Adenovirus E4orf4 protein induces PP2A-dependent growth arrest in Saccharomyces cerevisiae and interacts with the anaphase-promoting complex/cyclosome.

Adenovirus early region 4 open reading frame 4 (E4orf4) protein has been reported to induce p53-independent, protein phosphatase 2A (PP2A)-dependent apoptosis in transformed mammalian cells. In this report, we show that E4orf4 induces an irreversible growth arrest in Saccharomyces cerevisiae at the G2/M phase of the cell cycle. Growth inhibition requires the presence of yeast PP2A-Cdc55, and is accompanied by accumulation of reactive oxygen species. E4orf4 expression is synthetically lethal with mutants defective in mitosis, including Cdc28/Cdk1 and anaphase-promoting complex/cyclosome (APC/C) mutants. Although APC/C activity is inhibited in the presence of E4orf4, Cdc28/Cdk1 is activated and partially counteracts the E4orf4-induced cell cycle arrest. The E4orf4-PP2A complex physically interacts with the APC/C, suggesting that E4orf4 functions by directly targeting PP2A to the APC/C, thereby leading to its inactivation. Finally, we show that E4orf4 can induce G2/M arrest in mammalian cells before apoptosis, indicating that E4orf4-induced events in yeast and mammalian cells are highly conserved.

Selection of apoptosis-deficient adenovirus E4orf4 mutants in Saccharomyces cerevisiae.

Adenovirus E4orf4 protein has been shown to induce p53-independent, protein phosphatase 2A (PP2A)-dependent apoptosis in transformed cells. Furthermore, E4orf4 also induces toxicity in Saccharomyces cerevisiae in a PP2A-dependent manner (D. Kornitzer and T. Kleinberger, submitted for publication). In this work, we utilized yeast cells to select for nonapoptotic E4orf4 mutants which, in turn, were shown to possess a diminished ability to bind PP2A. The success of this selection system will provide additional apoptosis-relevant mutants for E4orf4 research and strongly supports the relevance of E4orf4-induced toxicity in S. cerevisiae to E4orf4-induced apoptosis in mammalian cells.

Increased vascular endothelial growth factor 165 binding to kinase insert domain-containing receptor after infection of human endothelial cells by recombinant adenovirus encoding the Vegf(165) gene.

BACKGROUND: The angiogenic effect of vascular endothelial growth factor (VEGF(165)) is mediated mainly through the high-affinity tyrosine kinase receptor VEGF-R2 (KDR/flk-1). This study examined the effects of VEGF overexpression by primary human endothelial cells (ECs), which do not express VEGF under physiological conditions, on cell proliferation, VEGF binding to the kinase insert domain-containing receptor (KDR), and KDR expression. METHODS AND RESULTS: Human primary ECs and SMCs were infected by recombinant adenoviral vector encoding VEGF(165) (rAdVEGF). Proliferation rate, bromodeoxyuridine incorporation, (125)I-labeled VEGF(165) binding to the KDR receptor, and KDR expression were tested in the infected cells and in cells supplemented with VEGF protein. Enhanced proliferation and a significant increase in (125)I-VEGF(165) binding to the KDR receptor were induced by rAdVEGF infection of ECs (autocrine effect) as well as by addition of recombinant VEGF(165) to noninfected cells. Infection of ECs by rAdVEGF led to posttranscriptional upregulation of the KDR receptor, whereas KDR mRNA expression levels remained unchanged. Similar effects were observed with supplemented recombinant VEGF(165) to noninfected ECs; nevertheless, this phenomenon occurred only with high VEGF(165) concentrations (10 ng/mL). CONCLUSIONS: The effect of VEGF(165) on proliferation and upregulation of KDR receptor expression demonstrated an autocrine phenomenon of EC sensitization. The fact that high concentrations of VEGF may be achieved in vivo by local continuous overexpression of VEGF(165) by gene transfer emphasizes the potential advantage of gene transfer over protein supplementation for therapeutic angiogenesis.

The adenovirus E4-ORF4 splicing enhancer protein interacts with a subset of phosphorylated SR proteins.

