The 3-herpesviridae includes widespread, important human pathogens like Epstein-Barr virus which causes infectious mononucleosis and malignant tumors of lymphoid and epithelial tissues and Kaposi Sarcoma virus, which causes Kaposi sarcoma tumors, especially among immunocompromised individuals such as AIDS patients and transplant recipients. 3-Herpesviruses encode homologs of the anti-apoptotic, cellular Bcl-2 protein (c-Bcl-2), presumably to facilitate viral propagation and oncogenicity by blocking cell death. A key mechanism by which anti-apoptotic Bcl-2 homologs prevent apoptosis is by sequestering pro-apoptotic Bcl-2 homologs. Although viral Bcl-2 (v-Bcl-2) and c-Bcl-2 homologs share very low sequence identity, they have very similar three-dimensional structures. Previous structural, biochemical and mutagenic analyses showed that a hydrophobic groove on the surface of anti-apoptotic Bcl-2 homologs, including most v-Bcl-2 homologs, binds the amphipathic BH3 helical domains of pro-apoptotic Bcl-2 homologs. Despite this shared general mechanism for binding BH3 domains, the different Bcl-2 homologs show unique specificity toward various BH3 domains, arising from differences in their amino acid sequences. 3-herpesviruses have been shown to also inhibit autophagy, a vital cellular process by which organelles, large protein aggregates, stable proteins, as well as intracellular pathogens are enclosed in double-membrane vesicles and targeted for lysosomal degradation. Autophagy is normally modulated by the interaction of c-Bcl-2 homologs with a key autophagy effector protein, Beclin 1, and the down-regulation of autophagy by 3-herpesviruses is mediated by the interaction of v-Bcl-2 homologs with Beclin 1. Recently, Beclin 1 was also shown to contain a BH3 domain that constitutes the primary determinant of binding to Bcl-XL and other c-Bcl-2 homologs. Our preliminary data indicates that additional regions of Beclin 1 contiguous with the BH3 domain are also involved in interaction with Bcl-2 homologs. Further, the various c- and v-Bcl-2 homologs bind to overlapping, but not identical, sites on Beclin 1. The goal of this grant is to characterize the differences and also to verify the similarities, in the interaction of Beclin 1 with v-Bcl-2 and c-Bcl-2 homologs. Interactions will first be quantified by biochemical measurements of binding affinity. Structure determination and analyses will provide details of each interaction, identifying key specificity determinants, which will be confirmed by quantifying binding affinity of selected mutants. Finally, ABT737, a peptido-mimetic that inhibits binding of certain c-Bcl-2 homologs and BH3 domains, will be tested for its ability to disrupt the interaction of v-Bcl-2 homologs and Beclin 1. Thus, this research will provide a better understanding of the molecular mechanisms of herpesvirus pathogenicity, the cellular process of autophagy, the interaction between herpesviral and host proteins, and information on how to selectively modify these interactions either by mutagenesis or by small molecule inhibitors, both for the purposes of further research as well as for designing potential therapeutics to treat this medically important group of pathogens.
Important 3-Herpesviruses human pathogens include EBV, which causes infectious mononucleosis and malignant tumors of lymphoid and epithelial tissues, and KSHV, which causes Kaposi sarcoma tumors, especially among immuno-compromised individuals such as AIDS patients and transplant recipients. These viruses have evolved to down-regulate autophagy, a host defense mechanism known to degrade numerous pathogens. This application aims to investigate atomic details of the mechanism by which 3-herpesviruses inhibit autophagy, providing a better understanding of 3-herpesviral pathogenicity. ? ? ?
