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

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.

Herpesviral Interaction with Autophagy

“…Similar to cellular Bcl-2, we and others found that the vBcl-2 encoded by KSHV and γ-HV68 effectively attenuates autophagy through a direction interaction with Beclin1, surprisingly in a manner more potently than their cellular counterpart (66,67,69,70). Structural analyses further implicated that the two functions of vBcl-2, anti-apoptosis and anti-autophagy, engage the similar if not the same structural cassette, the hydrophobic BH3-binding groove on the surface of vBcl-2 (66,69). Yet, the affinity of vBcl-2 to Beclin1 is apparently higher than to pro-apoptotic molecules, suggesting that is recently found to induce autophagy to facilitate viral lytic replication, as blunting cellular autophagy reduces the virus's ability to reactivate from latency (78).…”

“…Bad) activate it (67,68). Similar to cellular Bcl-2, we and others found that the vBcl-2 encoded by KSHV and γ-HV68 effectively attenuates autophagy through a direction interaction with Beclin1, surprisingly in a manner more potently than their cellular counterpart (66,67,69,70). Structural analyses further implicated that the two functions of vBcl-2, anti-apoptosis and anti-autophagy, engage the similar if not the same structural cassette, the hydrophobic BH3-binding groove on the surface of vBcl-2 (66,69).…”

The Epstein–Barr virus Bcl-2 homolog, BHRF1, blocks apoptosis by binding to a limited amount of Bim

“…2A is the alignment of the BH1 sequences of Bcl-xL and BHRF1. Structural studies have suggested that the hydrophobic groove of BHRF1 is inaccessible; however, mutational analysis has indicated the region is important for its function (12,21,22). To test whether the BH1 region of BHRF1 is required for protection, BHRF1 was mutated at residues analogous to those responsible for hydrophilic interactions between Bcl-xL and Bim (1), specifically, L98, G99, and R100.…”

Mechanisms of apoptosis regulation by viral oncogenes in infection and tumorigenesis

“…Indeed, adenovirus is not the only virus family to encode a BAX and BAK antagonist. Poxviruses and herpesviruses have since been found to encode BAX and/or BAK BH3-interacting and inhibitory proteins [46][47][48][49][50][51][52] some of which are BCL-2 homologs, and some not. Still other viruses encode BCL-2 homologs whose mechanism of action has yet to be determined.…”