Induction of apoptosis by human Nbk/Bik, a BH3-containing protein that interacts with E1B 19K.

The E1B 19-kilodalton protein (19K protein) is a potent apoptosis inhibitor and the adenovirus homolog of Bcl-2 (E. White, Genes Dev. 10:1-15, 1996). To obtain a better understanding of the biochemical mechanism by which the E1B 19K protein regulates apoptosis, proteins that interact with 19K have been identified; one of these is Bax (J. Han, P. Sabbatini, D. Perez, L. Rao, D. Mohda, and E. White, Genes Dev. 10:461-477, 1996), and another is Bak (S. N. Farrow, J. H. M. White, I. Martinou, T. Raven, K.-T. Pun, C. J. Grinham, J.-C. Martinou, and R. Brown, Nature (London) 374:731-733, 1995). Bax and Bak are Bcl-2 family members which contain Bcl-2 homology regions 1, 2, and 3 (BH1, BH2, and BH3), which interact with E1B 19K and Bcl-2 and promote apoptosis. Like Bax and Bak, Nbk was cloned from a yeast two-hybrid screen for proteins that interact with E1B 19K. Nbk contained BH3 but not BH1 or BH2. It also interacted with Bcl-2 but not with Bax. Both Bcl-2 and E1B 19K interacted with Nbk in vitro, and this interaction was highly specific. In vivo, the Nbk and E1B 19K proteins may colocalize with cytoplasmic and nuclear membranes. Nbk expression functionally antagonized 19K-mediated inhibition of apoptotic cell death and completely prevented transformation by E1A and E1B 19K. Nbk was sufficient for induction of apoptosis in the presence of mutant p53 and thus low levels of Bax, suggesting that Nbk functions independently of Bax to induce apoptosis. Nbk may therefore represent a novel death regulator which contains only a BH3 that interacts with and antagonizes apoptosis inhibitors such as the E1B 19K protein.     

The E1B19K oncoprotein complexes with Beclin 1 to regulate autophagy in adenovirus-infected cells

The mechanisms underlying adenovirus-mediated autophagy are currently unknown. Recently, members of the Bcl-2 protein family have been associated with autophagy. It was also reported that the Bcl-2 homology-3 (BH3) domain encompassed by both Beclin 1 and Bcl-2-like proteins is essential for their pro-autophagy or anti-autophagy functions. Here, we report for the first time that E1B19K, the adenovirus BH3 domain protein, interacts with Beclin 1 to initiate autophagy. Using immunoprecipitation assays we showed that expression of E1B19K in the host cell disrupted the physical interactions between Beclin 1 and Bcl-2 proteins. The displacement of Bcl-2 was coincident with the recruitment of PI3KC3 to the Beclin 1/E1B19K complexes. As a result of the changes in the components of the Beclin 1 interactome, there was activation of PI3KC3, as showed by the identification of PI3K-mediated lipid phosphorylation, and subsequent formation of autophagosomes. Importantly, the BH3 functional domain of E1B19K protein was required for the heterodimerization with Beclin 1. We also showed that transfer of E1B19K was sufficient to trigger autophagy in cancer cells. Consistent with these data, mutant adenoviruses encompassing a deletion of the E1B19K gene produced a marked deficiency in the capability of the virus to induce autophagy as showed by examining the lipidation and cleavage of LC3-I as well as the subcellular localization of LC3-II, the decrease in the levels of p62, and the formation of autophagosomes. Our work offers new information on the mechanisms of action of the adenoviral E1B19K protein as partner of Beclin 1 and positive regulator of autophagy.     

Tumor necrosis factor-alpha induces Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K

