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Journal of Virology, October 2007, p. 11032-11045, Vol. 81, No. 20
0022-538X/07/$08.00+0     doi:10.1128/JVI.00734-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Fowlpox Virus Encodes a Bcl-2 Homologue That Protects Cells from Apoptotic Death through Interaction with the Proapoptotic Protein Bak{triangledown}

Logan Banadyga, Jenna Gerig, Tara Stewart, and Michele Barry*

Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

Received 4 April 2007/ Accepted 30 July 2007


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ABSTRACT
 
Poxviruses are renowned for encoding numerous immunomodulatory proteins capable of undermining potent immune defenses. One effective barrier against infection is apoptosis, a process controlled at the mitochondria by pro- and antiapoptotic members of the highly conserved Bcl-2 family of proteins. Although poxviruses are known to encode an array of effective inhibitors of apoptosis, members of the Avipoxvirus genus, which includes fowlpox virus, encode proteins with Bcl-2 homology. Here, we show that FPV039, a fowlpox virus protein with limited Bcl-2 homology, inhibited apoptosis in response to a variety of cytotoxic stimuli, including virus infection itself. Similar to other antiapoptotic Bcl-2 proteins, FPV039 localized predominantly to the mitochondria in both human and chicken cells and protected human cells from tumor necrosis factor alpha-induced loss of the mitochondrial membrane potential. In addition, coimmunoprecipitation revealed that FPV039 interacted constitutively with the proapoptotic Bcl-2 protein, Bak, in both human and chicken cells. Concordantly, FPV039 also inhibited apoptosis induced by the transient overexpression of Bak. To confirm these results in the context of virus infection, we generated a recombinant vaccinia virus lacking F1L, the endogenous apoptotic inhibitor in vaccinia virus, and expressing FPV039. In the context of vaccinia virus infection, FPV039 retained the ability to localize to the mitochondria and interacted with Bak. Moreover, FPV039 prevented the activation of Bak and protected infected cells from apoptosis induced by staurosporine and virus infection. Together, our data indicate that FPV039 is a functional Bcl-2 homologue that inhibits apoptosis by neutralizing the proapoptotic Bcl-2 family member Bak.


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INTRODUCTION
 
Apoptosis is a complex and highly structured mechanism of cell death that is evolutionarily conserved throughout all multicellular organisms and functions to eliminate redundant, damaged, infected, or otherwise unwanted cells (19, 42). The network of biochemical pathways that regulate apoptosis converges on the activation of caspases, a group of cysteine proteases that cleave various cellular proteins and lead to the systematic dismantling of the cell (49, 71). Although the activation of caspases and, concordantly, the commitment to cell death are multifaceted and largely circuitous, the mitochondrion has been established as an important organelle governing the apoptotic cascade (33, 34, 77). Within the mitochondrion is sequestered a pool of apoptogenic proteins, including cytochrome c, that when released into the cytosol activate caspases and induce apoptosis (33, 77). Thus, the ability to regulate the integrity of the mitochondrion is paramount in controlling apoptosis.

The highly conserved family of Bcl-2 proteins is ultimately responsible for regulating apoptosis at the mitochondrion (1, 17). Within this family exist both pro- and antiapoptotic members whose cooperative or antagonistic interactions collectively determine the fate of the cell (1, 17). These interactions depend on the presence of at least one of four highly conserved Bcl-2 homology (BH) domains as well as domains necessary for localization to the outer mitochondrial membrane. Antiapoptotic members of the Bcl-2 family possess four BH domains whereas Bak and Bax, proapoptotic members of the family, possess only BH domains 1, 2, and 3 (BH1, BH2, and BH3, respectively) (1, 17). BH3-only proteins, a second subset of proapoptotic Bcl-2 proteins, bear little homology to other Bcl-2 family members and contain only BH3 domains (65, 84). Structural analyses of these proteins have revealed that the BH1, BH2, and BH3 domains comprise {alpha}-helices that form a hydrophobic cleft capable of binding the BH3 {alpha}-helix of other Bcl-2 family members, thus forming the basis of the homo- and heterotypic interactions that characterize this family (57).

As upstream regulators of apoptosis, the BH3-only proteins are activated in response to a variety of cellular stresses, and they trigger apoptosis by repressing antiapoptotic Bcl-2 proteins and activating the proapoptotic Bcl-2 proteins, Bak and Bax (65, 84). Following activation, Bak and Bax undergo a series of conformational changes that result in their oligomerization (4, 35, 36, 81). Through a still largely undefined process, these oligomers induce the loss of the mitochondrial membrane potential and facilitate the release of apoptogenic proteins, such as cytochrome c (3, 20, 29). Bak and Bax are thus crucial to the induction of apoptosis; without Bax and Bak, mitochondrial membrane permeabilization and release of cytochrome c cannot occur (51, 82, 89). Conversely, antiapoptotic Bcl-2 family members, exemplified by Bcl-2 and Bcl-xL, interact at the mitochondria with Bak, Bax, and BH3-only proteins, to hold them in inactive conformers, thereby preventing apoptosis (1, 17). Together, the balance of interactions among the Bcl-2 family members determines whether or not a cell is to live or die (1, 17).

Apoptosis is a critical defense against virus infection, and many viruses, therefore, have evolved mechanisms to interfere with apoptosis (9, 60, 72). For example, a large number of viruses encode Bcl-2 homologues that function at the mitochondria to inhibit the release of cytochrome c (18, 41, 58). Additionally, some viruses encode mitochondrion-localized proteins that inhibit apoptosis but exhibit no obvious sequence homology to Bcl-2 proteins (25, 31, 80). Poxviruses, by virtue of their relatively large genomes, encode numerous immunomodulatory proteins, including several that are capable of inhibiting cytochrome c release (69). Our laboratory recently identified F1L as a unique vaccinia virus (VV) protein that localizes to the mitochondria and constitutively interacts with Bak to inhibit apoptosis, despite lacking obvious sequence homology to cellular Bcl-2 proteins (64, 70, 78-80). Another novel poxviral inhibitor of apoptosis, M11L, encoded by myxoma virus, also interacts constitutively with Bak to inhibit apoptosis (25, 75). M11L, like F1L, lacks obvious sequence homology to cellular Bcl-2 proteins; however, the three-dimensional structure of M11L bears striking similarity to that of cellular Bcl-2 proteins (22, 50). F1L orthologues are found only in other members of the Orthopoxvirus genus, whereas M11L orthologues are encoded by leporipoxviruses, capripoxviruses, suipoxviruses, and yatapoxviruses. As such, the majority of poxviruses lack proteins with obvious sequence homology to members of the Bcl-2 family.

Recent genomic analyses, however, have suggested that members of the highly divergent genus Avipoxvirus encode proteins with significant sequence homology to cellular Bcl-2 proteins (2, 74). Fowlpox virus, the prototypical avipoxvirus, is responsible for considerable economic loss in the worldwide poultry industry by causing mild to severe disease in chickens and turkeys (27, 73). Additionally, fowlpox virus, among other avipoxviruses, is being actively pursued as a vector for gene therapy and vaccination (10, 62). Genomic sequencing of fowlpox virus revealed a putative Bcl-2 homologue encoded by the open reading frame FPV039 (2). Like cellular Bcl-2 proteins, FPV039 possesses highly conserved BH1 and BH2 domains as well as a putative C-terminal transmembrane domain. Unlike cellular antiapoptotic Bcl-2 family members, however, FPV039 lacks obvious homology within the BH3 and BH4 domains, which are required for antiapoptotic function (40, 45, 88). Moreover, FPV039 is a smaller version of Bcl-2 than its cellular antiapoptotic counterparts (2). The lack of critical Bcl-2 homology domains and the relatively small size of FPV039 therefore distinguish it from cellular antiapoptotic Bcl-2 family members.

