The inhibition of apoptosis and Bax activation by mitochondrial anti-apoptotic proteins encoded by vaccinia virus and ectromelia virus

Vaccinia virus, the prototypic member of the orthopoxvirus genus, encodes the mitochondrial-localized protein F1L that protects cells from apoptosis and inhibits cytochrome c release. F1L is a 26kDa protein that interacts with the pro-apoptotic Bcl-2 family member Bak and inhibits activation of Bak during cell death. In addition to Bak, the pro-apoptotic protein Bax is also capable of initiating cytochrome c release, suggesting that vaccinia virus infection might also modulate Bax. Here we show that F1L inhibits the activity of Bax by inhibiting oligomerization and N-terminal activation of Bax. F1L expression also inhibited the subcellular redistribution of Bax to the mitochondria and Bax insertion into the outer mitochondrial membrane. The ability of F1L to inhibit Bax activation does not require Bak, as F1L expression inhibited cytochrome c release and Bax activation in Bak-deficient cells. No interaction between Bax and F1L was detected during infection, suggesting that F1L functions upstream of Bax activation. Notably, F1L was capable of interacting with the BH3-only protein BimL as shown by co-immunoprecipitation, and F1L expression inhibited BimL-induced apoptosis. Vaccinia virus-induced apoptosis was also partially inhibited in Bim-deficient cells, suggesting that Bim is an important mediator of vaccinia virus-induced apoptosis. We have also investigated the F1L orthologue from the related orthopoxvirus ectromelia virus, which is a natural pathogen of mice. This F1L orthologue, EVM025, is a 55 kDa protein that also inhibits apoptosis. EVM025 exhibits a large N-terminal extension consisting of an eight amino acid region repeated 30 times. While the function of this repeat region is unknown, EVM025 was expressed as a 55kDa protein, and deletion of the gene encoding EVM025 from the ectromelia virus genome abrogated anti-apoptotic activity conferred by ectromelia virus infection. While the amino acid sequence of EVM025 exhibits a number of differences from F1L, a truncated version of EVM025 lacking the N-terminal repeat region still inhibited Bax activation and also constitutively interacted with Bak. These studies suggest that in addition to interacting with Bak, F1L and EVM025 can function to indirectly inhibit the activation of Bax, likely by interfering with the pro-apoptotic activity of BH3-only proteins such as BimL.