Structural Insight into African Swine Fever Virus A179L-Mediated Inhibition of Apoptosis

Abstract

Programmed cell death is a tightly controlled process critical for the removal of damaged or infected cells. Pro- and antiapoptotic proteins of the Bcl-2 family are pivotal mediators of this process. African swine fever virus (ASFV) is a large DNA virus, the only member of the Asfarviridae family, and harbors A179L, a putative Bcl-2 like protein. A179L has been shown to bind to several proapoptotic Bcl-2 proteins; however, the hierarchy of binding and the structural basis for apoptosis inhibition are currently not understood. We systematically evaluated the ability of A179L to bind proapoptotic Bcl-2 family members and show that A179L is the first antiapoptotic Bcl-2 protein to bind to all major death-inducing mammalian Bcl-2 proteins. We then defined the structural basis for apoptosis inhibition of A179L by determining the crystal structures of A179L bound to both Bid and Bax BH3 motifs. Our findings provide a mechanistic understanding for the potent antiapoptotic activity of A179L by identifying it as the first panprodeath Bcl-2 binder and serve as a platform for more-detailed investigations into the role of A179L during ASFV infection.IMPORTANCE Numerous viruses have acquired strategies to subvert apoptosis by encoding proteins capable of sequestering proapoptotic host proteins. African swine fever virus (ASFV), a large DNA virus and the only member of the Asfarviridae family, encodes the protein A179L, which functions to prevent apoptosis. We show that A179L is unusual among antiapoptotic Bcl-2 proteins in being able to physically bind to all core death-inducing mammalian Bcl-2 proteins. Currently, little is known regarding the molecular interactions between A179L and the proapoptotic Bcl-2 members. Using the crystal structures of A179L bound to two of the identified proapoptotic Bcl-2 proteins, Bid and Bax, we now provide a three-dimensional (3D) view of how A179L sequesters host proapoptotic proteins, which is crucial for subverting premature host cell apoptosis.

Keywords: ASFV; Bax family; Bcl-2; X-ray crystallography; apoptosis.

FIG 1

A179L interactions with peptides from proapoptotic Bcl-2 family members. The affinity of recombinant A179L for BH3 motif peptides (26-mers, except for a Bax 28-mer and a Bid 34-mer) was assessed using isothermal titration calorimetry (ITC). Raw heats of titration for A179L:Bid BH3 (A), A179L:Bax BH3 (B), and A179L:Noxa BH3 (C) interactions.

FIG 2

Crystal structures of A179L:Bid BH3, A179L:Bax BH3, and comparison with other BH3-complexes of Bcl-2 proteins. The view in all panels is into the hydrophobic binding groove formed by helices α2 to α5. (A) A179L (green) in complex with the Bid BH3 motif (magenta). A179L helices are labeled α1 to α8. (B) Bcl-xL (sand) in complex with the Bid BH3 motif (magenta) (37). (C) Variola virus F1L (cyan) in complex with Bid BH3 motif (magenta) (20). (D) A179L (green) in complex with the Bax BH3 motif (yellow). (E) Bcl-2 (brown) in complex with Bax BH3 motif (yellow) (36). (F) EBV BHRF1 (blue) in complex with Bim BH3 (orange) (40).

FIG 3

Sequence alignment of A179L with Bcl-2 family members. The alignment was generated using MUSCLE with sequences from ASFV A179L (Uniprot accession number P42485), EBV BHRF1 (P03182), and human Bcl-x_L (Q07817) and Bcl-2 (P10415).

FIG 4

Detailed views of the A179L:BH3 peptide interfaces. (Left) The A179L surface, backbone, and floor of the binding groove are shown in gray, green, and orange, respectively, while Bid BH3 is shown in cyan. The four key hydrophobic residues of Bid (I83, L87, I90, M94) protruding into the binding groove and the conserved salt bridge formed by A179L R86 and Bid D92 are labeled, as well as residues involved in additional ionic interactions and hydrogen bonds. (Right) The A179L surface, backbone, and floor of the binding groove are shown in gray, green, and orange, respectively, while Bid BH3 is shown in yellow. The four key hydrophobic residues of Bid (L59, L63, I66, L70) protruding into the binding groove and the conserved salt bridge formed by A179L R86 and Bid D68 are labeled, as well as residues involved in additional ionic interactions and hydrogen bonds.