SR proteins purified from uninfected HeLa cells inhibit adenovirus IIIa pre-mRNA splicing by binding to the intronic IIIa repressor element (3RE). In contrast, SR proteins purified from late adenovirus-infected cells are functionally inactivated as splicing repressor proteins by a virus-induced dephosphorylation. We have shown that the adenovirus E4-ORF4 protein, which binds the cellular protein phos phatase 2A (PP2A) and activates IIIa splicing in vitro and in vivo, induces SR protein dephosphorylation. Here we show that E4-ORF4 interacts with only a subset of SR proteins present in HeLa cells. Thus, E4-ORF4 interacts efficiently with SF2/ASF and SRp30c, but not with other SR proteins. Interestingly, E4-ORF4 interacts with SF2/ASF through the latter's RNA recognition motifs. Furthermore, E4-ORF4 interacts preferentially with the hyperphosphorylated form of SR proteins found in uninfected HeLa cells. E4-ORF4 mutant proteins that fail to bind strongly to PP2A or SF2/ASF do not relieve the repressive effect of HeLa SR proteins on IIIa pre-mRNA splicing in transient transfection experiments, suggesting that an interaction between all three proteins is required for E4-ORF4-induced SR protein dephosphorylation.

Caspase activation by adenovirus e4orf4 protein is cell line specific and Is mediated by the death receptor pathway.

Adenovirus E4orf4 protein has been shown to induce transformed cell-specific, protein phosphatase 2A-dependent, and p53-independent apoptosis. It has been further reported that the E4orf4 apoptotic pathway is caspase-independent in CHO cells. Here, we show that E4orf4 induces caspase activation in the human cell lines H1299 and 293T. Caspase activation is required for apoptosis in 293T cells, but not in H1299 cells. Dominant negative mutants of caspase-8 and the death receptor adapter protein FADD/MORT1 inhibit E4orf4-induced apoptosis in 293T cells, suggesting that E4orf4 activates the death receptor pathway. Cytochrome c is released into the cytosol in E4orf4-expressing cells, but caspase-9 is not required for induction of apoptosis. Furthermore, E4orf4 induces accumulation of reactive oxygen species (ROS) in a caspase-8- and FADD/MORT1-dependent manner, and inhibition of ROS generation by 4,5-dihydroxy-1, 3-benzene-disulfonic acid (Tiron) inhibits E4orf4-induced apoptosis. Thus, our results demonstrate that E4orf4 engages the death receptor pathway to generate at least part of the molecular events required for E4orf4-induced apoptosis.

Adenovirus E4orf4 protein interacts with both Balpha and B' subunits of protein phosphatase 2A, but E4orf4-induced apoptosis is mediated only by the interaction with Balpha.

Adenovirus E4orf4 protein is a multifunctional viral regulator, which is involved in down regulation of virally-modulated signal transduction, in control of alternative splicing of viral mRNAs, and in induction of apoptosis in transformed cells. It has been previously shown that E4orf4 interacts with protein phosphatase 2A through the phosphatase Balpha subunit. It was further shown that PP2A is required for performing the various E4orf4 functions. We report here that E4orf4 interacts with multiple isoforms of the PP2A-B' subunit, as well as with Balpha. We map the interaction sites of the B subunits on E4orf4 and show that they overlap but are not identical. We identify a dominant negative E4orf4 mutant, which disrupts the PP2A holoenzyme. We show that induction of apoptosis by E4orf4, which we previously reported to require the interaction with Balpha, is not affected by the interaction with B'. Our results suggest that the interaction of E4orf4 with various PP2A subpopulations may mediate the different E4orf4 functions.

Induction of apoptosis by adenovirus E4orf4 protein.

Adenovirus E4orf4 protein is a multifunctional viral regulator that induces p53-independent apoptosis in transformed cells, but not in normal cells. E4orf4-induced apoptosis can occur without activation of known caspases, although E4orf4 induces caspase activity in some cell lines. The interaction of E4orf4 with a specific subpopulation of protein phosphatase 2A (PP2A) molecules that contain B subunits, but not with those that contain B' subunits, is required for induction of apoptosis. This review suggests the potential use of E4orf4 in cancer therapy, and discusses whether E4orf4-induced apoptosis plays a role in the viral life cycle. Future research directions are also highlighted.

