E4orf4 Protein Research Articles

Identification of the Adenovirus E4orf4 Protein Binding Site on the B55α and Cdc55 Regulatory Subunits of PP2A: Implications for PP2A Function, Tumor Cell Killing and Viral Replication

Adenovirus E4orf4 protein induces the death of human cancer cells and Saccharomyces cerevisiae. Binding of E4orf4 to the B/B55/Cdc55 regulatory subunit of protein phosphatase 2A (PP2A) is required, and such binding inhibits PP2AB55 activity leading to dose-dependent cell death. We found that E4orf4 binds across the putative substrate binding groove predicted from the crystal structure of B55α such that the substrate p107 can no longer interact with PP2AB55α. We propose that E4orf4 inhibits PP2AB55 activity by preventing access of substrates and that at high E4orf4 levels this inhibition results in cell death through the failure to dephosphorylate substrates required for cell cycle progression. However, E4orf4 is expressed at much lower and less toxic levels during a normal adenovirus infection. We suggest that in this context E4orf4 largely serves to recruit novel substrates such as ASF/SF2/SRSF1 to PP2AB55 to enhance adenovirus replication. Thus E4orf4 toxicity probably represents an artifact of overexpression and does not reflect the evolutionary function of this viral product.

Adenovirus E4orf4 Protein-Induced Death of p53 −/− H1299 Human Cancer Cells Follows a G 1 Arrest of both Tetraploid and Diploid Cells due to a Failure To Initiate DNA Synthesis

The adenovirus E4orf4 protein selectively kills human cancer cells independently of p53 and thus represents a potentially promising tool for the development of novel antitumor therapies. Previous studies suggested that E4orf4 induces an arrest or a delay in mitosis and that both this effect and subsequent cell death rely largely on an interaction with the B55 regulatory subunit of protein phosphatase 2A. In the present report, we show that the death of human H1299 lung carcinoma cells induced by expression of E4orf4 is typified not by an accumulation of cells arrested in mitosis but rather by the presence of both tetraploid and diploid cells that are arrested in G1 because they are unable to initiate DNA synthesis. We believe that these E4orf4-expressing cells eventually die by various processes, including those resulting from mitotic catastrophe.

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

The adenovirus E4orf4 protein must bind protein phosphatase 2A (PP2A) for its functions. The E4orf4 binding site in PP2A was mapped to the α1,α2 helices of the B55α subunit. The E4orf4 binding site in PP2A-B55α lies above the substrate binding site and does not overlap it. 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.

E4orf4 induces PP2A- and Src-dependent cell death in Drosophila melanogaster and at the same time inhibits classic apoptosis pathways

The adenovirus E4orf4 protein regulates the progression of viral infection, and when expressed alone in mammalian tissue culture cells it induces protein phosphatase 2A (PP2A)-B55- and Src-dependent cell death, which is more efficient in oncogene-transformed cells than in normal cells. This form of cell death is caspase-independent, although it interacts with classic caspase-dependent apoptosis. PP2A-B55-dependent E4orf4-induced toxicity is highly conserved in evolution from yeast to mammalian cells. In this work we investigated E4orf4-induced cell death in a whole multicellular organism, Drosophila melanogaster. We show that E4orf4 induced low levels of cell killing, caused by both caspase-dependent and -independent mechanisms. Drosophila PP2A-B55 (twins/abnormal anaphase resolution) and Src64B contributed additively to this form of cell death. Our results provide insight into E4orf4-induced cell death, demonstrating that in parallel to activating caspase-dependent apoptosis, E4orf4 also inhibited this form of cell death induced by the proapoptotic genes reaper, head involution defective, and grim. The combination of both induction and inhibition of caspase-dependent cell death resulted in low levels of tissue damage that may explain the inefficient cell killing induced by E4orf4 in normal cells in tissue culture. Furthermore, E4orf4 inhibited JNK-dependent cell killing as well. However, JNK inhibition did not impede E4orf4-induced toxicity and even enhanced it, indicating that E4orf4-induced cell killing is a distinctive form of cell death that differs from both JNK- and Rpr/Hid/Grim-induced forms of cell death.