Tumor necrosis factor (TNF)-alpha-mediated death signaling induces oligomerization of proapoptotic Bcl-2 family member Bax into a high molecular mass protein complex in mitochondrial membranes. Bax complex formation is associated with the release of cytochrome c, which propagates death signaling by acting as a cofactor for caspase-9 activation. The adenovirus Bcl-2 homologue E1B 19K blocks TNF-alpha-mediated apoptosis by preventing cytochrome c release, caspase-9 activation, and apoptosis of virus-infected cells. TNF-alpha induces E1B 19K-Bax interaction and inhibits Bax oligomerization. Oligomerized Bax may form a pore to release mitochondrial proteins, analogous to the homologous pore-forming domains of bacterial toxins. E1B 19K can also bind to proapoptotic Bak, but the functional significance is not known. TNF-alpha signaling induced Bak-Bax interaction and both Bak and Bax oligomerization. E1B 19K was constitutively in a complex with Bak, and blocked the Bak-Bax interaction and oligomerization of both. The TNF-alpha-mediated cytochrome c and Smac/DIABLO release from mitochondria was inhibited by E1B 19K expression in adenovirus-infected cells. Since either Bax or Bak is essential for death signaling by TNF-alpha, the interaction between E1B 19K and both Bak and Bax may be required to inhibit their cooperative or independent oligomerization to release proteins from mitochondria which promote caspase activation and cell death.     

E1B 19,000-Molecular-Weight Protein Interacts with and Inhibits CED-4-Dependent, FLICE-Mediated Apoptosis

Genetic studies of the nematode Caenorhabditis elegans (C. elegans) have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-x_L) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-x_L have functional similarity, we determined if E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did not prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homologue of CED-4, to inhibit caspase activation and apoptosis.     

E1B 19K Inhibits Fas-mediated Apoptosis through FADD-dependent Sequestration of FLICE

E1B 19K, the adenovirus Bcl-2 homologue, is a potent inhibitor of apoptosis induced by various stimuli including Fas and tumor necrosis factor-α. Fas and TNFR-1 belong to a family of cytokine-activated receptors that share key components in their signaling pathways, Fas-associating protein with death domain (FADD) and FADD-like interleukin-1β–converting enzyme (FLICE), to induce an apoptotic response. We demonstrate here that E1B 19K and Bcl-x_L are able to inhibit apoptosis induced by FADD, but not FLICE. Surprisingly, apoptosis was abrogated by E1B 19K and Bcl-x_L when FADD and FLICE were coexpressed. Immunofluorescence studies demonstrated that FADD expression produced large insoluble death effector filaments that may represent oligomerized FADD. E1B 19K expression disrupted FADD filament formation causing FADD and FLICE to relocalize to membrane and cytoskeletal structures where E1B 19K is normally localized. E1B 19K, however, does not detectably bind to FADD, nor does it inhibit FADD and FLICE from being recruited to the death-inducing signaling complex (DISC) when Fas is stimulated. Thus, E1B 19K may inhibit Fas-mediated cell death downstream of FADD recruitment of FLICE but upstream of FLICE activation by disrupting FADD oligomerization and sequestering an essential component of the DISC.     

Bak and Bax function to limit adenovirus replication through apoptosis induction

Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level.     

Enhanced TRAIL sensitivity by E1A expression in human cancer and normal cell lines: inhibition by adenovirus E1B19K and E3 proteins

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines that induces apoptosis in a variety of cancer cells, but not in normal cells. However, more and more tumor cells remain resistant to TRAIL, which limited its application for cancer therapy. Expression of the adenovirus serotype 5 (Ad5) E1A sensitizes tumor cells to apoptosis by TNF-alpha, Fas-ligand, and TRAIL. Here we asked whether E1A overcomes this resistance and enhances TRAIL-induced apoptosis in the tumor cells. Our results revealed that the tumor cell lines, HeLa and HepG2, with infection by Ad-E1A, were highly sensitive to TRAIL-induced apoptosis. Importantly, we found that in normal primary human lung fibroblast cells (HLF) TRAIL is capable of inducing apoptosis in combination with E1A as efficiently as in some tumor cell lines. The adenovirus type 5 encoding proteins, E1B19K and E3 gene products, have been shown to inhibit E1A and TRAIL-induced apoptosis of HLF cells by using the recombinant adenovirus AdDeltaE1B55K, with mutation of E1B55K, containing E1B19K and complete E3 region. Further results demonstrated that the expression of DR5 and TRAIL was down-regulated in the AdDeltaE1B55K co-infected HLF cells. These findings suggest that TRAIL may play an important role in limiting virus infections and the ability of adenovirus to inhibit killing may prolong acute and persistent infections. The results from this study have also suggested the possibility that the combination of E1A with TRAIL could be used in the treatment of human malignancy, or in the selection of the optimal adenovirus mutant as effective delivering vector for cancer therapy.     