Given the unique homology between FPV039 and cellular Bcl-2 proteins, we sought to determine what role FPV039 played in the modulation of apoptosis. We report that FPV039 localized to the mitochondria, where it maintained the integrity of the mitochondria by constitutive interaction with the proapoptotic protein Bak. FPV039 expressed in the context of viral infection retained interaction with Bak and rendered cells resistant to apoptosis. Furthermore, infection with a recombinant VV expressing FPV039 prevented Bak conformational change, a prerequisite for cytochrome c release and apoptotic death of the cell.


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MATERIALS AND METHODS
 
Cells and viruses. HeLa, CV-1, HEK 293T, HuTK–-143B, and wild-type Jurkat cells were obtained from the ATCC and maintained as previously described (78). Chicken leghorn male hepatoma (LMH) cells were obtained from the ATCC and maintained at 37°C and 5% CO2 in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (GIBCO Invitrogen Corp.), 50 U of penicillin (GIBCO Invitrogen Corp.) per ml, 50 µg of streptomycin (GIBCO Invitrogen Corp.) per ml, and 200 µM glutamine (GIBCO Invitrogen Corp.) (47). Jurkat cells overexpressing Bcl-2 were generated and maintained as previously described (6). Bak- and Bax-deficient Jurkat cells (Jurkat Bak–/–/Bax–/–) were generously provided by H. Rabinowich (University of Pittsburgh School of Medicine, Pittsburgh, PA) (76). VV strain Copenhagen expressing the enhanced green fluorescent protein, VV(Cop)-EGFP, was provided by G. McFadden (University of Florida, Gainesville, FL). VV strain Copenhagen lacking the F1L open reading frame (VV{Delta}F1L) and VV strain Western Reserve expressing a Flag-tagged F1L (VV-Flag-F1L) were generated as described previously (78, 80). Viruses were propagated in baby green monkey kidney cells and isolated as previously described (66).

Generation of VV{Delta}F1L-Flag-FPV039(1-176) and VV{Delta}F1L-Flag-FPV039(1-94). CV-1 cells were infected at a multiplicity of infection (MOI) of 0.05 with VV{Delta}F1L and transfected with 5 µg of either pSC66-Flag-FPV039(1-176) (containing residues 1 to 176 of FPV039) or pSC66-Flag-FPV039(1-94) using Lipofectin (Invitrogen Life Technologies). Recombinant viruses were selected by growth on HuTK–-143B cells in the presence of 5'-bromodeoxyuridine (Sigma-Aldrich) and were plaque purified using 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (Rose Scientific Ltd.) to visualize recombinant viruses (23). The presence of FPV039(1-176) or FPV039(1-94) was confirmed by PCR, and protein expression was confirmed by Western blotting using the anti-Flag M2 antibody (Sigma-Aldrich).

Plasmid construction. Wild-type FPV039(1-176) was amplified from fowlpox virus DNA (provided by Chris Upton, University of Victoria, Victoria, BC, Canada) using the forward primer 5'-AAGCTTATGGCTAGTAATATGAAA-3' containing an HindIII restriction site and the reverse primer 5'-GGATTACATATAAAAGGAACATAT-3' containing a BamHI restriction site. FPV039(1-141), lacking the mitochondrion targeting sequence, was generated via PCR using the forward primer 5'-AAGCTTATGGCTAGTAATATGAAA-3' containing an HindIII restriction site and the reverse primer 5'-GGATCCTTAATATATACGAATTCCATTCCAATA-3' containing a BamHI restriction site. FPV039(142-176), comprising only the mitochondrion targeting sequence, was generated via PCR using the forward primer 5'-GAATTCTCAAAAACTATTCGTATATT-3' containing an EcoRI restriction site and the reverse primer 5'-GGATCCTTACATATAAAAGGAACATAT-3' containing a BamHI restriction site. FPV039({Delta}41-54), which lacks the 14 internal amino acids from positions 41 to 54, was generated by overlapping PCR. Segments of DNA coding for the N-terminal portion of FPV039 were generated from pEGFP-FPV039(1-176) using the FPV039(1-176) forward primer and the internal reverse primer 5'-TTCATTATAATTGGATATAGAATCTATTACAAA-3'; segments of DNA coding for the C-terminal portion of FPV039 were also generated from pEGFP-FPV039(1-176) using the FPV039(1-176) reverse primer and the internal forward primer 5'-AATTATAATGAATTTGATATA-3'. The resulting PCR products were combined, and an additional PCR was performed using the FPV039(1-176) forward and reverse primers. A full-length Flag-tagged version of FPV039, Flag-FPV039(1-176), was generated via PCR using the forward primer 5'-GTCGACATGGACTACAAAGACGATGACGACAAGGCTAGTAGTAATATGAAA-3' containing an SalI restriction site and Flag tag and the reverse primer 5'-GCGGCCGCTTACATATAAAAGGAACATAT-3' containing an NotI restriction site. All PCR products were cloned into pGemT vector (Promega), and constructs were sequenced to ensure the absence of errors. FPV039(1-176), FPV039(1-141), FPV039(142-176), and FPV039({Delta}41-54) were subcloned into pEGFP-C3 (Clontech) to generate N-terminal EGFP versions of each FPV039 construct. FPV039(1-94), which contains a stop codon (TAA) in place of the Tyr-95 immediately after the putative BH1 domain, was generated by PCR and subcloned into pEGFP-C3. Flag-FPV039(1-176) and Flag-FPV039(1-94) were subcloned into the pSC66 vector, which places Flag-FPV039(1-176) and Flag-FPV039(1-94) under the control of a poxviral promoter (23).

Confocal microscopy. For live cell analysis, 1 x 106 HeLa cells or LMH cells were grown on 18-mm glass coverslips in modified 3.5-cm cell culture dishes and transiently transfected with 2 µg of DNA using Lipofectamine 2000 (GIBCO Invitrogen Corp.). Mitochondria were labeled with 15 ng/ml of MitoTracker Red CMXRos (Invitrogen Life Technologies). Live cells were examined using an LSM510 laser scanning confocal microscope at 543 nm to detect MitoTracker fluorescence and at 489 nm to detect EGFP fluorescence.

Measurement of mitochondrial membrane potential. Changes in the mitochondrial membrane potential were quantified by staining with tetramethylrhodamine ethyl ester (TMRE) (Invitrogen Life Technologies) (24, 53). HeLa cells (1 x 106) were transfected with pEGFP-C3, pEGFP-HuBcl-2, pEGFP-F1L, pEGFP-FPV039(1-176), pEGFP-FPV039(1-94), pEGFP-FPV039(1-141), or pEGFP-FPV039(142-176) using Lipofectamine 2000, and apoptosis was induced by treating cells with 10 ng/ml of tumor necrosis factor alpha (TNF-{alpha}) (Roche Diagnostics) and 5 µg/ml of cycloheximide (ICN Biomedicals Inc.) for 6 h or by transfecting cells with 2 µg of pcDNA3-HA-Bak (where HA is hemagglutinin) for 18 h. Following treatment, cells were stained with 0.2 µM TMRE (24, 53). Similar experiments were performed in the context of viral infection. Jurkat cells or Jurkat Bcl-2-overexpressing cells (1 x 106) were infected for 8 h at an MOI of 10. Cells were analyzed by two-color flow cytometry (FACScan; Becton Dickinson) with TMRE fluorescence measured through the FL-2 channel equipped with a 585-nm filter (42-nm band-pass filter), and EGFP fluorescence was measured through the FL-1 channel equipped with a 489 nm filter (42-nm band-pass filter). Data were acquired on 20,000 cells per sample with fluorescence signals at logarithmic gain, and analysis was performed using CellQuest software. The percentage of killing was calculated as the number of EGFP+ TMRE– cells divided by the total number of EGFP+ cells, and standard deviations were generated from three independent experiments.

