Structural studies of poxvirus mediated inhibition of host cell apoptosis

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

In multi-cellular organisms, apoptosis plays an important role in homeostasis. However, apoptosis also plays a major role as a powerful immune response. Because of its profound impact on cellular integrity, multiple mechanisms evolved to ensure appropriate triggering or inhibition. Viral invasion of a host cell is one event capable of triggering apoptosis. DNA viruses posses a large genome, encoding molecular responses against many pathways inducing apoptosis. One of them is the expression of viral Bcl-2 homologs that interfere with the Bcl-2 mediated signaling designed to activate Bak and Bax, two proteins essential to permeabillize the outer mitochondrial membrane. Cellular Bcl-2 proteins harbour four characteristic Bcl-2 Homology (BH) domains. A noteworthy feature of their viral counterparts is the typically low level of sequence identity between viral and cellular Bcl-2 proteins. Most poxviral vBcl-2 proteins do not harbor readily identifiable BH domains. Despite this observation, these viral proteins efficiently prevent intrinsic mediated apoptosis. Previous studies identified two potential poxviral Bcl-2 proteins: DPV022 and SPPV14. Strong evidence suggests that they could display the same activity of the previously identified M11L and F1L. However, whether or not DPV022 and SPPV14 adopt a Bcl-2 fold remains unclear. I determined the oligomeric state of each protein and assessed the binding affinity of DPV022 to BH3-peptides. Using the identified ligand binding profile, I determined three structures of complexes between DPV022 with Bim, Bak and Bax. These structures revealed features suggesting a common ancestor that was shared with cellular Bcl-2 proteins. Based on my findings I postulate the existence of at least two classes of poxviral Bcl-2 proteins: monomeric proteins, which bind numerous ligands with high affinity, and dimeric vBcl-2 proteins with less ligands and lower affinities Although several attempts were made, determination of a crystal structure of SPPV14 was unsuccessful. In its absence a homology model was generated on the basis of the determined crystal structure of DPV022 and the structure of M11L. The final model revealed essential binding interactions shared with DPV022 and cellular Bcl-2. In its totality the work presented in this thesis enabled a better understanding of the poxviral Bcl-2 proteins, the potential molecular origin and their molecular mechanism of action.