Crystal Structure of African Swine Fever Virus A179L with the Autophagy Regulator Beclin

Abstract

Subversion of programmed cell death-based host defence systems is a prominent feature of infections by large DNA viruses. African swine fever virus (ASFV) is a large DNA virus and sole member of the Asfarviridae family that harbors the B-cell lymphoma 2 or Bcl-2 homolog A179L. A179L has been shown to bind to a range of cell death inducing host proteins including proapoptotic Bcl-2 proteins as well as the autophagy regulator Beclin. Here we report the crystal structure of A179L bound to the Beclin BH3 motif. A179L engages Beclin using the same canonical ligand binding groove that is utilized to bind to pro-apoptotic Bcl-2 proteins. The mode of binding of Beclin to A179L mirrors that of Beclin binding to human Bcl-2 and Bcl-xL as well as murine herpesvirus 68. Introduction of bulky hydrophobic residues into the A179L ligand binding groove via site directed mutagenesis ablates binding of Beclin to A179L, leading to a loss of ability of A179L to modulate autophagosome formation in Vero cells during starvation. Our findings provide a mechanistic understanding for the potent autophagy inhibitory activity of A179L and serve as a platform for more detailed investigations into the role of autophagy during ASFV infection.

Structural insight into tanapoxvirus‐mediated inhibition of apoptosis

“…Viral Bcl‐2 homologs have been identified in herpesviridae , include those from Epstein–Barr virus BHRF1 [13,14] and Kaposi sarcoma virus KsBcl‐2 [15–17]. Other major virus families that contain members encoding for prosurvival Bcl‐2 proteins include the asfarviridae with African swine fever virus‐encoded A179L [18–20], and grouper iridovirus‐encoded GIV66 [21,22] from the iridoviridae . However, the largest number of Bcl‐2 homologs is found in poxviridae [12] such as vaccinia and variola virus F1L [23–25] and myxomavirus M11L [26–28].…”

The structural basis of Bcl-2 mediated cell death regulation in hydra

“…To determine the structural basis for HyBcl-2-4 mediated inhibition of apoptosis we next determined the crystal structures of the HyBcl-2-4:HyBak1 BH3 and HyBcl-2-4:HyBax BH3 complexes. Crystallization of HyBcl-2-4 complexes was initially unsuccessful, and in order to facilitate crystallization we employed a strategy where Maltose-binding protein (MBP) was fused via a short linker sequence to the N-terminus of HyBcl-2-4 to provide additional opportunities for crystal contact formation [ 19 ]. The resulting MBP-HyBcl-2-4:HyBak1 BH3 crystals diffracted to a resolution of 2.2 Å ( Table 1 ).…”

“…Recombinant MBP-HyBcl-2-4 was expressed in BL21 DE3 Codon Plus RIPL cells using the auto-induction method [ 18 ] for 24 h at 30°C with shaking, and expressed as previously described [ 19 ]. Purified protein was concentrated (Amicon ® Ultra 15, 30 kDa, Merck, Burlington, Massachusetts, US) to a final concentration of 10 mg/ml.…”