Crystal Structures of Epstein–Barr Virus Bcl-2 Homolog BHRF1 Bound to Bid and Puma BH3 Motif Peptides

Abstract

Apoptosis is a powerful defense mechanism used by multicellular organisms to counteract viral infection. In response to premature host cell suicide, viruses have evolved numerous countermeasures to ensure cell viability to optimize their replication by encoding proteins homologous in structure and function to cellular pro-survival Bcl-2 proteins. Epstein–Barr virus (EBV), a member of the Gammaherpesviridae, encodes the Bcl-2 homolog BHRF1, a potent inhibitor of Bcl-2-mediated apoptosis. BHRF1 acts by directly targeting Bid and Puma, two proapoptotic proteins of the Bcl-2 family. Here, we determined the crystal structures of BHRF1 bound to peptides spanning the Bcl-2 binding motifs (Bcl-2 homology 3 motif, BH3) of Bid and Puma. BHRF1 engages BH3 peptides using the canonical ligand-binding groove of its Bcl-2 fold and maintains a salt bridge between an Arg residue with a conserved Asp residue in the BH3 motif mimicking the canonical ionic interaction seen in host Bcl-2:BH3 motif complexes. Furthermore, both Bid and Puma utilize a fifth binding pocket in the canonical ligand binding groove of BHRF1 to provide an additional hydrophobic interaction distinct from the interactions previously seen with Bak and Bim. These findings provide a structural basis for EBV-mediated suppression of host cell apoptosis and reveal the flexibility of virus encoded Bcl-2 proteins in mimicking key interactions from the endogenous host signaling pathways.

Epstein–Barr Virus Encoded BCL2, BHRF1, Downregulates Autophagy by Noncanonical Binding of BECN1

“…While this article was in preparation, another group reported the 2.7 Å structure of BHRF1 bound to BID residues 76–109 (PDB ID:7P33). Our structure agrees with this structure and superimposes with an rmsd of 0.300 Å over 149 Cα atoms. Our detailed structural analysis of the BHRF1:BID BH3D complex structure relative to other BHRF1:BH3D complexes, and also relative to structures of other antiapoptotic BCL2 proteins bound to the BID BH3D, helps identify structural determinants important for selective binding to BHRF1 and also explains why the BECN1 BH3D alone binds poorly to BHRF1.…”

“…We investigated the impact of exogenously expressed BHRF1 on cellular autophagy levels in human breast adenocarcinoma MCF7 cells (Figure 8). MCF7 cells lack detectable endogenous expression of BECN1, resulting in very low basal levels of autophagy 36,39,74 even in starvation conditions unless BECN1 is ectopically expressed (Figure 8B,C). Expression of full-length, wild-type BHRF1 and BECN1 was comparable in starvation and nutrient-rich conditions (Figure 8A).…”