Tamar Kleinberger

The adenovirus E4orf4 protein targets PP2A to the ACF chromatin-remodeling factor and induces cell death through regulation of SNF2h-containing complexes

The adenovirus E4 open-reading-frame 4 (E4orf4) protein regulates the progression of viral infection and when expressed individually it induces non-classical apoptosis in transformed cells. Here we show that E4orf4 associates with the ATP-dependent chromatin-remodeling factor ACF that consists of a sucrose non fermenting-2h (SNF2h) ATPase and an Acf1 regulatory subunit. Furthermore, E4orf4 targets protein phosphatase 2A (PP2A) to this complex and to chromatin. Obstruction of SNF2h activity inhibits E4orf4-induced cell death, whereas knockdown of Acf1 results in enhanced E4orf4-induced toxicity in both mammalian and yeast cells, and Acf1 overexpression inhibits E4orf4′s ability to downregulate early adenovirus gene expression in the context of viral infection. Knockdown of the Acf1 homolog, WSTF, inhibits E4orf4-induced cell death. Based on these results we suggest that the E4orf4–PP2A complex inhibits ACF and facilitates enhanced chromatin-remodeling activities of other SNF2h-containing complexes, such as WSTF–SNF2h. The resulting switch in chromatin remodeling determines life versus death decisions and contributes to E4orf4 functions during adenovirus infection.

Adenovirus E4orf4 Protein Downregulates MYC Expression through Interaction with the PP2A-B55 Subunit

ABSTRACT The adenovirus E4 open reading frame 4 (E4orf4) protein is a multifunctional viral regulator that is involved in the temporal regulation of viral gene expression by modulating cellular and viral genes at the transcription and translation levels and by controlling alternative splicing of adenoviral late mRNAs. When expressed individually, E4orf4 induces apoptosis in transformed cells. Using oligonucleotide microarray analysis, validated by quantitative real time PCR, we found that MYC (also known as c-Myc) is downregulated early after the induction of E4orf4 expression. As a result, Myc protein levels are reduced in E4orf4-expressing cells. MYC downregulation is observed both when E4orf4 is expressed individually and within the context of viral infection. E4orf4 reduces MYC transcription but does not affect transcriptional elongation or RNA stability. An interaction with the PP2A-B55 subunit is required for the downregulation of MYC by E4orf4. Since Myc overexpression was previously shown to inhibit adenovirus replication, the downregulation of Myc by E4orf4 would contribute to efficient virus infection.

YND1 Interacts with CDC55 and Is a Novel Mediator of E4orf4-induced Toxicity

Adenovirus E4orf4 (early region 4 open reading frame 4) protein induces protein phosphatase 2A-dependent non-classical apoptosis in mammalian cells and irreversible growth arrest in Saccharomyces cerevisiae. Oncogenic transformation sensitizes cells to E4orf4-induced cell death. To uncover additional components of the E4orf4 network required for induction of its unique mode of apoptosis, we used yeast genetics to select gene deletions conferring resistance to E4orf4. Deletion of YND1, encoding a yeast Golgi apyrase, conferred partial resistance to E4orf4. However, Ynd1p apyrase activity was not required for E4orf4-induced toxicity. Ynd1p and Cdc55p, the yeast protein phosphatase 2A-B subunit, contributed additively to E4orf4-induced toxicity. Furthermore, concomitant overexpression of one and deletion of the other was detrimental to yeast growth, demonstrating a functional interaction between the two proteins. YND1 and CDC55 also interacted genetically with CDC20 and CDH1/HCT1, encoding activating subunits of the anaphase-promoting complex/cyclosome. In addition to their functional interaction, Ynd1p and Cdc55p interacted physically, and this interaction was disrupted by E4orf4, which remained associated with both proteins. The results suggested that Ynd1p and Cdc55p share a common downstream target whose balanced modulation by the two E4orf4 partners is crucial to viability. Disruption of this balance by E4orf4 may lead to cell death. NTPDase-4/Lalp70/UDPase, the closest mammalian homologue of Ynd1p, associated with E4orf4 in mammalian cells, suggesting that the results in yeast are relevant to the mammalian system.

Structure- and Modeling-based Identification of the Adenovirus E4orf4 Binding Site in the Protein Phosphatase 2A B55α Subunit

