The Modulation of Apoptosis by Poxviruses

Abstract

Apoptosis serves as a powerful defense against damaged and unwanted cells. The family of Bcl-2 proteins plays a key role in the regulation of apoptosis at the mitochondria. Members of the Bcl-2 family include pro- and anti-apoptotic members that collectively determine the state of the cell. Apoptosis is an effective defense against viral infection, and many viruses, including poxviruses, encode proteins to prevent or delay apoptosis. In this thesis, we demonstrate that ectromelia virus, the causative agent of lethal mousepox, encodes an inhibitor of apoptosis, EVM025. EVM025 is the orthologue of VACV F1, previously shown to inhibit apoptosis. EVM025 contains a large N-terminal repeat and a C-terminal domain that is essential for localization to the mitochondria. Cells infected with ECTV lacking EVM025 undergo apoptosis, highlighting the importance of the anti-apoptotic activity of EVM025. We further demonstrate that expression of EVM025 is crucial to prevent apoptosis triggered by various stimulations. Expression of EVM025 prevents the activation of Bak and Bax and maintains mitochondrial membrane integrity upon infection with ECTV. Our virulence studies have established that ECTV∆025 is highly attenuated in mice, with virus only being detected in the draining lymph node (D-LN) following footpad inoculation. ECTV∆025 was unable to escape the D-LN and replicate in other organs such as spleen, kidney and liver. Our studies show that the T cell response is required to limit the spread of the virus beyond the D-LN. The vaccinia virus Copenhagen (VACV) E3 protein is essential for virus replication in a wide range of cells. E3 dampens various innate immune responses by inhibiting PKR, thus maintaining efficient protein translation over the course of a virus infection cycle. In addition to being an inhibitor of PKR, E3 has also been associated with the inhibition of cellular apoptosis. In this thesis we confirmed that infection with VACVΔE3L triggers the intrinsic apoptotic pathway, and leads to the cleavage of Caspase-3 and eventual cell death. However, our data suggest that E3 does not directly inhibit the intrinsic apoptotic pathway; instead, it suppresses apoptosis indirectly, by promoting the accumulation of the VACV anti-apoptotic F1 protein. F1 mRNA is present in cells infected VACV∆E3L but the protein product does not detectably accumulate, suggesting a block at the translational level. F1 expression is restored by ectopic expression of E3 or by knocking out PKR, reversing the pro-apoptotic phenotype of VACV∆E3L.