Conformational analysis of Bak by flow cytometry. Jurkat cells (1 x 106), Jurkat Bcl-2 cells, or Jurkat Bak–/–/Bax–/– cells were infected with VV-EGFP, VV{Delta}F1L, VV{Delta}F1L-Flag-FPV039(1-176), or VV{Delta}F1L-Flag-FPV039(1-94) for 4 h at an MOI of 10. Following infection, cells were exposed to 250 nM staurosporine (Sigma-Aldrich) for 2 h and then fixed in 0.25% paraformaldehyde. Cells were permeabilized with 500 µg/ml digitonin and stained with 2 µg/ml anti-Bak antibody (Ab-1; Oncogene Research Products) (35, 36) or 2 µg/ml of an isotype control antibody specific for NK1.1 (PK136) (48) and counterstained with phycoerythrin-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories Inc.). Antibody staining was analyzed by flow cytometry (FACScan; Becton Dickinson) with fluorescence measured through the FL-2 channel equipped with a 585-nm filter (42-nm band-pass filter).

Immunoprecipitations and immunoblotting. HEK 293T cells (1 x 106) were transfected with 2 µg of pEGFP-C3, pEGFP-F1L, pEGFP-FPV039(1-176), pEGFP-FPV039(1-94), or pEGFP-FPV039({Delta}41-54) and cotransfected with 2 µg of pcDNA3-HA-Bak using Lipofectamine 2000. Cells were lysed in 2% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate) (Sigma) supplemented with EDTA-free proteinase inhibitor (Roche Diagnostics), followed by precipitation using goat anti-GFP antibody (Luc Berthiaume, University of Alberta, Edmonton, Alberta, Canada). Similar experiments were performed in the context of viral infection. HeLa or LMH cells (7 x 106) were infected with VV-EGFP, VV{Delta}F1L, VV{Delta}F1L-Flag-FPV039(1-176), or VV{Delta}F1L-Flag-FPV039(1-94) at an MOI of 5 for 6 h or, in the case of VV{Delta}F1L-Flag-FPV039(1-94), 16 h. Cells were lysed in 2% CHAPS (Sigma) supplemented with EDTA-free proteinase inhibitor (Roche Diagnostics), followed by precipitation using rabbit anti-BakNT (Upstate) or mouse anti-Flag M2 (Sigma) antibody.

Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose or polyvinylidene difluoride membranes. The following antibodies were used for detection: mouse anti-GFP (1:5,000; Cedarlane Laboratories Ltd.), mouse anti-HA (clone 12CA5) (1:4,000; Roche Diagnostics), rabbit anti-BakNT (1:2,000; Upstate), mouse anti-Bak (1:500; Pharmingen), mouse anti-Flag M2 (1:5,000; Sigma), and mouse anti-Flag-horseradish peroxidase (HRP) (1:2,000; Sigma). Proteins were visualized using enhanced chemiluminescence according to the manufacturer's directions (GE Healthcare).


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RESULTS
 
Fowlpox virus encodes a Bcl-2 homologue. Genomic sequencing of fowlpox virus revealed a putative Bcl-2 homologue encoded by the open reading frame FPV039 (2). Similar to most antiapoptotic Bcl-2 proteins, FPV039 is predicted to contain a series of {alpha}-helices that closely align with those determined from the structure of Bcl-2 (Fig. 1A). In addition, FPV039 possesses conserved BH1 and BH2 domains; however, FPV039 lacks obvious BH3 and BH4 domains and is unusually short, owing to deletion of an unstructured loop region present in Bcl-2 (Fig. 1A and B). Alignment of the BH1 domain of FPV039 with several cellular Bcl-2 proteins indicates that FPV039 possesses a highly conserved BH1 domain characterized by the presence of the NWGR sequence (amino acids 82 to 85) (Fig. 1B). Likewise, the BH2 domain of FPV039 bears sequence homology to the same domain in cellular Bcl-2 proteins (Fig. 1B). Importantly, G84 and R85 of the BH1 domain and W128 of the BH2 domain in FPV039 correspond to the same residues in cellular Bcl-2 proteins that are critical for antiapoptotic function (37, 87). Although FPV039 lacks key residues usually conserved within the BH3 domain, {alpha}-helix 2 of FPV039 corresponds with an {alpha}-helix in Bcl-2 that defines the BH3 domain (Fig. 1A). Similarly, {alpha}-helix 1 in Bcl-2, which contains the BH4 domain, also aligns with an {alpha}-helical region in FPV039, suggesting that despite lacking obvious sequence homology, FPV039 may be structurally and functionally homologous to Bcl-2. These discrepancies in sequence conservation between FPV039 and Bcl-2 may highlight substantial, and possibly critical, functional differences.


Figure 1
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  		FIG. 1. Alignment of FPV039 with human Bcl-2. (A) FPV039 is predicted to contain eight {alpha}-helices (JPred) and demonstrates significant homology to human Bcl-2 within the BH1 and BH2 domains. Predicted {alpha}-helices are indicated ({alpha}1 to {alpha}8), and the mitochondrial targeting sequences of Bcl-2 and FPV039 are underlined (dashed line). Identical amino acids are highlighted in black, and similar amino acids are highlighted in gray (ClustalW). (B) Alignment of BH1, BH2, BH3, and BH4 domains from various Bcl-2 family members with FPV039. Identical amino acids are highlighted in black, and similar amino acids are highlighted in gray (ClustalW).

FPV039 is a tail-anchored protein that localizes to the mitochondria. Proapoptotic Bcl-2 proteins possess C-terminal "tail anchors" that typically target them to the endoplasmic reticulum membrane or the outer mitochondrial membrane (11, 61). Sequence analysis of FPV039 demonstrated the presence of a 26-amino-acid hydrophobic domain flanked by positively charged lysines at positions 141 and 169 of FPV039, suggesting that FPV039 may localize to intracellular membranes (Fig. 1A). Therefore, to determine the subcellular localization pattern of FPV039, HeLa cells were transiently transfected with pEGFP, pEGFP-F1L, or pEGFP-FPV039(1-176) (Fig. 2A) and analyzed by confocal microscopy (Fig. 2B). Cells transfected with pEGFP alone showed a diffuse fluorescence pattern indicative of uniform distribution throughout the cell that did not display colocalization with MitoTracker, a mitochondrion-specific dye that labels the mitochondria (Fig. 2B, frames a to c). As a control, cells transfected with pEGFP-F1L, which expresses an EGFP-tagged version of the VV-encoded F1L protein (64), showed a punctate fluorescence pattern that overlapped with MitoTracker (Fig. 2B, frames d to f). Similarly, cells expressing wild-type EGFP-FPV039(1-176) displayed a punctate fluorescence pattern that colocalized with MitoTracker, indicating that FPV039 also localized to the mitochondria (Fig. 2B, frames g to i). Deletion of the region in FPV039 predicted to encode an {alpha}-helical BH3 domain in other antiapoptotic Bcl-2 proteins did not affect localization of EGFP-FPV039({Delta}41-54) to the mitochondria (Fig. 2B, frames j to l) since this construct retains the C-terminal transmembrane domain.


Figure 2
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  		FIG. 2. FPV039 localizes to mitochondria. (A) Schematic representation of FPV039 wild-type and mutant constructs appended to EGFP or Flag. (B) HeLa cells were transiently transfected, and mitochondrial localization was visualized by EGFP colocalization with MitoTracker. (C) Chicken LMH cells were transiently transfected, and mitochondrial localization was visualized by EGFP colocalization with MitoTracker.