Role of E1B55K in E4orf6/E1B55K E3 Ligase Complexes Formed by Different Human Adenovirus Serotypes

The E4orf6 protein of serotypes representing all human adenovirus species forms Cullin-based E3 ubiquitin ligase complexes that facilitate virus infection by inducing degradation of cellular proteins that impede efficient viral replication. This complex also includes the viral E1B55K product believed to bind and introduce substrates for ubiquitination. Heterogeneity in the composition of these ligases exists, as some serotypes form Cul5-based complexes whereas others utilize Cul2. Significant variations in substrate specificities also exist among serotypes, as some degrade certain substrates very efficiently whereas others induce more modest or little degradation. As E1B55K is believed to function as the substrate acquisition component of the ligase, we undertook studies to compare the ability of representative E1B55K proteins to bind substrates with the efficacy of degradation by their respective E4orf6-based ligases. Interestingly, although efficient degradation in some cases corresponded to the ability of E1B55K to bind to or relocalize substrates, there were several examples of substrates that bound efficiently to E1B55K but were not degraded and others in which substrates were degraded even though binding to E1B55K was low or undetectable. These results suggest that transient interactions with E1B55K may be sufficient for efficient substrate degradation and that binding alone is not sufficient, implying that the orientation of the substrate in the ligase complex is probably crucial. Nevertheless, we found that the substrate specificity of certain E4orf6-based ligases could be altered through the formation of hybrid complexes containing E1B55K from another serotype, thus confirming identification of E1B55K as the substrate acquisition component of the complex.

Transducing properties of a pre-structured α-helical DPT-peptide containing a short canine adenovirus type 2 E4orf4 PP2A1-binding sequence

BackgroundInduction of the death pathway resulting from the specific interaction of the PP2A1 phosphatase with adenoviral E4orf4 protein is a promising approach for cancer therapy. With the aim of deregulating tumor pathways, and mimicking E4orf4 anti-cancer signal, we have previously proposed the DPT technology concept, based on design of specific PP1/PP2A interacting penetrating peptides. MethodsUsing biochemical, structural and cell survival experiments, we have characterized new DPT-peptides containing short PP2A binding sequences. ResultsWe identified overlapping sequences, located within the N-terminal domain E4orf423-46 of canine adenoviral E4orf4 protein, that interact with the PP2A-Bα subunit of PP2A1 holoenzyme. We characterized DPT-E4orf44 and TAT-E4orf44, two bi-partite cell penetrating peptides containing the 12 PP2A1 binding residues of the canine type 2 E4orf427-38 sequence, respectively fused to the DPT-sh1 and TAT shuttle sequences. Surprisingly DPT-E4orf44, in contrast to inactive TAT-E4orf44, adopted a well defined α-helical structure and co-precipitated PP2A1 from HeLa cell extracts. DPT-E4orf44 also internalized streptavidin-HRP and inhibited survival of HeLa cells more efficiently than TAT, TAT-E4orf44 or the previously published anti-tumor TAT-derived peptide shepherdin. DPT-E4orf44 also efficiently inhibited the survival of human adherent transformed cells, including wild type and p53 mutated colonic HCT116 cells, without affecting survival of human non-transformed fibroblasts. ConclusionsWe characterized the transducing properties of a new α-helical DPT-E4orf44 peptide containing a short PP2A-interacting sequence from canine Adenoviral E4orf4 protein. General significanceOur results suggest that α-helical structured DPT peptides specifically interacting with PP2A could be a valuable anti-cancer drug design scaffold.

Aggresome Formation by the Adenoviral Protein E1B55K Is Not Conserved among Adenovirus Species and Is Not Required for Efficient Degradation of Nuclear Substrates

Much of the work on the basic molecular biology of human adenoviruses has been carried out on a very limited number of the more than 60 serotypes, primarily the highly related species C viruses adenovirus type 5 (Ad5) and Ad2 and, to some extent, Ad12 of species A. Until recently, it has been widely assumed that insights obtained with these model viruses were representative of all human adenoviruses. Recent studies on the E3 ubiquitin ligase formed by the viral E1B55K and E4orf6 proteins with a cellular Cullin-based complex indicated that although all species form such a functional complex, significant variations exist in terms of complex composition and the substrates that are degraded. In the present report we conducted a comprehensive analysis of the localization of E1B55K products from representatives of six of the seven adenovirus species in the presence and the absence of the corresponding E4orf6 protein. We found that although in some species E1B55K localized in aggresomes, such was not always the case, suggesting that these structures are not necessary for the efficient degradation of substrates. In addition, differences were evident in the localization of E1B55K, although all forms readily associated with PML. Finally, Ad5 E1B55K was seen to localize in close proximity to Rab11, a marker for the endosomal recycling compartment, and both focused at the microtubule organizing center. These findings suggest that E1B55K from some species may employ the transport system utilized by the membrane recycling pathway to assemble aggresomes and the possibility that this structure might then affect recycling of cell surface components.

