Viral modulators of intrinsic apoptosis

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular Sciences, College of Science, Health and Engineering, La Trobe University, Bundoora.

Apoptosis is a crucial process required by multicellular organisms to remove damaged, unwanted or diseased cells. The main mediators of intrinsic apoptosis are the BCL-2 family. Upon viral infection cells undergo apoptosis to inhibit productive viral replication, and to prevent this, viruses express homologues of the BCL-2 family. These include KsBCL-2 from Human Herpes virus 8 (HHV8), and the F1L proteins from Vaccinia Virus (VV) and Variola virus (VAR), the causative agent of small pox. To date the interactions between these viral BCL-2 homologues and their cellular counterparts have not been well studied. This thesis characterises structural and biochemical aspects of the three aforementioned viral BCL-2s (KsBCL-2, VV F1L and VAR F1L). KsBCL-2 binding interactions have been disputed in the literature and are explored further here utilising surface plasmon resonance techniques. Crystallisation attempts of KsBCL-2 resulted in the structure of a contaminant Escherichia coli protein, YodA, which is also described. Although VV F1L interactions with the BCL-2 family have been studied, it has been reported that the N-terminal region may be capable of inhibiting active caspase 9. Analysing the 3D solution structure of VV F1L via SAXS and probing interactions with both intrinsic and extrinsic apoptogenic molecules does not provide evidence that VV F1Ls N-terminal region, nor its entirety, is able to inhibit caspase 9. Lastly F1L from small pox was shown to act in a novel un-predictable fashion when compared to the VV homologue through in vitro and in vivo studies. This is further explored using structural information, but no distinct differences were observed.