To determine whether the C-terminal domain of FPV039 is necessary for mitochondrial localization of FPV039, three mutated constructs were generated (Fig. 2A). Cells transfected with pEGFP-FPV039(1-94), which is truncated immediately after the BH1 domain and therefore lacks the downstream BH2 domain and putative transmembrane domain, showed a diffuse cytoplasmic distribution, which did not colocalize with MitoTracker (Fig. 2B, frames m to o). These results suggested that the C-terminal 82 amino acids were important for the mitochondrial localization of FPV039. A second mutant construct, pEGFP-FPV039(1-141), which lacks only the 35 C-terminal amino acids was used to refine the domain required for mitochondrial localization. Cells transfected with pEGFP-FPV039(1-141) displayed diffuse localization throughout the cell and failed to colocalize with MitoTracker (Fig. 2B, frames p to r). In contrast, cells transfected with pEGFP-FPV039(142-176), a construct comprised of only the C-terminal 35 amino acids appended to the C terminus of EGFP, displayed a punctate green fluorescence pattern similar to pEGFP-F1L and pEGFP-FPV039(1-176) (Fig. 2B, frames s to u). Colocalization with MitoTracker indicated that the C-terminal 35 amino acids of FPV039 are necessary and sufficient for promoting localization to the mitochondria.

Because fowlpox virus is a natural pathogen of various poultry species (27, 73), including chickens, we wanted to confirm the localization of FPV039 in a physiologically relevant context. Chicken LMH cells were transfected with pEGFP-FPV039(1-176), pEGFP-FPV039(1-94), pEGFP-FPV039(1-141), or pEGFP-FPV039(142-176) (Fig. 2A) and analyzed by confocal microscopy (Fig. 2C). The colocalization observed in LMH cells precisely reflected the colocalization of the same constructs observed in HeLa cells (Fig. 2B and C). EGFP-FPV039(1-176) and EGFP-FPV039(142-176) localized predominantly to the mitochondria (Fig. 2C, frames a to c and j to l). Conversely, EGFP-FPV039(1-94) and EGFP-FPV039(1-141) exhibited a cytoplasmic distribution that did not colocalize with the mitochondria (Fig. 2C, frames d to i). Together, these data indicate that FPV039 is capable of localizing to the mitochondria in human and chicken cells.

FPV039 inhibits TNF-{alpha}-induced loss of mitochondrial membrane potential. The apparent sequence homology between FPV039 and cellular Bcl-2 proteins coupled with the observed localization of FPV039 to the mitochondria suggested that FPV039 may play a role in modulating apoptosis. To determine whether FPV039 was sufficient to inhibit apoptosis, HeLa cells were transfected with pEGFP, pEGFP-HuBcl-2, pEGFP-F1L, pEGFP-FPV039(1-176), or pEGFP-FPV039(142-176). Transiently transfected cells were subsequently treated with TNF-{alpha} to induce apoptosis, and uptake of TMRE, a dye that fluorescently labels healthy, respiring mitochondria, was used as an indicator of apoptosis (Fig. 3A) (24, 53). To ensure the phenomena we were observing were specific to cells expressing the transfected proteins, EGFP-expressing cells were analyzed for TMRE fluorescence. After TNF-{alpha} treatment, 53% of cells expressing EGFP alone exhibited loss of the mitochondrial membrane potential, whereas less than 1% of cells transfected with EGFP-HuBcl-2 and only 6% of cells expressing EGFP-F1L showed a drop in TMRE fluorescence (Fig. 3A). These data agree with previous studies and indicate that both Bcl-2 and the VV-encoded protein, F1L, protect cells from TNF-{alpha}-induced apoptosis (64). Similarly, expression of EGFP-FPV039(1-176) also inhibited apoptosis, with only 2% of transfected cells exhibiting a loss of mitochondrial membrane potential after TNF-{alpha} treatment (Fig. 3A). Conversely, cells expressing EGFP-FPV039(142-176) provided no protection, with 51% of transfected cells exhibiting a loss in TMRE fluorescence (Fig. 3A).


Figure 3
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  		FIG. 3. Expression of FPV039 protects cells from TNF-{alpha}-induced apoptosis. (A) HeLa cells were transfected with either pEGFP, pEGFP-HuBcl-2, pEGFP-F1L, pEGFP-FPV039(1-176), or pEGFP-FPV039(141-176) and treated with 10 ng/ml TNF-{alpha} and 5 µg/ml cycloheximide for 6 h, and apoptosis was assessed by measuring TMRE fluorescence. Standard deviations were calculated from three independent experiments. (B) Expression levels of the various transfected proteins. HeLa cells were transfected, and protein levels were determined by Western blotting (WB) with anti-GFP.

To ensure that differences in protein expression levels were not influencing the ability of the various EGFP-tagged constructs to inhibit apoptosis, expression levels were analyzed by Western blotting. HeLa cells transfected with either pEGFP or pEGFP-FPV039(142-176) showed the highest levels of protein expression (Fig. 3B); however, neither construct was capable of inhibiting apoptosis. EGFP-HuBcl-2, EGFP-F1L, and EGFP-FPV039(1-176) were all expressed at lower levels, but all of these constructs protected against TNF-{alpha}-induced apoptosis. Two additional mutant constructs, pEGFP-FPV039(1-94), which encodes a protein truncated after the BH1 domain, and pEGFP-FPV039(1-141), which encodes a protein lacking the C-terminal transmembrane domain, also did not protect against the TNF-{alpha}-induced collapse of the mitochondrial membrane potential, but expression levels of both proteins were significantly decreased in this experiment compared to EGFP-FPV039(1-176) and EGFP-FPV039(142-176) (data not shown). Together, these data clearly demonstrate that FPV039 is an antiapoptotic protein capable of inhibiting TNF-{alpha}-induced apoptosis.

FPV039 interacts with the proapoptotic protein Bak and inhibits Bak-induced apoptosis. Loss of the mitochondrial membrane potential and subsequent induction of apoptosis is governed by the Bcl-2 family of proteins and requires the activity of at least one of two proapoptotic proteins, Bak or Bax (51, 82, 89). Two other poxviral antiapoptotic proteins, M11L and F1L, which, unlike FPV039, display little sequence homology to members of the Bcl-2 family, constitutively interact with and inhibit Bak activity (75, 78). Accordingly, we wanted to ascertain if FPV039 also interacted with Bak. HEK 293T cells were cotransfected with pEGFP, pEGFP-F1L, pEGFP-FPV039(1-176), pEGFP-FPV039(1-94), or pEGFP-FPV039({Delta}41-54) and HA-tagged Bak and lysed in 2% CHAPS, a detergent which preserves the conformational integrity of Bcl-2 family members (43, 44). Immunocomplexes were precipitated with an anti-GFP antibody, and subsequent Western blotting was performed with an anti-HA or anti-Bak antibody (Fig. 4). In agreement with previous studies, an interaction between EGFP-F1L and HA-Bak was detected (Fig. 4A) (78). Using this approach, an interaction between EGFP-FPV039(1-176) and HA-Bak was also observed (Fig. 4A). Significantly, no coimmunoprecipitation was observed in cells transfected with EGFP or HA-Bak alone, confirming the specificity of the interaction between EGFP-FPV039(1-176) and HA-Bak (Fig. 4A). To ensure that we were precipitating equal amounts of the EGFP-tagged proteins, we subjected the lysates and immunoprecipitations to Western blotting with an anti-GFP antibody, and the results indicated that each EGFP-tagged protein was expressed and precipitated at approximately equal levels (Fig. 4A and B). Although EGFP alone was highly expressed and readily precipitated, this protein exhibited no interaction with HA-Bak.


