Molecular mechanism by which Epstein-Barr Virus-encoded BHRF1 blocks BECN1-mediated autophagy - PROJECT ABSTRACT
Autophagy is a conserved eukaryotic, cellular catabolic pathway responsible for the sequestration and
degradation of intracellular macromolecules and assemblies that are unnecessary, defective or harmful to
cells. Beclin 1 / BECN1, a highly conserved, key autophagy protein serves as an interaction hub to integrate
numerous signals that regulate autophagy. Defects or deficiencies in BECN1-mediated autophagy play a role
in a wide range of diseases such as cancer, cardiovascular diseases, neurodegenerative diseases, embryonic
defects, as well as several infectious diseases, especially those caused by viruses. BECN1 is a complex,
multi-domain, conformationally-flexible protein and the mechanisms by which its diverse interactions regulate
autophagy is poorly understood. The long-term goal of the proposed research is to understand how the BECN
interactome regulates autophagy and cellular homeostasis. Epstein-Barr virus (EBV), an obligate intracellular
ɣ-herpesvirus (ɣHV), infects more than 95% of the global adult population. EBV establishes latent and lytic
infections, causing infectious mononucleosis and is associated with many diverse malignancies of lymphoid
and epithelial tissues. EBV initially blocks autophagy, and later, after lytic cycle induction, up-regulates
autophagy incorporating autophagic membranes into the final EBV envelope, but the mechanism(s) by which
EBV modulates autophagy is unknown. Like other ɣHVs, EBV encodes an anti-apoptotic BCL2 homolog,
BHRF1. BCL2s encoded by other ɣHVs bind to a non-conserved BH3 domain (BH3D) within BECN1 to block
autophagy, thereby helping ɣHVs evade autophagic degradation. Our hypothesis is that BHRF1 binds to and
impacts the function of BECN1 domains beyond the BH3D essential for up-regulating autophagy, and this
interaction causes substantial conformational changes in both BECN1 and BHRF1. The objective of this
proposal is to obtain a structural and mechanistic understanding by which BHRF1 binds to and regulates
human BECN1, while simultaneously elucidating binding-induced structural changes in BHRF1. This objective
will be accomplished by three specific aims: (1) To demonstrate that BHRF1 blocks BECN1-mediated
autophagy, and identify the minimal BECN1 region required to bind BHRF1. (2) To elucidate the mechanism by
which BHRF1-binding blocks BECN1-mediated autophagy. (3) To understand BECN1-binding associated
structural changes in BHRF1. A wide range of computational, structural, biophysical, biochemical, cellular and
molecular biology methods will be used to accomplish these aims. This research will provide the first
biochemical and structural information of the mechanism by which EBV BHRF1 blocks autophagy; the role of
BECN1 domains beyond the BH3D in binding BCL2s; and conformational changes and allosteric sites in
BHRF1. This detailed information will lay the groundwork to elucidating similarities and differences in the
binding of BECN1 to BHRF1 and human BCL2s to potentially identify “drug-able” sites on BHRF1 to enable
development of therapeutics that selectively target EBV BHRF1.