Background: The adenovirus E4orf4 protein must bind protein phosphatase 2A (PP2A) for its functions. Results: The E4orf4 binding site in PP2A was mapped to the α1,α2 helices of the B55α subunit. Conclusion: The E4orf4 binding site in PP2A-B55α lies above the substrate binding site and does not overlap it. Significance: A novel functional significance was assigned to the α1,α2 helices of the PP2A-B55α subunit. The adenovirus E4orf4 protein regulates the progression of viral infection and when expressed outside the context of the virus it induces nonclassical, cancer cell-specific apoptosis. All E4orf4 functions known to date require an interaction between E4orf4 and protein phosphatase 2A (PP2A), which is mediated through PP2A regulatory B subunits. Specifically, an interaction with the B55α subunit is required for induction of cell death by E4orf4. To gain a better insight into the E4orf4-PP2A interaction, mapping of the E4orf4 interaction site in PP2A-B55α has been undertaken. To this end we used a combination of bioinformatics analyses of PP2A-B55α and of E4orf4, which led to the prediction of E4orf4 binding sites on the surface of PP2A-B55α. Mutation analysis, immunoprecipitation, and GST pulldown assays based on the theoretical predictions revealed that the E4orf4 binding site included the α1 and α2 helices described in the B55α structure and involved at least three residues located in these helices facing each other. Loss of E4orf4 binding was accompanied by reduced contribution of the B55α mutants to E4orf4-induced cell death. The identified E4orf4 binding domain lies above the previously described substrate binding site and does not overlap it, although its location could be consistent with direct or indirect effects on substrate binding. This work assigns for the first time a functional significance to the α1,α2 helices of B55α, and we suggest that the binding site defined by these helices could also contribute to interactions between PP2A and some of its cellular regulators.

NTPDASE4 Gene Products Cooperate with the Adenovirus E4orf4 Protein through PP2A-Dependent and -Independent Mechanisms and Contribute to Induction of Cell Death

ABSTRACT The adenovirus E4orf4 protein induces nonclassical apoptosis in mammalian cells through at least two complementing pathways regulated by the interactions of E4orf4 with protein phosphatase 2A (PP2A) and Src kinases. In Saccharomyces cerevisiae cells, which do not express Src, E4orf4 induces PP2A-dependent toxicity. The yeast Golgi apyrase Ynd1 was found to contribute to E4orf4-mediated toxicity and to interact with the PP2A-B55α regulatory subunit. In addition, a mammalian Ynd1 orthologue, the NTPDASE4 gene product Golgi UDPase, was shown to physically interact with E4orf4. Here we report that knockdown of NTPDASE4 suppressed E4orf4-induced cell death. Conversely, overexpression of the NTPDASE4 gene products Golgi UDPase and LALP70 enhanced E4orf4-induced cell killing. We found that similarly to results obtained in yeast, the apyrase activity of mammalian UDPase was not required for its contribution to E4orf4-induced toxicity. The interaction between E4orf4 and UDPase had two consequences: a PP2A-dependent one, resulting in increased UDPase levels, and a PP2A-independent outcome that led to dissociation of large UDPase-containing protein complexes. The present report extends our findings in yeast to E4orf4-mediated death of mammalian cells, and combined with previous results, it suggests that the E4orf4-NTPDase4 pathway, partly in association with PP2A, may provide an alternative mechanism for the E4orf4-Src pathway to contribute to the cytoplasmic death function of E4orf4. IMPORTANCE The adenovirus E4orf4 protein contributes to regulation of the progression of virus infection from the early to the late phase, and when expressed alone, it induces a unique caspase-independent programmed cell death which is more efficient in cancer cells than in normal cells. The interactions of E4orf4 with cellular proteins that mediate its functions, such as PP2A and Src kinases, are highly conserved in evolution. The results presented here reveal that the NTPDASE4 gene product Golgi UDPase, first discovered to contribute to E4orf4 toxicity in Saccharomyces cerevisiae, associates with E4orf4 and plays a role in induction of cell death in mammalian cells. Details of the functional interaction between E4orf4, PP2A, and the UDPase are described. Identification of the evolutionarily conserved mechanisms underlying E4orf4 activity will increase our understanding of the interactions between the virus and the host cell and will contribute to our grasp of the unique mode of E4orf4-induced cell death.

The adenovirus E4orf4 protein induces a unique mode of cell death while inhibiting classical apoptosis

The adenovirus E4 open reading frame 4 (E4orf4) protein is a multifunctional viral regulator that contributes to temporal regulation of the progression of viral infection. When expressed outside the context of the virus, E4orf4 induces p53-independent cell death in transformed cells. Oncogenic transformation of primary cells in tissue culture sensitizes them to cell killing by E4orf4,1 indicating that E4orf4 research may have implications for cancer therapy. It has been further reported that E4orf4 induces a caspase-independent, non-classical apoptotic pathway that maintains crosstalk with classical caspase-dependent pathways.2,3 An investigation into the mechanisms involved in E4orf4-induced cell death revealed that E4orf4 interacts with the heterotrimeric protein phosphatase 2A (PP2A) through direct association with its regulatory B subunits, and the interaction mediated by the PP2A Bα/B55 subunit is required for inducing cell death.1 Furthermore, E4orf4 recruits PP2A to a new substrate, the ACF chromatin remodeling factor, which contributes to E4orf4 functions.4 E4orf4 has also been reported to associate with members of the Src kinase family, leading to its Tyr phosphorylation and to deregulation of Src signaling, resulting in enhanced cell death.2 We showed previously that the interaction between E4orf4 and PP2A and its toxic consequences were conserved from yeast to mammals,5 indicating a high degree of evolutionary conservation of the underlying mechanisms. This finding suggested the feasibility of using various model organisms for studying E4orf4-induced cell death. Indeed, our work in yeast revealed a novel E4orf4 partner, Ynd1, a Golgi UDPase that contributes to E4orf4 toxicity.6