E1B 19K Blocks Bax Oligomerization and Tumor Necrosis Factor Alpha-Mediated Apoptosis

Tumor necrosis factor alpha (TNF-alpha)-mediated death signaling causes the recruitment of monomeric pro- apoptotic Bax into a 500-kDa protein complex. The adenovirus Bcl-2 homologue, E1B 19K, inhibits TNF-alpha-mediated apoptosis, interacts with Bax, and blocked the formation of the 500-kDa Bax complex. TNF-alpha and truncated Bid induced Bax-Bax cross-linking, indicative of oligomerization, and E1B 19K expression during infection inhibited this TNF-alpha-mediated Bax oligomerization. TNF-alpha signaled conformation changes at the Bax amino and carboxy termini. Exposure of the Bax amino terminus facilitates E1B 19K-Bax binding, which prevented exposure of the carboxy-terminal Bax Bcl-2 homology region 2 epitope. Inhibition of Bax oligomerization by E1B 19K is an activity that bears striking similarity to the means by which bacterial immunity proteins block pore formation by bacterial toxins which have structural homology to Bax.     

Latent Epstein-Barr virus can inhibit apoptosis in B cells by blocking the induction of NOXA expression

Latent Epstein-Barr virus (EBV) has been shown to protect Burkitt's lymphoma-derived B cells from apoptosis induced by agents that cause damage to DNA, in the context of mutant p53. This protection requires expression of the latency-associated nuclear proteins EBNA3A and EBNA3C and correlates with their ability to cooperate in the repression of the gene encoding the pro-apoptotic, BH3-only protein BIM. Here we confirm that latent EBV in B cells also inhibits apoptosis induced by two other agents--ionomycin and staurosporine--and show that these act by a distinct pathway that involves a p53-independent increase in expression of another pro-apoptotic, BH3-only protein, NOXA. Analyses employing a variety of B cells infected with naturally occurring EBV or B95.8 EBV-BAC recombinant mutants indicated that the block to NOXA induction does not depend on the well-characterized viral latency-associated genes (EBNAs 1, 2, 3A, 3B, 3C, the LMPs or the EBERs) or expression of BIM. Regulation of NOXA was shown to be at least partly at the level of mRNA and the requirement for NOXA to induce cell death in this context was demonstrated by NOXA-specific shRNA-mediated depletion experiments. Although recombinant EBV with a deletion removing the BHRF1 locus--that encodes the BCL2-homologue BHRF1 and three microRNAs--partially abrogates protection against ionomycin and staurosporine, the deletion has no effect on the EBV-mediated block to NOXA accumulation.     

Multiple BH3 mimetics antagonize antiapoptotic MCL1 protein by inducing the endoplasmic reticulum stress response and up-regulating BH3-only protein NOXA

BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-X(L), or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.     

Characterization of an anti-apoptotic glycoprotein encoded by Kaposi's sarcoma-associated herpesvirus which resembles a spliced variant of human survivin

We have investigated the expression and function of a novel protein encoded by open reading frame (ORF) K7 of Kaposi's sarcoma-associated herpesvirus (KSHV). Computational analyses revealed that K7 is structurally related to survivin-DeltaEx3, a splice variant of human survivin that protects cells from apoptosis by an undefined mechanism. Both K7 and survivin-DeltaEx3 contain a mitochondrial-targeting sequence, an N-terminal region of a BIR (baculovirus IAP repeat) domain and a putative BH2 (Bcl-2 homology)-like domain. These suggested that K7 is a new viral anti-apoptotic protein and survivin-DeltaEx3 is its likely cellular homologue. We show that K7 is a glycoprotein, which can inhibit apoptosis and anchor to intracellular membranes where Bcl-2 resides. K7 does not associate with Bax, but does bind to Bcl-2 via its putative BH2 domain. In addition, K7 binds to active caspase-3 via its BIR domain and thus inhibits the activity of caspase-3. The BH2 domain of K7 is crucial for the inhibition of caspase-3 activity and is therefore essential for its anti-apoptotic function. Furthermore, K7 bridges Bcl-2 and activated caspase-3 into a protein complex. K7 therefore appears to be an adaptor protein and part of an anti-apoptotic complex that presents effector caspases to Bcl-2, enabling Bcl-2 to inhibit caspase activity. These data also suggest that survivin-DeltaEx3 might function by a similar mechanism to that of K7. We denote K7 as vIAP (viral inhibitor-of-apoptosis protein).     