Induction of apoptosis by adenovirus E4orf4 protein is specific to transformed cells and requires an interaction with protein phosphatase 2A.

We previously have shown that adenovirus type 5 E4orf4 protein associates with protein phosphatase 2A (PP2A) and induces apoptosis in transformed cells in a p53-independent manner. Here we show that the interaction between E4orf4 and PP2A is required for induction of apoptosis by the viral protein. This conclusion is supported by a mutation analysis of E4orf4 protein, showing a correlation between the ability to bind PP2A and to induce apoptosis, and by the observation that transfection of an antisense construct of the PP2A-B55 subunit reduces expression of the PP2A-B55 subunit and inhibits induction of apoptosis by E4orf4, but not by p53. The mutant analysis also indicates that even a low level of interaction with PP2A is sufficient to initiate the E4orf4 apoptotic pathway. In addition, E4orf4 inhibits cellular transformation by various oncogenes, and this function is coupled to its ability to induce apoptosis. Furthermore, expression of oncogenes in primary cell cultures sensitizes these cells to induction of apoptosis by E4orf4. Our results suggest that E4orf4 is a potentially useful tool for cancer gene therapy.

Adenovirus type 5 E4 open reading frame 4 protein induces apoptosis in transformed cells.

Adenovirus type 5 E4 open reading frame 4 (E4orf4) protein has been previously shown to counteract transactivation of the junB and c-fos genes by cyclic AMP plus E1A protein and to interact with protein phosphatase 2A (PP2A). Here, we show that the wild-type E4orf4 protein induces apoptosis in the E1A-expressing 293 cells, in NIH 3T3 cells transformed with v-Ras, and in the lung carcinoma cell line H1299. The induction of apoptosis is not accompanied by enhanced levels of p53 in 293 cells and occurs in the absence of p53 in H1299 cells, indicating involvement of a p53-independent pathway. A mutant E4orf4 protein that had lost the ability to induce apoptosis also lost its ability to bind PP2A. We suggest that E4orf4 antagonizes continuous signals to proliferate, like those given by E1A or v-Ras, and that the conflicting signals lead to the induction of cell death.

Adenovirus E4orf4 protein binds to protein phosphatase 2A, and the complex down regulates E1A-enhanced junB transcription.

Adenovirus E4orf4 protein was previously shown to counteract transactivation of junB by cyclic AMP (cAMP) and E1A protein. It was also shown to cause hypophosphorylation of E1A and c-Fos proteins. Here we show that the E4orf4 protein associates with protein phosphatase 2A. All three subunits of the phosphatase are present in the complex, and the B subunit interacts directly with the viral protein. The complex possesses a phosphatase activity typical of protein phosphatase 2A, and the phosphatase mediates the E4orf4-induced down regulation of junB transcription. Thus, adenovirus E4orf4 protein recruits protein phosphatase 2A into a signal transduction pathway initiated by cAMP and E1A protein.

Adenovirus E4orf4 protein reduces phosphorylation of c-Fos and E1A proteins while simultaneously reducing the level of AP-1.

Adenovirus E1A protein and cyclic AMP cooperate to induce transcription factor AP-1 and viral gene expression in mouse S49 cells. We report that a protein encoded within the viral E4 gene region acts to counterbalance the induction of AP-1 DNA-binding activity by E1A and cyclic AMP. Studies with mutant adenoviruses demonstrated that in the absence of E4orf4 protein, AP-1 DNA-binding activity is induced to substantially higher levels than in wild-type virus-infected cells. The induction is the result of increased production of JunB and c-Fos proteins. Hyperphosphorylated forms of c-Fos and E1A proteins accumulate in the absence of functional E4orf4 protein. We propose that the E4orf4 protein acts to inhibit the activity of a cellular kinase that phosphorylates both the E1A and c-Fos proteins. Phosphorylation-dependent alterations in the activity of c-Fos, E1A, or some unidentified protein might, then, lead to decreased synthesis of AP-1 components. This E4 function likely plays an important role in natural infections, since a mutant virus unable to express the E4orf4 protein is considerably more cytotoxic than the wild-type virus.

