Subversion of host cell apoptosis is an important survival strategy for viruses to ensure their own proliferation and survival. Certain viruses express proteins homologous in sequence, structure and function to mammalian pro-survival B-cell lymphoma 2 (Bcl-2) proteins, which prevent rapid clearance of infected host cells. In vaccinia virus (VV), the virulence factor F1L was shown to be a potent inhibitor of apoptosis that functions primarily be engaging pro-apoptotic Bim. Variola virus (VAR), the causative agent of smallpox, harbors a homolog of F1L of unknown function. We show that VAR F1L is a potent inhibitor of apoptosis, and unlike all other characterized anti-apoptotic Bcl-2 family members lacks affinity for the Bim Bcl-2 homology 3 (BH3) domain. Instead, VAR F1L engages Bid BH3 as well as Bak and Bax BH3 domains. Unlike its VV homolog, variola F1L only protects against Bax-mediated apoptosis in cellular assays. Crystal structures of variola F1L bound to Bid and Bak BH3 domains reveal that variola F1L forms a domain-swapped Bcl-2 fold, which accommodates Bid and Bak BH3 in the canonical Bcl-2-binding groove, in a manner similar to VV F1L. Despite the observed conservation of structure and sequence, variola F1L inhibits apoptosis using a startlingly different mechanism compared with its VV counterpart. Our results suggest that unlike during VV infection, Bim neutralization may not be required during VAR infection. As molecular determinants for the human-specific tropism of VAR remain essentially unknown, identification of a different mechanism of action and utilization of host factors used by a VAR virulence factor compared with its VV homolog suggest that studying VAR directly may be essential to understand its unique tropism.

Premature programmed cell death or apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Tanapoxvirus (TANV) is a large double‐stranded DNA virus belonging to the poxviridae that causes mild monkeypox‐like infections in humans and primates. TANV encodes for a putative apoptosis inhibitory protein 16L. We show that TANV16L is able to bind to a range of peptides spanning the BH3 motif of human proapoptotic Bcl‐2 proteins and is able to counter growth arrest of yeast induced by human Bak and Bax. We then determined the crystal structures of TANV16L bound to three identified interactors, Bax, Bim and Puma BH3. TANV16L adopts a globular Bcl‐2 fold comprising 7 α‐helices and utilizes the canonical Bcl‐2 binding groove to engage proapoptotic host cell Bcl‐2 proteins. Unexpectedly, TANV16L is able to adopt both a monomeric and a domain‐swapped dimeric topology where the α1 helix from one protomer is swapped into a neighbouring unit. Despite adopting two different oligomeric forms, the canonical ligand binding groove in TANV16L remains unchanged from monomer to domain‐swapped dimer. Our results provide a structural and mechanistic basis for tanapoxvirus‐mediated inhibition of host cell apoptosis and reveal the capacity of Bcl‐2 proteins to adopt differential oligomeric states whilst maintaining the canonical ligand binding groove in an unchanged state. Database Structural data are available in the Protein Data Bank (PDB) under the accession numbers http://6TPQ, http://6TQQ and http://6TRR.

BHRF1 is a pro-survival Bcl-2 homologue encoded by Epstein-Barr virus (EBV) that plays a key role in preventing premature host cell death during viral infection and may contribute to the development of malignancies associated with chronic EBV infections. The anti-apoptotic action of BHRF1 is based on its ability to sequester pro-apoptotic Bcl-2 family proteins, in particular Bim and Bak. These interactions have been previously studied in three dimensions by determining crystal structures of BHRF1 in complex with both Bim and Bak BH3 domains. Screening of a library of peptidomimetic compounds based on the benzoylurea scaffold that mimics critical Bim BH3 domain side chains against BHRF1 led to the identification of an inhibitor of BHRF1 that displays micromolar affinity. Single crystals were obtained from the co-crystallization of recombinant BHRF1 protein with this peptidomimetic compound. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=66.8, b=91.1, c=151.9 Å. Diffraction data were collected to 2.11 Å resolution on the MX2 beamline at the Australian Synchrotron.

22Premature programmed cell death or apoptosis of cells is a strategy utilized by multicellular 23 organisms to counter microbial threats. Tanapoxvirus (TPV) is a large double-stranded DNA 24 virus belonging to the poxviridae that causes mild Monkeypox-like infections in humans and 25 primates. TPV encodes for a putative apoptosis inhibitory protein 16L. We now show that 26 TPV16L is able to bind to a range of peptides spanning the BH3 motif of human pro-27 apoptotic Bcl-2 proteins, and is able to counter growth arrest of yeast induced by human Bak 28 and Bax. We then determined the crystal structures of TPV16L bound to three identified 29 interactors, Bax, Bim and Puma BH3. TPV16L adopts a globular Bcl-2 fold comprising 7 α-30 helices, and utilizes the canonical Bcl-2 binding groove to engage pro-apoptotic host cell Bcl-31 2 proteins. Unexpectedly, TPV16L is able to adopt both a monomeric as well as a domain-32 swapped dimeric topology where the α1 helix from one protomer is swapped into a 33 neighbouring unit. Despite adopting two different oligomeric forms, the canonical ligand 34 binding groove in TPV16L remains unchanged from monomer to domain-swapped dimer. 35Our results provide a structural and mechanistic basis for tanapoxvirus mediated inhibition of 36 host cell apoptosis, and reveal the capacity of Bcl-2 proteins to adopt differential oligomeric 37 states whilst maintaining the canonical ligand binding groove in an unchanged state. 38 39 40