TNF-alpha signals apoptosis through a bid-dependent conformational change in Bax that is inhibited by E1B 19K

The adenovirus E1B 19K gene product is an inhibitor of apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) during viral infection. We report that E1B 19K inhibited neither caspase-8 activation nor caspase-8-dependent Bid cleavage by TNF-alpha. Rather, TNF-alpha induced a tBid-dependent conformational change in Bax that allowed an interaction between E1B 19K and conformationally altered Bax, which caused inhibition of cytochrome c release and caspase-9 activation. E1B 19K expression interrupted caspase-3 processing, permitting cleavage to remove the p12 subunit but not the prodomain consistent with caspase-8 and not caspase-9 enzymatic activity. Thus, E1B 19K blocks TNF-alpha-mediated death signaling by inhibiting a specific form of Bax that interrupts caspase activation downstream of caspase-8 and upstream of caspase-9.     

SILKWORM 30K PROTEIN INHIBITS ECDYSONE‐INDUCED APOPTOSIS BY BLOCKING THE BINDING OF ULTRASPIRACLE TO ECDYSONE RECEPTOR‐B1 IN CULTURED Bm5 CELLS

This study investigates the mechanism through which increased 30K protein inhibits ecdysone‐induced apoptosis in the Bm5 silkworm ovarian cell line. Treatment of Bm5 cells with 20‐hydroxyecdysone (20E) after transfection with the pIZT/V5‐His control vector triggered apoptosis, but 20E treatment did not trigger apoptosis in Bm5 cells transfected with the pIZT/30K/V5‐His vector. To confirm its inhibitory effect on apoptosis, 30K protein was first purified from Escherichia coli transformed with a 30K expression vector and used to generate specific antibodies in mice. Anti‐30K antiserum was used to confirm synthesis of the 30K protein in pIZT/30K/V5‐His‐transfected Bm5 cells and to detect 30K protein binding to the ecdysone receptor‐B1 (EcR‐B1). Anti‐30K antiserum was used to immunoprecipitate protein complexes containing 30K from Bm5 cells transfected with pIZT/30K/V5‐His vector and treated with 20E. We observed that 30K proteins bound primarily to the EcR‐B1 and not to ultraspiracle (USP). Reciprocal immunoprecipitation of EcR‐B1‐containing complexes from Bm5 cells transfected with control pIZT/V5‐His vector and treated with 20E showed that EcR‐B1 bound to USP in the absence of 30K but did not bind to USP in pIZT/30K/V5‐His‐transfected Bm5 cells. These results demonstrate that 30K proteins block USP binding to EcR‐B1 through formation of a 30K/EcR‐B1 complex, resulting in inhibition of 20E‐induced Bm5 cell apoptosis.     

Bim: a novel member of the Bcl-2 family that promotes apoptosis.

Certain members of the Bcl-2 family inhibit apoptosis while others facilitate this physiological process of cell death. An expression screen for proteins that bind to Bcl-2 yielded a small novel protein, denoted Bim, whose only similarity to any known protein is the short (nine amino acid) BH3 motif shared by most Bcl-2 homologues. Bim provokes apoptosis, and the BH3 region is required for Bcl-2 binding and for most of its cytotoxicity. Like Bcl-2, Bim possesses a hydrophobic C-terminus and localizes to intracytoplasmic membranes. Three Bim isoforms, probably generated by alternative splicing, all induce apoptosis, the shortest being the most potent. Wild-type Bcl-2 associates with Bim in vivo and modulates its death function, whereas Bcl-2 mutants that lack survival function do neither. Significantly, Bcl-xL and Bcl-w, the two closest homologues of Bcl-2, also bind to Bim and inhibit its activity, but more distant viral homologues, adenovirus E1B19K and Epstein-Barr virus BHRF-1, can do neither. Hence, Bim appears to act as a 'death ligand' which can only neutralize certain members of the pro-survival Bcl-2 sub-family.     

