediated degradation of target proteins. The implications of this oligomerisation with regards to OV008 and other poxviral ankyrin/F-box interactions with the SCF-1 complex and possible host target proteins are discussed.