Figure 4
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  		FIG. 4. FPV039 interacts with the proapoptotic protein Bak. (A) HEK 293T cells were cotransfected with either pEGFP, pEGFP-F1L, or pEGFP-FPV039(1-176) and pcDNA3-HA-Bak. Cellular lysates were immunoprecipitated (IP) with anti-GFP and subjected to Western blotting (WB) with anti-GFP and anti-HA to detect interaction. Lysates were Western blotted with anti-HA or anti-GFP to determine expression of HA-Bak and EGFP-tagged proteins, respectively. (B) Deletion of the BH3 and BH2 domains of FPV039 abrogates interaction with Bak. HEK 293T cells were cotransfected with either pEGFP-FPV039(1-176), pEGFP-FPV039(1-94), or pEGFP-FPV039({Delta}41-54) and pcDNA3-HA-Bak. Interaction between FPV039 and Bak was assessed by immunoprecipitation (IP) with anti-GFP and Western blotting (WB) with anti-BakNT or anti-GFP. Lysates were Western blotted with anti-BakNT or anti-GFP to determine expression of HA-Bak and EGFP-tagged proteins, respectively.

Two mutant FPV039 constructs were employed to refine the domains within FPV039 required for its interaction with Bak (Fig. 4B). EGFP-FPV039(1-94) failed to interact with HA-Bak, suggesting that the BH2 domain and the transmembrane domain play critical functional roles (Fig. 4B). Additionally, although FPV039 lacks a domain with obvious homology to the BH3 domain of human Bcl-2 (Fig. 1A and B), secondary structural analysis predicted an {alpha}-helix ({alpha}2) in FPV039 (amino acids 41 to 54) that aligns with the BH3 {alpha}-helix of human Bcl-2 (Fig. 1A), suggesting that this region may be important for interaction with Bak. To determine if amino acids 41 to 54 of FPV039 were critical for interaction with Bak, we generated an EGFP-tagged version of FPV039 lacking amino acids 41 to 54, EGFP-FPV039({Delta}41-54). No interaction by coimmunoprecipitation was detected between Bak and EGFP-FPV039({Delta}41-54), indicating that, despite lacking homology to the BH3 domain of several antiapoptotic Bcl-2 proteins, this region was required for Bak interaction (Fig. 4B). Together, these data demonstrate that, like cellular Bcl-2 proteins and viral proteins, such as M11L and F1L, FPV039 also interacts with Bak (1, 17, 75, 78).

Since FPV039 clearly interacted with Bak, we next wanted to determine whether FPV039 could inhibit apoptosis induced by overexpression of Bak. Ectopic overexpression of Bak saturates the mitochondria with Bak, artificially inducing its activation and leading to apoptosis (15). HeLa cells were cotransfected with pEGFP, pEGFP-HuBcl-2, pEGFP-F1L, pEGFP-FPV039(1-176), or pEGFP-FPV039(142-176) and HA-Bak. Apoptosis was quantified by staining cells with TMRE to determine the percent loss of the mitochondrial membrane potential, and EGFP-expressing cells were analyzed by flow cytometry. EGFP expression alone was unable to prevent the loss of the mitochondrial membrane potential induced by HA-Bak, with almost 30% of cells exhibiting decreased TMRE fluorescence (Fig. 5A). A loss of mitochondrial membrane potential in cells expressing EGFP-HuBcl-2 was undetectable, and less than 5% of cells expressing EGFP-F1L exhibited a drop in TMRE fluorescence, indicating that both Bcl-2 and F1L protected against Bak-induced apoptosis (Fig. 5A). EGFP-FPV039(1-176) also inhibited Bak-induced apoptosis, with approximately only 1% of transfected cells showing a decrease in TMRE fluorescence (Fig. 5A). The activity of wild-type FPV039 contrasted sharply with that of EGFP-FPV039(142-176), where greater than 30% of cells transfected with this construct lost their mitochondrial membrane potential as a result of HA-Bak overexpression (Fig. 5A).


Figure 5
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  		FIG. 5. FPV039 inhibits Bak-induced apoptosis. (A) HeLa cells were cotransfected with either pEGFP, pEGFP-HuBcl-2, pEGFP-F1L, pEGFP-FPV039(1-176), or pEGFP-FPV039(141-176) and pcDNA3-HA-Bak. Eighteen hours posttransfection, apoptosis was assessed by TMRE fluorescence in EGFP-positive cells by two-color flow cytometry. The asterisk indicates complete protection when cells were transfected with pEGFP-HuBcl-2. Standard deviations were calculated from three independent experiments. (B) Expression levels of the various transfected proteins. HeLa cells were transfected, and protein levels were determined by Western blotting (WB) with anti-GFP and anti-BakNT.

Protein expression levels were analyzed via Western blotting to ensure that differences in the protective abilities of the EGFP-tagged proteins were not a result of differences in expression of either the EGFP-tagged proteins or HA-Bak. EGFP and EGFP-FPV039(142-176) were robustly expressed, but neither prevented apoptosis induced by HA-Bak (Fig. 5B). EGFP-HuBcl-2, EGFP-F1L, and EGFP-FPV039(1-176) were expressed at slightly varied levels, which correlated with the ability to inhibit apoptosis (Fig. 5A and B). HA-Bak was equally expressed in all samples. Collectively, these data imply that FPV039 is capable of interacting with the proapoptotic protein Bak and preventing the induction of apoptosis.

FPV039 interacts with endogenous Bak during VV infection. To further elucidate the antiapoptotic mechanism of FPV039 during virus infection, we constructed two recombinant viruses using the previously generated VV strain Copenhagen lacking its natural apoptotic inhibitor, F1L, VV{Delta}F1L (78). Using homologous recombination, we generated VV{Delta}F1L-Flag-FPV039(1-176), which expresses wild-type FPV039, and VV{Delta}F1L-Flag-FPV039(1-94), which expresses a truncated FPV039 lacking the 82 amino acids immediately C-terminal of the BH1 domain (Fig. 2A).

To determine if FPV039 was able to interact with Bak in the context of infection, we infected HeLa cells with VV{Delta}F1L, VV-Flag-F1L, VV{Delta}F1L-Flag-FPV039(1-176), or VV{Delta}F1L-Flag-FPV039(1-94) and immunoprecipitated cell lysates with an anti-Flag antibody. Subsequent Western blotting of the precipitates using an anti-Bak antibody revealed that Flag-tagged FPV039(1-176) interacted with endogenous Bak during infection (Fig. 6A). Conversely, Flag-tagged FPV039(1-94), which lacks the BH2 region and the C-terminal transmembrane domain, was unable to interact with Bak during virus infection, despite being precipitated and expressed at equal or greater levels (Fig. 6A). As a control, Flag-F1L also interacted with Bak, as previously shown (78), but no interaction was observed in the lysates from cells infected with VV{Delta}F1L, confirming the specificity of the interaction between the Flag-tagged proteins and endogenous Bak (Fig. 6A). Western blot analysis of these samples with anti-Flag antibodies revealed that each Flag-tagged protein was precipitated equally and that equal levels were expressed in the lysates (Fig. 6A). Notably, Western blotting of lysates infected with VV{Delta}F1L-Flag-FPV039(1-94) showed a pattern indicative of ubiquitination (Fig. 6A and B), which we routinely observed for this mutant as well as FPV039(1-176) (data not shown). To confirm the interaction, we performed the reciprocal immunoprecipitation using anti-Bak and again showed that both F1L and FPV039 interacted with Bak during infection (Fig. 6B). Western blotting of infected cell lysates indicated that endogenous Bak and each Flag-tagged protein were expressed at equal levels (Fig. 6B). Importantly, the interactions observed between virally expressed FPV039 and endogenous Bak reflect those observed between ectopically expressed EGFP-tagged proteins and HA-Bak (Fig. 4).