Identification of three functions of the adenovirus e4orf6 protein that mediate p53 degradation by the E4orf6-E1B55K complex

Complexes containing adenovirus E4orf6 and E1B55K proteins play critical roles in productive infection. Both proteins interact directly with the cellular tumor suppressor p53, and in combination they promote its rapid degradation. To examine the mechanism of this process, degradation of exogenously expressed p53 was analyzed in p53-null human cells infected with adenovirus vectors encoding E4orf6 and/or E1B55K. Coexpression of E4orf6 and E1B55K greatly reduced both the level and the half-life of wild-type p53. No effect was observed with the p53-related p73 proteins, which did not appear to interact with E4orf6 or E1B55K. Mutant forms of p53 were not degraded if they could not efficiently bind E1B55K, suggesting that direct interaction between p53 and E1B55K may be required. Degradation of p53 was independent of both MDM2 and p19ARF, regulators of p53 stability in mammalian cells, but required an extended region of E4orf6 from residues 44 to 274, which appeared to possess three separate biological functions. First, residues 39 to 107 were necessary to interact with E1B55K. Second, an overlapping region from about residues 44 to 218 corresponded to the ability of E4orf6 to form complexes with cellular proteins of 19 and 14 kDa. Third, the nuclear retention signal/amphipathic arginine-rich alpha-helical region from residues 239 to 253 was required. Interestingly, neither the E4orf6 nuclear localization signal nor the nuclear export signal was essential. These results suggested that if nuclear-cytoplasmic shuttling is involved in this process, it must involve another export signal. Degradation was significantly blocked by the 26S proteasome inhibitor MG132, but unlike the HPV E6 protein, E4orf6 and E1B55K were unable to induce p53 degradation in vitro in reticulocyte lysates. Thus, this study implies that the E4orf6-E1B55K complex may direct p53 for degradation by a novel mechanism.     

Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins.

Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these cytotoxic effects and that other viral gene products are not involved. Finally, placing E1A expression under the control of a strong promoter did not alter the requirement for E1B in the transformation of primary cells. One possibility is that the E1B 19K protein is required to overcome the cytotoxic effects of E1A protein expression and thereby enable primary cells to become transformed.     

Functional complementation of the adenovirus E1B 19-kilodalton protein with Bcl-2 in the inhibition of apoptosis in infected cells.

Expression of the adenovirus E1A oncogene induces apoptosis which impedes both the transformation of primary rodent cells and productive adenovirus infection of human cells. Coexpression of E1A with the E1B 19,000-molecular-weight protein (19K protein) or the Bcl-2 protein, both of which have antiapoptotic activity, is necessary for efficient transformation. Induction of apoptosis by E1A in rodent cells is mediated by the p53 tumor suppressor gene, and both the E1B 19K protein and the Bcl-2 protein can overcome this p53-dependent apoptosis. The functional similarity between Bcl-2 and the E1B 19K protein suggested that they may act by similar mechanisms and that Bcl-2 may complement the requirement for E1B 19K expression during productive infection. Infection of human HeLa cells with E1B 19K loss-of-function mutant adenovirus produces apoptosis characterized by enhanced cytopathic effects (cyt phenotype) and degradation of host cell chromosomal DNA and viral DNA (deg phenotype). Failure to inhibit apoptosis results in premature host cell death, which impairs virus yield. HeLa cells express extremely low levels of p53 because of expression of human papillomavirus E6 protein. Levels of p53 were substantially increased by E1A expression during adenovirus infection. Therefore, E1A may induce apoptosis by overriding the E6-induced degradation of p53 and promoting p53 accumulation. Stable Bcl-2 overexpression in HeLa cells infected with the E1B 19K- mutant adenovirus blocked the induction of the cyt and deg phenotypes. Expression of Bcl-2 in HeLa cells also conferred resistance to apoptosis mediated by tumor necrosis factor alpha and Fas antigen, which is also an established function of the E1B 19K protein. A comparison of the amino acid sequences of Bcl-2 family members and that of the E1B 19K protein indicated that there was limited amino acid sequence homology between the central conserved domains of E1B 19K and Bcl-2. This domain of the E1B 19K protein is important in transformation and regulation of apoptosis, as determined by mutational analysis. The limited sequence homology and functional equivalency provided further evidence that the Bcl-2 and E1B 19K proteins may possess related mechanisms of action and that the E1B 19K protein may be the adenovirus equivalent of the cellular Bcl-2 protein.