Targeting γ-Herpesvirus 68 Bcl-2-mediated Down-regulation of Autophagy

Abstract

γ‐herpesviruses (γHVs) are common human pathogens that encode homologs of the anti‐apoptotic cellular Bcl‐2 proteins, which are critical to viral reactivation and oncogenic transformation. Bcl‐XL, a cellular Bcl‐2 homolog, and the γHV68 Bcl‐2 homolog, M11, both bind to a BH3 domain within the key autophagy effector BECN1 with comparable affinities, resulting in the suppression of BECN1‐mediated autophagy. Despite this similarity, the different residues lining the binding site of M11 and Bcl‐XL dictate varying affinities for BH3 domains from diverse proteins. Here we delineate BECN1 differential specificity determinants for binding to M11 or Bcl‐XL by quantifying autophagy levels in cells expressing different BECN1 mutants and either M11 or Bcl‐XL, and we show that a G120E+D121A BECN1 mutant selectively prevents suppression of BECN1‐mediated autophagy by Bcl‐XL, but not by M11. We use isothermal titration calorimetry to identify a BECN1 BH3 domain‐derived peptide that selectively binds to M11, but not to Bcl‐XL. The X‐ray crystal structure of this peptide bound to M11 reveals the mechanism by which the M11 BH3 domain‐binding groove accommodates this M11‐specific peptide. Lastly, we have used this information to develop a cell‐permeable peptide inhibitor that selectively inhibits M11‐mediated, but not Bcl‐XL‐mediated suppression of autophagy. We are now testing the ability of this inhibitor to selectively inhibit M11‐mediated suppression of apoptosis. This work was funded by NIH grants P20 RR015566 and P30 GM103332‐01, and NSF grant EPS‐0814442 to SS and CC; NIH grant R21 AI078108 and NSF grant HRD‐0811239 to SS.

Viruses and the autophagy pathway

“…Other viral proteins inhibiting through Beclin 1 binding include Bcl-2 homologs, such as the KSHV orf16 protein and the MHV-68 M11 protein. (Ku et al, 2008; Su et al, 2014) In addition to ICP34.5′s Beclin 1 binding, HSV also encodes the US11 protein, which inhibits autophagy through direct interaction with the PKR kinase, indicating that HSV encodes at least two proteins capable of inhibiting autophagy, which may speak to the importance of autophagy as an antiviral against this particular virus. (Lussignol et al, 2013)…”

Conformational Flexibility Enables the Function of a BECN1 Region Essential for Starvation-Mediated Autophagy

“…While the BECN1 IDR is poorly conserved amongst homologs, BECN1 residues 136–450, are highly conserved, hence, the C-terminal part of this region is sometimes also called the evolutionarily-conserved domain (ECD) (34, 36). Residues 105–130 within the IDR constitute a BCL2 Homology 3 domain (BH3D), a functional domain necessary and sufficient for binding to diverse BCL2 homologs (26, 37, 38). …”

BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy

“…20 Furthermore, mutations in BCL2, BCL2L1 or viral BCL2s that block interaction with BECN1 fail to inhibit autophagy, 21,22 and mutations in BECN1 that block its interaction with BCL2 family members confer resistance to their antiautophagy effects. 21,23 The crystal structures of BCL2 family members, including BCL2L1 and viral BCL2, bound to the BH3 domain of BECN1 have been solved 22,[24][25][26] and predict that mutations that block the antiautophagy activity of BCL2 family members are crucial for physical interaction with BECN1.…”