Figure 6
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  		FIG. 6. FPV039 interacts with the proapoptotic protein Bak during virus infection. (A) HeLa cells were either mock infected or infected with VV{Delta}F1L, VV-Flag-F1L, VV{Delta}F1L-Flag-FPV039(1-176), or VV{Delta}F1L-Flag-FPV039(1-94) at an MOI of 5. Cell lysates were subjected to immunoprecipitation (IP) with anti-Flag M2 and subjected to Western blotting (WB) with anti-BakNT to detect endogenous Bak or with anti-Flag-HRP to detect the Flag-tagged proteins. Cell lysates were Western blotted with anti-Flag-HRP or anti-BakNT. (B) Reciprocal immunoprecipitations (IP) were performed with anti-BakNT followed by Western blotting (WB) with mouse anti-Bak or anti-Flag-HRP. Cell lysates were Western blotted with anti-Flag-HRP or anti-BakNT.

We next sought to confirm the ability of FPV039 to interact with chicken Bak, a presumably important and natural partner. LMH cells were infected with VV{Delta}F1L, VV-Flag-F1L, or VV{Delta}F1L-Flag-FPV039(1-176), and the lysates were immunoprecipitated with either an anti-Flag (Fig. 7B) or anti-Bak (Fig. 7C) antibody. Because of the paucity of reagents available to study Bcl-2 proteins in chickens, we relied on rabbit anti-human BakNT, an antibody generated against the human Bak homologue (26). Overall, chicken Bak and human Bak share approximately 60% amino acid identity; however, the immunogen used to generate rabbit anti-human BakNT was a 16-amino-acid peptide of human Bak that shares approximately 80% identity with chicken Bak (Fig. 7A). Indeed, anti-BakNT detected a single band from LMH whole-cell lysates running at the predicted size of chicken Bak (data not shown). Immunoprecipitation of cell lysates with an anti-Flag antibody, followed by Western blotting with anti-BakNT, revealed an interaction between chicken Bak and both Flag-F1L and Flag-FPV039(1-176) (Fig. 7B). No interaction was observed in the lysates from cells infected with VV{Delta}F1L (Fig. 7B). Western blotting the immunoprecipitates with anti-Flag revealed that Flag-F1L and Flag-FPV039(1-176) were precipitated in equal amounts, and Western blotting of infected cell lysates showed that the Flag-tagged proteins and endogenous chicken Bak were equally expressed (Fig. 7B). To confirm the interaction, a reciprocal immunoprecipitation was performed. Infected cell lysates were immunoprecipitated with anti-BakNT and subsequently Western blotted with an anti-Flag antibody (Fig. 7C). As expected, both Flag-F1L and Flag-FPV039(1-176) interacted with chicken Bak, and no interaction was observed in lysates from cells infected with VV{Delta}F1L (Fig. 7C). Anti-BakNT precipitated approximately equal amounts of Bak in mock-, VV-Flag-F1L-, and VV{Delta}F1L-Flag-FPV039(1-176)-infected cells. More Bak was immunoprecipitated in VV{Delta}F1L-infected cells, but, as expected, no protein coprecipitated (Fig. 7C). Both Flag-tagged proteins and endogenous Bak were expressed at equal levels (Fig. 7C). The results in chicken cells precisely mirror those obtained in human cells and lend further support to the idea that FPV039 is a functional Bcl-2 homologue.


Figure 7
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  		FIG. 7. FPV039 interacts with endogenous levels of Bak in chicken cells. (A) Amino acid alignment of the N-terminal portion of chicken Bak and human Bak, with the immunogen used to generate the human Bak-specific antibody, rabbit anti-BakNT, indicated by underlining. Identical amino acids are highlighted in black, and similar amino acids are highlighted in gray (ClustalW). (B) LMH cells were mock infected or infected with VV{Delta}F1L, VV-Flag-F1L, or VV{Delta}F1L-Flag-FPV039(1-176). Cellular lysates were immunoprecipitated (IP) with anti-Flag and Western blotted (WB) with anti-Flag or anti-BakNT. Both F1L and FPV039 interact with endogenous levels of chicken Bak. Lysates were Western blotted with anti-Bak or anti-Flag to determine expression of endogenous Bak and Flag-tagged proteins, respectively. (C) LMH lysates were subjected to reciprocal immunoprecipitation (IP) with anti-BakNT followed by Western blotting (WB) with anti-BakNT or anti-Flag to detect an interaction. Lysates were Western blotted with anti-BakNT or anti-Flag to determine protein expression. The immunoglobulin light chain (Ig LC) is indicated by an arrow.

FPV039 inhibits apoptosis induced by virus infection. We have previously shown that the VV protein F1L is a potent inhibitor of apoptosis and that, in the absence of F1L, VV infection induces apoptosis (78). To ascertain whether FPV039 could functionally replace F1L as an inhibitor of VV-induced apoptosis, Jurkat cells were infected with either VV-EGFP, VV{Delta}F1L, VV{Delta}F1L-Flag-FPV039(1-176), or VV{Delta}F1L-Flag-FPV039(1-94), and the ability of these viruses to inhibit apoptosis was quantified using two-color flow cytometric analysis. Apoptosis was measured by quantifying the loss of the inner mitochondrial membrane potential using TMRE staining in EGFP-positive cells since each virus expressed EGFP. Using this experimental approach, the majority of cells infected with VV-EGFP, which encodes F1L, and VV{Delta}F1L-Flag-FPV039(1-176) maintained their mitochondrial membrane potential following infection for 8 h (Fig. 8a and c). Conversely, 23% of cells infected with VV{Delta}F1L, which induces apoptosis, exhibited a loss of the inner mitochondrial membrane potential (Fig. 8 b) (78). Similar to F1L, expression of FPV039 inhibited apoptosis induced by VV lacking the antiapoptotic protein F1L. Infection with VV{Delta}F1L-Flag-FPV039(1-94), however, failed to completely protect cells from virus-induced apoptosis, perhaps offering partial protection (Fig. 8d). Jurkat cells overexpressing Bcl-2 were completely resistant to apoptosis induced by either VV{Delta}F1L or VV{Delta}F1L-Flag-FPV039(1-94) (Fig. 8f and h). Additionally, both VV-EGFP and VV{Delta}F1L-Flag-FPV039(1-176), but not VV{Delta}F1L or VV{Delta}F1L-Flag-FPV039(1-94), protected cells from apoptosis induced by staurosporine, a potent apoptotic stimulus (data not shown). Together, these data demonstrate that FPV039, despite lacking sequence homology to F1L, is capable of inhibiting apoptosis induced by VV infection.


Figure 8
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  		FIG. 8. FPV039 inhibits virus-induced apoptosis. Expression of FPV039 inhibits apoptosis induced by VV{Delta}F1L. Jurkat (a to d) or Jurkat cells overexpressing Bcl-2 (e to h) were infected with VV-EGFP, VV{Delta}F1L, VV{Delta}F1L-Flag-FPV039(1-176), or VV{Delta}F1L-Flag-FPV039(1-94) at an MOI of 10. After 8 h, apoptosis was quantified via two-color flow cytometry by measuring loss of the inner mitochondrial membrane potential as detected by TMRE fluorescence. Data were acquired on 20,000 cells per sample, and the data shown are representative of three or more independent experiments.