The Cytosolic Tail of the Golgi Apyrase Ynd1 Mediates E4orf4-Induced Toxicity in Saccharomyces cerevisiae

The adenovirus E4 open reading frame 4 (E4orf4) protein contributes to regulation of the progression of virus infection. When expressed individually, E4orf4 was shown to induce non-classical transformed cell-specific apoptosis in mammalian cells. At least some of the mechanisms underlying E4orf4-induced toxicity are conserved from yeast to mammals, including the requirement for an interaction of E4orf4 with protein phosphatase 2A (PP2A). A genetic screen in yeast revealed that the Golgi apyrase Ynd1 associates with E4orf4 and contributes to E4orf4-induced toxicity, independently of Ynd1 apyrase activity. Ynd1 and PP2A were shown to contribute additively to E4orf4-induced toxicity in yeast, and to interact genetically and physically. A mammalian orthologue of Ynd1 was shown to bind E4orf4 in mammalian cells, confirming the evolutionary conservation of this interaction. Here, we use mutation analysis to identify the cytosolic tail of Ynd1 as the protein domain required for mediation of the E4orf4 toxic signal and for the interaction with E4orf4. We also show that E4orf4 associates with cellular membranes in yeast and is localized at their cytoplasmic face. However, E4orf4 is membrane-associated even in the absence of Ynd1, suggesting that additional membrane proteins may mediate E4orf4 localization. Based on our results and on a previous report describing a collection of Ynd1 protein partners, we propose that the Ynd1 cytoplasmic tail acts as a scaffold, interacting with a multi-protein complex, whose targeting by E4orf4 leads to cell death.

The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response

The DNA damage response (DDR) is a conglomerate of pathways designed to detect DNA damage and signal its presence to cell cycle checkpoints and to the repair machinery, allowing the cell to pause and mend the damage, or if the damage is too severe, to trigger apoptosis or senescence. Various DDR branches are regulated by kinases of the phosphatidylinositol 3-kinase-like protein kinase family, including ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). Replication intermediates and linear double-stranded genomes of DNA viruses are perceived by the cell as DNA damage and activate the DDR. If allowed to operate, the DDR will stimulate ligation of viral genomes and will inhibit virus replication. To prevent this outcome, many DNA viruses evolved ways to limit the DDR. As part of its attack on the DDR, adenovirus utilizes various viral proteins to cause degradation of DDR proteins and to sequester the MRN damage sensor outside virus replication centers. Here we show that adenovirus evolved yet another novel mechanism to inhibit the DDR. The E4orf4 protein, together with its cellular partner PP2A, reduces phosphorylation of ATM and ATR substrates in virus-infected cells and in cells treated with DNA damaging drugs, and causes accumulation of damaged DNA in the drug-treated cells. ATM and ATR are not mutually required for inhibition of their signaling pathways by E4orf4. ATM and ATR deficiency as well as E4orf4 expression enhance infection efficiency. Furthermore, E4orf4, previously reported to induce cancer-specific cell death when expressed alone, sensitizes cells to killing by sub-lethal concentrations of DNA damaging drugs, likely because it inhibits DNA damage repair. These findings provide one explanation for the cancer-specificity of E4orf4-induced cell death as many cancers have DDR deficiencies leading to increased reliance on the remaining intact DDR pathways and to enhanced susceptibility to DDR inhibitors such as E4orf4. Thus DDR inhibition by E4orf4 contributes both to the efficiency of adenovirus replication and to the ability of E4orf4 to kill cancer cells.

Induction of Cancer-Specific Cell Death by the Adenovirus E4orf4 Protein

The adenovirus E4orf4 protein is a multifunctional viral regulator that contributes to temporal regulation of the progression of viral infection. When expressed alone, outside the context of the virus, E4orf4 induces p53-independent cell-death in transformed cells. Oncogenic transformation of primary cells in tissue culture sensitizes them to cell killing by E4orf4, indicating that E4orf4 research may have implications for cancer therapy. It has also been reported that E4orf4 induces a caspase-independent, non-classical apoptotic pathway, which maintains crosstalk with classical caspase-dependent pathways. Furthermore, several E4orf4 activities in the nucleus and in the cytoplasm and various protein partners contribute to cell killing by this viral protein. In the following chapter I summarize the current knowledge of the unique mode of E4orf4-induced cell death and its underlying mechanisms. Although several explanations for the cancer-specificity of E4orf4-induced toxicity have been proposed, a better grasp of the mechanisms responsible for E4orf4-induced cell death is required to elucidate the differential sensitivity of normal and cancer cells to E4orf4.