The Fowlpox Virus BCL-2 Homologue,FPV039, Interacts with Activated Bax and a Discrete Subset of BH3-Only Proteins To Inhibit Apoptosis

Apoptosis is a potent immune barrier against viral infection, and many viruses, including poxviruses, encode proteins to overcome this defense. Interestingly, the avipoxviruses, which include fowlpox and canarypox virus, are the only poxviruses known to encode proteins with obvious Bcl-2 sequence homology. We previously characterized the fowlpox virus protein FPV039 as a Bcl-2-like antiapoptotic protein that inhibits apoptosis by interacting with and inactivating the proapoptotic cellular protein Bak. However, both Bak and Bax can independently trigger cell death. Thus, to effectively inhibit apoptosis, a number of viruses also inhibit Bax. Here we show that FPV039 inhibited apoptosis induced by Bax overexpression and prevented both the conformational activation of Bax and the subsequent formation of Bax oligomers at the mitochondria, two critical steps in the induction of apoptosis. Additionally, FPV039 interacted with activated Bax in the context of Bax overexpression and virus infection. Importantly, the ability of FPV039 to interact with active Bax and inhibit Bax activity was dependent on the structurally conserved BH3 domain of FPV039, even though this domain possesses little sequence homology to other BH3 domains. FPV039 also inhibited apoptosis induced by the BH3-only proteins, upstream activators of Bak and Bax, despite interacting detectably with only two: BimL and Bik. Collectively, our data suggest that FPV039 inhibits apoptosis by sequestering and inactivating multiple proapoptotic Bcl-2 proteins, including certain BH3-only proteins and both of the critical “gatekeepers” of apoptosis, Bak and Bax.Apoptosis is a highly conserved form of programmed cell death that plays an important role in the immune defense against pathogens. The controlled and deliberate destruction of virally infected cells comprises a potent innate immune barrier against rampant viral replication and infection. As such, many viruses, including poxviruses, encode numerous proteins that inhibit a variety of steps in the biochemical pathways that lead to cell death (29, 69).The mitochondria, and the Bcl-2 family of proteins that preside over them, serve as an important control point in the regulation of apoptosis (87). United by the presence of one to four highly conserved Bcl-2 homology (BH) domains, the Bcl-2 family regulates the integrity of the outer mitochondrial membrane (OMM) and controls the release of apoptogenic molecules from the mitochondrial intermembrane space. Bak and Bax, the two proapoptotic Bcl-2 proteins, possess BH domains 1 to 3 and, upon activation, commit the cell to death (53, 77). Whereas Bak resides constitutively at the OMM, Bax exists in an inactive form in the cytoplasm and, upon apoptotic insult, undergoes a conformational change that exposes its C-terminal transmembrane domain and results in its relocalization to the OMM (10, 34, 41, 56). The attendant exposure of the N termini of both Bak and Bax precedes Bak and Bax homooligomerization, which facilitates mitochondrial damage and, ultimately, the release of cytochrome c (3, 4, 36, 37, 76). Cytochrome c, in turn, triggers the activation of caspases, a group of cysteine proteases responsible for dismantling the apoptotic cell (59). Bak and Bax are therefore crucial for the induction of apoptosis and, because either Bak or Bax alone is sufficient to facilitate the release of cytochrome c, both must be inactivated to effectively inhibit apoptosis (53, 77, 90). The activation of Bak and Bax is counteracted by the antiapoptotic members of the Bcl-2 family, including Bcl-2, Bcl-X_L, and Mcl-1. These three proteins, which possess all four BH domains, reside at the mitochondria and prevent apoptosis by directly interacting with and inhibiting Bak and Bax or the BH3-only proteins (87). The BH3-only proteins, which possess only the BH3 domain, act as sentinels responsive to a variety of cellular stresses, including virus infection (79). Upon receipt of an apoptotic stimulus, BH3-only proteins become activated and subsequently activate Bak and Bax or inhibit the antiapoptotic function of Bcl-2, Bcl-X_L, and Mcl-1 (15). Of the eight BH3-only proteins that are directly involved in the induction of apoptosis—namely, Bim, Bid, Puma, Bik, Bmf, Bad, Noxa, and Hrk—each displays a specific and characteristic ability to bind and inhibit Bcl-2 proteins (79).Like cellular antiapoptotic Bcl-2 proteins, viral inhibitors of apoptosis have evolved especially to interfere with the activation of Bak and Bax (18, 40). For example, E1B 19K, encoded by adenovirus, and M11L, encoded by myxoma virus, bind and inactivate both Bak and Bax to inhibit apoptosis (26, 49, 65, 67, 72). Similarly, ORF125, the antiapoptotic protein encoded by the poxvirus Orf virus, also inactivates Bak and Bax, but exactly how ORF125 mediates this inactivation remains unknown (78). Although interacting with Bak and Bax is ostensibly the most direct way to prevent apoptosis, several viral antiapoptotic proteins appear to inhibit apoptosis by functioning upstream of Bak and Bax at the level of the BH3-only proteins. The vaccinia virus protein F1L, for example, interacts with Bak but not Bax, yet F1L is nonetheless capable of inactivating Bax, likely a result of F1L interacting with the BH3-only protein and Bax activator, Bim (61, 70, 74). Moreover, the Bcl-2 homolog encoded by Kaposi''s sarcoma-associated herpesvirus, and BHRF-1, encoded by Epstein-Barr virus, each interact with a specific and distinct array of BH3-only proteins, yet neither protein interacts detectably with Bak or Bax (14, 27, 44). Thus, to effectively inhibit apoptosis, it may not be necessary for viral proteins to directly target Bak and Bax but, instead, to prevent the activation of Bak and Bax by interfering with the upstream BH3-only proteins (15).Recently, our lab has shown that FPV039, encoded by fowlpox virus, localizes to the mitochondria, where it inhibits apoptosis induced by a variety of stimuli (6). Interestingly, FPV039 is the only characterized poxvirus protein that shares obvious, albeit limited, sequence homology with cellular Bcl-2 proteins (1, 6). FPV039 possesses a highly conserved BH1 and BH2 domain but lacks an obvious BH3 and BH4 domain. Importantly, however, we predicted structural homology between the Bcl-2 BH3 domain and a corresponding region in FPV039, and we validated the prediction by showing that this cryptic FPV039 BH3 domain is functionally important (6). Indeed, the ability of FPV039 to interact with the proapoptotic protein Bak is dependent on this cryptic BH3 domain (6). Thus, despite lacking sequence conservation of a highly conserved BH3 domain, FPV039 is able to interact with, and inactivate, the proapoptotic protein Bak. Nevertheless, to completely inhibit apoptosis, both Bak and Bax must be inactivated.Accordingly, we wanted to determine whether FPV039, in addition to inactivating Bak, could inactivate Bax. We report here that FPV039 inhibited Bax activity and prevented critical steps in Bax activation. FPV039 did not appear to interact with endogenous inactive Bax; however, FPV039 was able to interact with active Bax. Moreover, FPV039 inhibited apoptosis induced by the BH3-only proteins despite interacting with only BimL and Bik. Together, these data strongly suggest FPV039 inhibits apoptosis by inactivating multiple proapoptotic Bcl-2 proteins, including the critical Bak and Bax, as well as a discrete subset of BH3-only proteins.