FPV039 inhibits the activation-associated conformational change of Bak. Given that FPV039 interacts with Bak (Fig. 4, 6, and 7) and inhibits apoptosis in response to a variety of different stimuli (Fig. 3, 5, and 8), we wanted to determine whether FPV039 functioned by inhibiting the activity of Bak. Bak oligomerization and disruption of mitochondrial integrity are preceded by a conformational change in Bak resulting in exposure of the N terminus, which serves as an indicator of Bak activation and impending apoptotic death (35, 36, 81). Therefore, to determine if FPV039 inhibited Bak activity, we assessed the activation-associated exposure of the N terminus of Bak. Jurkat cells were infected with either VV-EGFP, VV{Delta}F1L, VV{Delta}F1L-Flag-FPV039(1-176), or VV{Delta}F1L-Flag-FPV039(1-94), and the activation of Bak was induced by treatment with staurosporine. The exposure of the Bak N terminus was detected by staining with the conformational-specific antibody, anti-Bak Ab-1, and quantified by flow cytometry (35, 36). Untreated Jurkat cells displayed a low level of fluorescence, attributable to nonspecific antibody binding; however, upon staurosporine treatment, fluorescence intensity increased, indicating a change in Bak conformation and exposure of the N-terminal epitope recognized by the antibody (Fig. 9a). Infection with VV-EGFP inhibited the exposure of the Bak N terminus upon treatment with staurosporine, as previously shown (Fig. 9b) (78). Infection with VV{Delta}F1L, however, did not inhibit staurosporine-induced Bak activation (Fig. 9c) but was, instead, a potent inducer of apoptosis, capable of inducing Bak activation in the absence of staurosporine (Fig. 9c). In contrast, Jurkat cells infected with VV{Delta}F1L-Flag-FPV039(1-176) exhibited no increase in fluorescence intensity associated with Bak conformational change, whether treated with staurosporine or not (Fig. 9d), indicating that FPV039, like F1L, is capable of inhibiting the activation of Bak. This effect was not seen in Jurkat cells infected with VV{Delta}F1L-Flag-FPV039(1-94), which, whether exposed to staurosporine or not, exhibited an almost complete shift in fluorescence intensity (Fig. 9e). An increase in fluorescence intensity was not observed in Jurkat cells treated with staurosporine and stained with an isotype control antibody (Fig. 9f). To ensure that our assay was specifically measuring Bak, we repeated the experiments in Jurkat cells lacking both Bak and Bax (76). In each case, no detectable increase in fluorescence activity was observed, confirming the specificity of the anti-Bak Ab-1 antibody (Fig. 9g to k). Further, overexpression of Bcl-2 inhibited the exposure of the Bak N terminus in response to both infection and staurosporine treatment, as previously shown (Fig. 9l to p) (36, 78). Together, these data indicate that FPV039 is capable of inhibiting Bak conformational change and activation, providing a mechanistic explanation of the ability of FPV039 to inhibit apoptosis.


Figure 9
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  		FIG. 9. FPV039 expression inhibits Bak activation. Jurkat cells (a to f), Jurkat cells devoid of Bak and Bax (Bak–/–/Bax–/–) (g to k), or Jurkat cells overexpressing Bcl-2 (l to p) were mock infected or infected with VV-EGFP, VV{Delta}F1L, VV{Delta}F1L-Flag-FPV039(1-176), or VV{Delta}F1L-Flag-FPV039(1-94) at an MOI of 10 for 4 h and then treated with 250 nM staurosporine for 2 h to induce apoptosis. Exposure of the N terminus of Bak was monitored by flow cytometry using a confirmation-specific N-terminal anti-Bak antibody (Ab-1) or isotype control antibody (NK1.1). Untreated cells, shaded histogram; staurosporine treated cells, open histogram. Data are representative of at least three independent experiments. STS, staurosporine.


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DISCUSSION
 
Apoptosis comprises an important and powerful part of the innate and acquired immune system (19, 42). As obligate intracellular parasites, viruses depend on healthy host cells to complete their replication cycle and facilitate a productive infection; accordingly, many viruses encode proteins that interfere with apoptosis (9, 60). Poxviruses are renowned for encoding numerous immunomodulatory proteins capable of undermining or subverting the immune barriers to infection, including apoptosis (69). Notably, however, fowlpox virus and canarypox virus, both of the Avipoxvirus genus, encode proteins with obvious Bcl-2 sequence homology (2, 74). We now provide evidence indicating that FPV039 functions at the mitochondria to prevent the induction of apoptosis.

The highly conserved family of Bcl-2 proteins regulates the commitment to apoptosis at the mitochondria (1, 17). Not surprisingly, several viruses encode Bcl-2 homologues that specifically localize to the mitochondria and inhibit apoptosis (18, 41, 58). FPV039, encoded by fowlpox virus, was initially identified as a Bcl-2 homologue based on sequence analysis that revealed the presence of obvious BH1 and BH2 domains and a series of {alpha}-helices, similar to members of the cellular Bcl-2 family (Fig. 1A) (2). However, key amino acids typically found within the BH3 and BH4 domains of cellular Bcl-2 proteins are not conserved within FPV039 (Fig. 1) (2). As such, FPV039 differs markedly from cellular antiapoptotic Bcl-2 proteins, such as Bcl-2 and Bcl-xL, which typically possess all four BH domains (1, 17). In general, virus-encoded Bcl-2 (vBcl-2) homologues exhibit considerably less conservation within the BH3 and BH4 domains (18, 41, 58). For example, the Kaposi's sarcoma herpesvirus (KSHV) Bcl-2 homologue possesses conserved BH1 and BH2 domains but possesses poorly conserved BH3 and BH4 domains (14). Similarly, A179L, encoded by African swine fever virus, also possesses highly conserved BH1 and BH2 domains but lacks BH3 and BH4 domains (54, 86). In fact, of all vBcl-2 proteins identified, FPV039, KSHV Bcl-2, and A179L possess the most highly conserved BH1 domains and are the only vBcl-2 proteins to retain the entire NWGR sequence that is unfailingly conserved and functionally critical for cellular Bcl-2 proteins (Fig. 1B) (40).

Similar to Bcl-2, FPV039 is comprised of a series of {alpha}-helices that closely align with those determined from the structure of Bcl-2 (Fig. 1A) (56). The cellular multidomain Bcl-2 proteins adopt a similar fold comprised of at least eight {alpha}-helices, and the conserved BH domains, in turn, correspond to or overlap with these {alpha}-helices (56, 57). BH1, BH2, and BH3 contribute to the formation of a hydrophobic pocket that binds the {alpha}-helical BH3 domain of other Bcl-2 proteins, thus facilitating the homo- and heterotypic interactions that occur among all Bcl-2 family members (56, 57). Interestingly, two of the predicted {alpha}-helices possessed by FPV039 correspond with the BH3- and BH4-containing {alpha}-helices of Bcl-2 (Fig. 1A), suggesting that these {alpha}-helices in FPV039 are structurally homologous. Indeed, the three-dimensional structure of the KSHV Bcl-2 homologue exhibits a hydrophobic pocket similar to that possessed by cellular Bcl-2 and Bcl-xL, even though KSHV Bcl-2 lacks obvious BH3 and BH4 domains (46). Additionally, deletion of amino acids 41 to 54 in FPV039, which align with the BH3 domain of human Bcl-2, resulted in the inability of FPV039 to interact with Bak, providing evidence that this region within FPV039 is critical for function (Fig. 4B).

The absence of conserved BH3 and BH4 domain sequences in FPV039 may reflect a divergence on the part of FPV039 from negative regulation. Peptides corresponding to several BH3 domains are intrinsically proapoptotic and, like BH3-only proteins, capable of inducing apoptosis (65, 84). As such, strict conservation of the BH3 domain may be a disadvantage to the virus. Moreover, the BH3 domain plays an important role in the binding specificity of Bcl-2 proteins, and modification of this domain may enable FPV039 to interact more promiscuously with Bcl-2 family members or, conversely, increase the specificity of interaction. The lack of BH4 sequence conservation in FPV039 may reflect the loss of an additional phosphorylation site typically present in the BH4 domain of Bcl-2, which negatively regulates Bcl-2 (38). Furthermore, similar to other vBcl-2 homologues, FPV039 lacks the unstructured "loop" domain, normally present between the BH3 and BH4 domains of Bcl-2 and Bcl-xL, making FPV039 a comparably smaller protein (Fig. 1A). This loop acts as a negative regulatory domain, sensitive to inhibitory phosphorylation (55, 85) and caspase-3-mediated cleavage, which converts Bcl-2 and Bcl-xL from antiapoptotic to proapoptotic proteins (7, 13, 16, 32). Several vBcl-2 homologues bypass this level of control and are resistant to caspase cleavage (8, 18, 41, 58). Indeed, the various levels of negative regulation imposed on cellular Bcl-2 family members has likely placed a strong evolutionary pressure on vBcl-2 proteins, resulting in the preservation of only the minimal functional elements and the loss of regulatory elements. The absence of complete sequence homology in functional vBcl-2 homologues suggests that a strict conservation of amino acids is not necessary for these proteins to inhibit apoptosis.

