BH3 domains define selective inhibitory interactions with BHRF-1 and KSHV BCL-2

The Epstein-Barr and Kaposi's sarcoma gamma-herpesviruses (KSHVs) are associated with certain cancers, and encode B-cell leukemia/lymphoma 2 (BCL-2) homologs, BHRF-1 and KSHV BCL-2, respectively. Little is known, however, about the molecular interactions allowing viral BCL-2 homologs to mediate their anti-apoptotic function. Cellular anti-apoptotic proteins, such as BCL-2 and MCL-1, prevent death via selective interactions with pro-death BH3-only proteins. To investigate whether BHRF-1 and KSHV BCL-2 function similarly, we made recombinant BHRF-1 and KSHV BCL-2 proteins. We identified the individual binding patterns for BHRF-1 and KSHV BCL-2 to BH3 domains. These studies surprisingly showed that KSHV BCL-2 is more closely related to MCL-1 than to BCL-2, a result confirmed by sequence analysis. GST-BHRF-1 and GST-KSHV BCL-2 bound BH3-only family proteins from human cells. BHRF-1 protected mammalian cells from growth factor withdrawal, etoposide and adriamycin. We found that both BCL-2 and BHRF-1 sequestered pro-death BH3-only proteins under growth factor-deficient conditions. Finally, we tested the ability of a panel of BH3 peptides to inhibit BHRF-1 and KSHV BCL-2 function in a mitochondrial model of apoptosis. We found that each could be inhibited by the select group of BH3 peptides identified in our binding assay. Our studies define the biochemical interactions underlying BHRF-1 and KSHV BCL-2 anti-apoptotic function, and identify peptides that are prototypic inhibitors of this function.