A protein kinase is present in a complex with adenovirus E1A proteins.

A kinase activity can be immunoprecipitated in a complex that includes adenovirus E1A proteins. In vitro, this activity phosphorylated other E1A-associated proteins, as well as added E1A and histone H1 proteins. The E1A-associated kinase activity was cleared from extracts with an antibody to cyclin A, but not with antibody to cyclin B. The formation of a complex that included the kinase activity required amino acids 30-60 and 122-129 on the E1A proteins, sequences needed for association of E1A proteins with cyclin A and the retinoblastoma protein and implicated in control of cell growth. The complex of E1A-associated proteins included a 33-kDa ATP-binding protein, similar in size to a cyclin A-associated cdc2 kinase family member. Sucrose gradient analysis revealed two distinct E1A-containing complexes with the kinase activity. We suggest that E1A proteins may affect cellular proliferation by interacting with a member of the cdc2 kinase family and thereby influencing its activity.

Carcinogen-mediated co-activation of two independent genes in Chinese hamster cells.

Following carcinogen treatment, elevated expression levels of dihydrofolate reductase (dhfr) were measured by labeling cells with fluorescent methotrexate which binds to this enzyme. Fractionation of carcinogen-treated, Simian virus 40 (SV40)-transformed Chinese hamster embryo cells (C060) into subpopulations differing in their levels of dhfr expression revealed co-expression, at enhanced levels, of dhfr and SV40 T antigen in the same cells. The increased expression of dhfr was amplification independent, while the T antigen coding sequences were amplified. The co-expression of dhfr and the bacterial chloramphenicol acetyltransferase gene linked to the early SV40 regulatory region was measured in CHO cells stably transformed by pSV2CAT-SVgpt (CC24). Both these sequences were expressed at higher levels in treated cells and the elevated expression levels were observed in the same subpopulation of cells, although no increase in their gene copy number was detected. The concomitant activation of enhanced expression of two independent genes in the same cells suggests that cellular factors governing gene expression are activated in the carcinogen-treated cells. The implications of these findings to cellular control mechanisms and to the carcinogenic process are discussed.

Carcinogen-induced trans activation of gene expression.

We report a new mechanism of carcinogen action by which the expression of several genes was concomitantly enhanced. This mechanism involved the altered activity of cellular factors which modulate the expression of genes under their control. The increased expression was regulated at least in part on the transcriptional level and did not require amplification of the overexpressed genes. This phenomenon was transient; it was apparent as early as 24 h after carcinogen treatment and declined a few days later.

Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells.

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.

Mapping of SV40 DNA replication origin region binding sites for the SV40 T antigen by protection against exonuclease III digestion.

Incubation of 32P-5' end-labeled DNA fragments of less than 500 bp with excess amounts of the 3' leads to 5', double strand-dependent nuclease Exonuclease III generally results in single-stranded products of slightly more than half the size of the uncleaved substrate. When such restriction fragments of known size and sequence containing the lac operator were incubated with purified lac repressor, Exonuclease III cleavage was blocked at the 3' borders of the operator on each strand. It was possible to define the DNA sequence between the two boundaries of repressor-mediated exonuclease blockade by electrophoresing the single-stranded, protected products in urea-containing polyacrylamide gels in parallel with a dimethylsulfate modification-cleavage digest of the end-labeled, uncleaved substrate. The same approach was applied to an analysis of sites of large SV40 T antigen protection in the vicinity of the origin of SV40 DNA replication. Three discrete boundaries of apparent protection were observed--one on the "late" side of the origin and two on the "early" side. These sequences may constitute the 3' borders of discrete T antigen-binding sites in the origin region. Alternatively, one or more of these blockade points may signify regions of the genome which undergo conformational changes resulting in Exonuclease III resistance due to vicinal T antigen binding.