The subcellular location of FPV039, along with the predicted secondary structure and sequence homology to cellular Bcl-2, clearly suggested that, like cellular Bcl-2 proteins, FPV039 functions through interaction with members of the Bcl-2 family, which tightly regulates the mitochondrial events leading to apoptosis (1, 17). Specifically, Bak and Bax, the two proapoptotic proteins absolutely required for the induction of apoptosis, represent a crucial lynchpin in the regulation of cell death (51, 82, 89), and vBcl-2 proteins inhibit apoptosis by interfering with the proapoptotic activity of Bak and Bax (18, 41, 58). Accordingly, we have shown that FPV039 constitutively interacts with both human and chicken Bak (Fig. 4, 6, and 7). Additionally, FPV039 inhibits apoptosis induced by Bak overexpression, as well as TNF-{alpha} treatment, in mammalian cells (Fig. 3 and 5). Moreover, FPV039 expression prevented the activation of Bak, a key step in the progression to Bak oligomerization and permeabilization of the outer mitochondrial membrane (35, 36, 81), thereby retaining Bak in an inactive and unresponsive state, unable to facilitate the induction of apoptosis (Fig. 9). Such an inhibitory mechanism has been observed previously for the adenovirus Bcl-2 homologue, EIB 19K, which also constitutively interacts with Bak and inhibits Bak activation in response to a variety of cytotoxic stimuli (26, 68). Although interaction with proapoptotic Bcl-2 family members such as Bak represents the most direct mechanism for a viral protein to prevent apoptosis, other vBcl-2 proteins, including KSHV Bcl-2, have not yet demonstrated an ability to interact with Bak but are capable of inhibiting apoptosis (14). Given that Bak is a key player in the release of mitochondrion-associated proapoptotic proteins, it is likely that other vBcl-2 proteins will also function through interaction with Bak.

Poxviruses are renowned for manipulating multiple aspects of the host immune response and encode several other antiapoptotic proteins that, similar to FPV039, mediate their effect by interfering with Bcl-2 family members (69). Two well-characterized poxvirus proteins, M11L from myxoma virus and F1L from VV, bear no sequence homology to each other and lack obvious sequence homology to cellular Bcl-2 proteins, yet they function analogously to Bcl-2 proteins in their ability to inhibit apoptosis (25, 28, 59, 67, 70, 75, 78, 80). Both F1L and M11L constitutively interact with and inhibit the activity of Bak (59, 75, 78). Likewise, a recently characterized protein, ORFV125, encoded by the orf virus, inhibits apoptosis and prevents the activation of Bak (83). FPV039, like F1L and M11L, inhibits apoptosis by interacting with Bak and inhibiting Bak activation (59, 75, 78). Indeed, we have shown that FPV039 can functionally replace F1L in VV to inhibit virus-induced apoptosis (Fig. 8). Despite the lack of sequence homology to cellular Bcl-2 proteins, the recently solved three-dimensional structure of M11L demonstrates a strikingly similar structure to Bcl-xL, explaining the basis for the molecular interaction with Bak (22, 50). In addition to interacting with Bak, M11L also interacts with the proapoptotic protein Bax, suggesting that FPV039 may perform a similar function to efficiently inhibit apoptosis (67). Conversely, in a manner similar to F1L, FPV039 may not directly interact with Bax but, instead, may inhibit Bax activity by binding directly to members of the BH3-only family of proteins, which serve as activators of Bak and Bax (65, 84). Interacting with, and inhibiting the activity of, BH3-only proteins might be an equally protective mechanism by which FPV039 inhibits apoptosis. E1B 19K, encoded by adenovirus, interacts with Bik (39), and we have recently shown that F1L interacts with Bim (70), thus supporting the possibility that inhibitors of apoptosis, whether homologous to Bcl-2 proteins or not, can prevent cell death by interacting with BH3-only proteins. Further studies are therefore required to define the antiapoptotic mechanism of FPV039 and the range of interactions FPV039 exhibits. The fact that FPV039, F1L, and M11L all inhibit apoptosis by modulating Bak yet lack sequence homology clearly implicates Bak as a critical protein in the apoptotic cascade.

It is possible that the differences among FPV039, F1L, and M11L can be accounted for by convergent evolution, whereby the ancestral genes for these proteins were all acquired independently and subsequently evolved to inhibit apoptosis via similar mechanisms. Interestingly, however, FPV039, F1L, and M11L are all found at a similar location within the genome and are flanked by a gene encoding the dUTPase (2, 12, 30), suggesting that these three genes, despite bearing no sequence similarity, may be the products of divergent evolution from a common progenitor. A similar phenomenon has been observed in human and murine cytomegalovirus. Both human and murine cytomegaloviruses encode viral mitochondrial inhibitors of apoptosis that lack sequence similarity but are encoded by open reading frames in homologous positions in the viral genome (52). The lack of sequence homology between FPV039, F1L, and M11L, however, does not obviously preclude the presence of structural homology, given that M11L adopts a Bcl-2-like structure (22, 50). Additionally, recent structural data indicate that N1L, a VV protein linked to NF-{kappa}B regulation (21), structurally resembles Bcl-2 family members and interacts with BH3 peptides, yet N1L possesses no obvious BH domains or other sequence similarity with cellular Bcl-2 proteins (5). Determination of the three-dimensional structure of F1L and FPV039 will help elucidate the evolutionary relatedness of FPV039, F1L, and M11L and determine if F1L and FPV039 physically resemble cellular Bcl-2 proteins, as we predict for FPV039 (Fig. 1).

Notably, fowlpox virus is currently being pursued as a potential vaccine vector and gene therapy tool (10, 62). Although fowlpox virus is incapable of replicating in mammalian cells and causing a productive infection, mammalian cells do support the transcription and translation of native or recombinantly inserted early genes (63). As such, understanding how FPV039 interacts with Bcl-2 proteins and inhibits apoptosis in a mammalian context may have significant implications in the development of vaccines and gene therapy vectors using fowlpox virus. In addition, understanding how FPV039, F1L, and M11L, three seemingly unrelated poxviral proteins, interact with Bcl-2 family members and inhibit apoptosis will provide significant advancements in our understanding of the pathways that govern cellular fate and will ultimately shed light on the mechanisms of Bcl-2 family regulation.


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ACKNOWLEDGMENTS
 
We thank C. Upton for supplying fowlpox virus DNA, H. Rabinowich for supplying Bak–/–/Bax–/– cells, and K. Kane for the NK1.1 antibody. We also thank S. Campbell for critical review of the manuscript.

L.B. and T.S. were supported by studentships from the Natural Sciences and Engineering Research Council of Canada. M.B. is a Senior Scholar of the Alberta Heritage Foundation for Medical Research, a Canadian Institutes for Medical Research (CIHR) New Investigator, and a Howard Hughes Medical Institute (HHMI) Scholar in Infection and Parasitology. Work in the authors’ laboratory was supported by grants from CIHR and HHMI.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, University of Alberta, 621 HMRC, Edmonton, Alberta, Canada T6G 2S2. Phone: (780) 492-0702. Fax: (780) 492-9828. E-mail: michele.barry{at}ualberta.ca Back

{triangledown} Published ahead of print on 8 August 2007. Back


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Journal of Virology, October 2007, p. 11032-11045, Vol. 81, No. 20
0022-538X/07/$08.00+0     doi:10.1128/JVI.00734-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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