Infection with viruses often protects the infected cell against external stimuli to apoptosis. Here we explore the balance of apoptosis induction and inhibition for infection with the modified vaccinia virus Ankara (MVA), using two MVA mutants with experimentally introduced deletions. Deletion of the E3L-gene from MVA transformed the virus from an inhibitor to an inducer of apoptosis. Noxa-deficient mouse embryonic fibroblasts (MEF) were resistant to MVA-DE3L-induced apoptosis. When the gene encoding F1L was deleted from MVA, apoptosis resulted that required Bak or Bax. MVA-DF1L-induced apoptosis was blocked by Bcl-2. When expressed in HeLa cells, F1L blocked apoptosis induced by forced expression of the BH3-only proteins, Bim, Puma and Noxa. Finally, biosensor analysis confirmed direct binding of F1L to BH3 domains. These data describe a molecular framework of how a cell responds to MVA infection by undergoing apoptosis, and how the virus blocks apoptosis by interfering with critical steps of its signal transduction.

Proteins of the Bcl-2 family are critical regulators of apoptosis. Proapoptotic members, like Bax, contain three of the four Bcl-2 homology regions (BH1-3), while BH3-only proteins, like Bim, possess only the short BH3 motif. Database searches revealed Bfk, an unusual novel member of the Bcl-2 family that contains a BH2 and BH3 region but not BH1 or BH4. Bfk is thus most closely related to Bcl-G L . It lacks a C-terminal membrane anchor and is cytosolic. Enforced expression of Bfk weakly promoted apoptosis and antagonized Bcl-2's prosurvival function. Like Bcl-G L , Bfk did not bind to any Bcl-2 family members, even though its BH3 motif can mediate association with prosurvival proteins. Low amounts of Bfk were found in stomach, ovary, bone marrow and spleen, but its level in the mammary gland rose markedly during pregnancy, suggesting that Bfk may play a role in mammary development.

The pathway to cell death in Caenorhabditis elegans is well established. In cells undergoing apoptosis, the Bcl-2 homology domain 3 (BH3)-only protein EGL-1 binds to CED-9 at the mitochondrial membrane to cause the release of CED-4, which oligomerises and facilitates the activation of the caspase CED-3. However, despite many studies, the biophysical features of the CED-4/CED-9 complex have not been fully characterised. Here, we report the purification of a soluble and stable 2 : 2 heterotetrameric complex formed by recombinant CED-4 and CED-9 coexpressed in bacteria. Consistent with previous studies, synthetic peptides corresponding to the BH3 domains of worm BH3-only proteins (EGL-1, CED-13) dissociate CED-4 from CED-9, but not from the gain-offunction CED-9 (G169E) mutant. Surprisingly, the ability of worm BH3 domains to dissociate CED-4 was specific since mammalian BH3-only proteins could not do so.

Studies of the cell death pathway in the nematode Caenorhabditis elegans provided the first evidence of the evolutionary conservation of apoptosis signalling. Here we show that the worm Bcl-2 homology domain-3 (BH3)-only protein EGL-1 binds mammalian pro-survival proteins very poorly, but can be converted into a high-affinity ligand for Bcl-2 and Bcl-x L by subtle mutation of the cysteine residue at position 62 within the BH3 domain. A 100-fold increase in affinity was observed following a single atom change (cysteine to serine substitution), and a further 10-fold increase by replacement with glycine. The low affinity of wild-type EGL-1 for mammalian pro-survival proteins and its poor expression correlates with its weak killing activity in mammalian cells whereas the high-affinity C62G mutant is a very potent killer of cells lacking Mcl-1. Cell killing by the C62S mutant with intermediate affinity only occurs when this EGL-1 BH3 domain is placed in a more stable context, namely that of Bim S , which allows higher expression, though the kinetics of cell death now vary depending on whether Mcl-1 is neutralized by Noxa or genetically deleted. These results demonstrate how levels of BH3-only proteins, target affinity and the spectrum of neutralization of pro-survival proteins all contribute to killing activity. Landmark studies in Caenorhabditis elegans revealed that four genes, CED-9, CED-4, CED-3 and EGL-1, are essential for the programmed cell death (apoptosis) of 131 out of 1090 somatic cells during hermaphrodite nematode development. 1 Of these, EGL-1, CED-3 and CED-4 encode pro-apoptotic proteins whereas CED-9 encodes a pro-survival protein. CED-9 is the nematode homologue of mammalian B-cell lymphoma-2 (Bcl-2) pro-survival proteins (including Bcl-2 itself as well as Bcl-x L , Bcl-w, Mcl-1 and A1), 2 while CED-4 is similar to the mammalian adapter protein APAF-1 3 acting as a positive regulator of caspases, in particular, CED-3. 4 EGL-1 is the nematode Bcl-2 homology domain-3 (BH3)-only protein equivalent to mammalian BH3-only proteins such as Bim, Bad and Noxa. [5][6][7] In nematode cells, CED-9 functions by sequestering CED-4 at the mitochondrial membrane. 8-10 Following a developmental cue, EGL-1 is transcriptionally upregulated and the protein binds CED-9, 5-7 releasing CED-4, allowing its oligomerization. 11 These CED-4 oligomers then translocate to the perinuclear region of the cell 12 where they facilitate CED-3 activation by bringing these caspases into close proximity. 4,9 Biochemical and structural studies have revealed that binding of the EGL-1 BH3 domain into a hydrophobic groove on CED-9 causes a conformational change at the CED-4:CED-9 interface that results in CED-4 dissociation. [13][14][15] The BH3 domains of mammalian BH3-only proteins bind into similar grooves on mammalian pro-survival molecules, but a conformational change as seen in CED-9 has yet to be observed. [16][17][18] The functional equivalence, and hence evolutionary conservation, of CED-9 and mammalian Bcl-2 was demonstrated in st...