Contribution of GADD45 Family Members to Cell Death Suppression by Cellular Bcl-xL and Cytomegalovirus vMIA

Programmed cell death, or apoptosis, is an evolutionarily conserved cellular process that controls the destruction of cells during tissue development (34) and immune selection (51), and it is considered to be one of the ancient mechanisms for eliminating cells infected by obligate intracellular pathogens, such as viruses (12). Cellular components of apoptosis machinery include tumor necrosis factor family death receptors that engage soluble or cell-associated ligands to initiate an extrinsic pathway and intracellular stress sensors that initiate an intrinsic pathway of cell death. These share enzymes involved in common steps, including cytosolic protease zymogen caspases that initiate and execute essential steps in the pathway as well as Bcl-2 family proteins that localize to mitochondria and enhance or inhibit apoptosis. Many viruses encode proteins that modulate critical steps in apoptosis, thereby promoting an environment commensurate with viral replication (6). DNA viruses, including adenoviruses, poxviruses, and herpesviruses, express antiapoptotic proteins that imitate cellular functions (12, 19, 22) targeting two major apoptosis checkpoints. One, carried out by FLICE (an acronym for caspase 8) inhibitory proteins, prevents autocatalytic activation of procaspase 8 in the extrinsic pathway of apoptosis that follows activation of death receptors at the plasma membrane. The other, carried out by antiapoptotic Bcl-2 family proteins, blocks mitochondrial membrane permeabilization, a common checkpoint for apoptotic signals originating within or outside the cell. By blocking apoptosis at these points, viruses may prevent cell death occurring as a result of cytokine or cellular immune surveillance as well as the intracellular antiviral response. Human cytomegalovirus (CMV) encodes a range of functions that modulate interaction with host cells (35). This includes two potent cell death suppressors that lack sequence homology with cellular proteins even though they interfere with commonly targeted steps, inhibitor of caspase 8 activation (vICA) and mitochondrial-localized inhibitor of apoptosis (vMIA). Both of these were initially identified through a functional screen of CMV open reading frames in a HeLa cell death suppression assay (23, 48). vICA homologs are broadly distributed in all characterized betaherpesviruses infecting primates as well as rodents (32). Human CMV vICA is dispensable for viral replication in cultured cells (23, 48). vMIA sequence homologs are found only in chimpanzee CMV, rhesus macaque CMV, and African green monkey CMV (32). Murine CMV encodes a newly annotated gene, m38.5, whose position is conserved relative to UL37 homologs in primate CMVs (9, 31). This murine CMV gene product localizes to mitochondria and suppresses apoptosis (31). Although UL37 was initially reported to be critical for viral growth (15, 43, 58), replication is minimally disrupted in human CMV strains that retain a functional copy of vICA (31). The mitochondrial checkpoint of apoptosis that connects external and internal cell death induction signals to downstream nuclear and cytoskeletal events is tightly regulated by the Bcl-2 family of proteins (5). The vMIA family of antiapoptotic proteins (23, 32) functions at a step that appears analogous to Bcl-xL despite the lack of any sequence homology. Human CMV vMIA is a potent suppressor of cell death induced by mitochondrial membrane-permeabilizing agents, reactive oxygen species, and preapoptotic chromatin condensation (3, 7, 55), providing insight into apoptotic signaling and cellular pathways integrating DNA damage with apoptosis checkpoints. Highly conserved regions within vMIA (26) coincide with an amino-terminal mitochondrial targeting domain (amino acids [aa] 1 to 34) and a carboxyl-terminal antiapoptotic domain (AAD) (115 to 147) (32), both of which are contained within a functional 69-aa deletion mutant (Δ35-112/Δ148-163) of the natural protein (26), a derivative that is remarkably similar to some natural primate CMV vMIA homologs (23, 32). The mitochondrial targeting domain is required for activity, which strongly suggests that vMIA-mediated cell death suppression requires proper localization; however, the role of the AAD is not understood. The antiapoptotic Bcl-2 family members Bcl-2 and Bcl-xL bind proapoptotic family members, such as Bax, Bak, and Bid, through a hydrophobic pocket formed by conserved hydrophobic and amphipathic α-helices (36, 40). Viral Bcl-2 homologs are thought to form similar structures and interactions (40). vMIA, though not homologous to Bcl-2 or Bcl-xL, contains a conserved amphipathic α-helical motif within the AAD that we found to be critical in cell death suppression. We identified cellular AAD-dependent binding proteins in order to better characterize the mechanism of cell death suppression and found a common role for GADD45 family members in cell death suppression by vMIA and Bcl-xL. This work suggests that vMIA and Bcl-2 family members share a common cellular partner.