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

Background: Cellular and -herpesvirus Bcl-2 homologs down-regulate autophagy. Results: A peptide designed to bind to the -herpesvirus68 Bcl-2, M11, but not cellular Bcl-2 homologs, abrogates M11-mediated down-regulation of autophagy. Conclusion: This peptide is a selective M11 inhibitor. Significance: Such selective inhibitors are important for understanding the role of -herpesvirus Bcl-2 homologs in viral reactivation and oncogenic transformation of host cells. -herpesviruses (HVs) are common human pathogens that encode homologs of the anti-apoptotic cellular Bcl-2 proteins, which are critical to viral reactivation and oncogenic transformation. The murine HV68 provides a tractable in vivo model for understanding general features of these important human pathogens. Bcl-X-L, a cellular Bcl-2 homolog, and the murine HV68 Bcl-2 homolog, M11, both bind to a BH3 domain within the key autophagy effector Beclin 1 with comparable affinities, resulting in the down-regulation of Beclin 1-mediated autophagy. Despite this similarity, differences in residues lining the binding site of M11 and Bcl-X-L dictate varying affinities for the different BH3 domain-containing proteins. Here we delineate Beclin 1 differential specificity determinants for binding to M11 or Bcl-X-L by quantifying autophagy levels in cells expressing different Beclin 1 mutants and either M11 or Bcl-X-L, and we show that a G120E/D121A Beclin 1 mutant selectively prevents down-regulation of Beclin 1-mediated autophagy by Bcl-X-L, but not by M11. We use isothermal titration calorimetry to identify a Beclin 1 BH3 domain-derived peptide that selectively binds to M11, but not to Bcl-X-L. 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. This information was used to develop a cell-permeable peptide inhibitor that selectively inhibits M11-mediated, but not Bcl-X-L-mediated, down-regulation of autophagy.