A Surface Groove Essential for Viral Bcl-2 Function During Chronic Infection In Vivo

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Abstract
Antiapoptotic Bcl-2 family proteins inhibit apoptosis in cultured cells by binding BH3 domains of proapoptotic Bcl-2 family members via a hydrophobic BH3 binding groove on the protein surface. We investigated the physiological importance of the BH3 binding groove of an antiapoptotic Bcl-2 protein in mammals in vivo by analyzing a viral Bcl-2 family protein. We show that the γ-herpesvirus 68 (γHV68) Bcl-2 family protein (γHV68 v-Bcl-2), which is known to inhibit apoptosis in cultured cells, inhibits both apoptosis in primary lymphocytes and Bax toxicity in yeast. Nuclear magnetic resonance determination of the γHV68 v-Bcl-2 structure revealed a BH3 binding groove that binds BH3 domain peptides from proapoptotic Bcl-2 family members Bax and Bak via a molecular mechanism shared with host Bcl-2 family proteins, involving a conserved arginine in the BH3 peptide binding groove. Mutations of this conserved arginine and two adjacent amino acids to alanine (SGR to AAA) within the BH3 binding groove resulted in a properly folded protein that lacked the capacity of the wild-type γHV68 v-Bcl-2 to bind Bax BH3 peptide and to block Bax toxicity in yeast. We tested the physiological importance of this v-Bcl-2 domain during viral infection by engineering viral mutants encoding a v-Bcl-2 containing the SGR to AAA mutation. This mutation resulted in a virus defective for both efficient reactivation of γHV68 from latency and efficient persistent γHV68 replication. These studies demonstrate an essential functional role for amino acids in the BH3 peptide binding groove of a viral Bcl-2 family member during chronic infection. Viruses can manipulate their hosts by expressing proteins that structurally and functionally resemble host cellular proteins. One important cellular process manipulated by viruses is apoptosis, a cell death program that is regulated by a family of Bcl-2-like proapoptotic and antiapoptotic proteins. Gammaherpesviruses encode Bcl-2 family proteins (v-Bcl-2) that may contribute to their ability to cause tumors and persist for the lifetime of their hosts. The authors solved the structure of the murine γ-herpesvirus 68 (γHV68) v-Bcl-2 and found that it is similar to cellular antiapoptotic proteins and that v-Bcl-2 uses the same mechanism as cellular Bcl-2 to bind to peptides from proapoptotic Bcl-2 family proteins. Furthermore, they found that a γHV68 virus expressing a mutated form of v-Bcl-2 that cannot bind to peptides from proapoptotic Bcl-2 family proteins is defective in its ability to cause chronic viral infection in mice. Thus, a specific structural feature and molecular mechanism of the v-Bcl-2 that is shared with host antiapoptotic Bcl-2 proteins is important for the function of this protein during viral infection. These findings enhance our understanding of the molecular mechanisms of chronic γ-herpesvirus infection, and suggest that targeting the functions of the v-Bcl-2 protein might have therapeutic benefit.