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways(1,2). However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time(3-5). Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein(6). Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured(7-9). The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

The Human Adenovirus Type 5 E1B 55 kDa Protein Obstructs Inhibition of Viral Replication by Type I Interferon in Normal Human Cells

Vectors derived from human adenovirus type 5, which typically lack the E1A and E1B genes, induce robust innate immune responses that limit their therapeutic efficacy. We reported previously that the E1B 55 kDa protein inhibits expression of a set of cellular genes that is highly enriched for those associated with anti-viral defense and immune responses, and includes many interferon-sensitive genes. The sensitivity of replication of E1B 55 kDa null-mutants to exogenous interferon (IFN) was therefore examined in normal human fibroblasts and respiratory epithelial cells. Yields of the mutants were reduced at least 500-fold, compared to only 5-fold, for wild-type (WT) virus replication. To investigate the mechanistic basis of such inhibition, the accumulation of viral early proteins and genomes was compared by immunoblotting and qPCR, respectively, in WT- and mutant-infected cells in the absence or presence of exogenous IFN. Both the concentration of viral genomes detected during the late phase and the numbers of viral replication centers formed were strongly reduced in IFN-treated cells in the absence of the E1B protein, despite production of similar quantities of viral replication proteins. These defects could not be attributed to degradation of entering viral genomes, induction of apoptosis, or failure to reorganize components of PML nuclear bodies. Nor was assembly of the E1B- and E4 Orf6 protein- E3 ubiquitin ligase required to prevent inhibition of viral replication by IFN. However, by using RT-PCR, the E1B 55 kDa protein was demonstrated to be a potent repressor of expression of IFN-inducible genes in IFN-treated cells. We propose that a primary function of the previously described transcriptional repression activity of the E1B 55 kDa protein is to block expression of IFN- inducible genes, and hence to facilitate formation of viral replication centers and genome replication.

Adenovirus Regulates Sumoylation of Mre11-Rad50-Nbs1 Components through a Paralog-Specific Mechanism

The Mre11-Rad50-Nbs1 (MRN) complex plays a key role in the DNA damage response, presenting challenges for DNA viruses and retroviruses. To inactivate this complex, adenovirus (Ad) makes use of the E1B-55K and E4-open reading frame 6 (ORF6) proteins for ubiquitin (Ub)-mediated, proteasome-dependent degradation of MRN and the E4-ORF3 protein for relocalization and sequestration of MRN within infected-cell nuclei. Here, we report that Mre11 is modified by the Ub-related modifier SUMO-2 and Nbs1 is modified by both SUMO-1 and SUMO-2. We found that Mre11 and Nbs1 are sumoylated during Ad5 infection and that the E4-ORF3 protein is necessary and sufficient to induce SUMO conjugation. Relocalization of Mre11 and Nbs1 into E4-ORF3 nuclear tracks is required for this modification to occur. E4-ORF3-mediated SUMO-1 conjugation to Nbs1 and SUMO-2 conjugation to Mre11 and Nbs1 are transient during wild-type Ad type 5 (Ad5) infection. In contrast, SUMO-1 conjugation to Nbs1 is stable in cells infected with E1B-55K or E4-ORF6 mutant viruses, suggesting that Ad regulates paralog-specific desumoylation of Nbs1. Inhibition of viral DNA replication blocks deconjugation of SUMO-2 from Mre11 and Nbs1, indicating that a late-phase process is involved in Mre11 and Nbs1 desumoylation. Our results provide direct evidence of Mre11 and Nbs1 sumoylation induced by the Ad5 E4-ORF3 protein and an important example showing that modification of a single substrate by both SUMO-1 and SUMO-2 is regulated through distinct mechanisms. Our findings suggest how E4-ORF3-mediated relocalization of the MRN complex influences the cellular DNA damage response.

Biophysical and Functional Analyses Suggest That Adenovirus E4-ORF3 Protein Requires Higher-order Multimerization to Function against Promyelocytic Leukemia Protein Nuclear Bodies

The early region 4 open reading frame 3 protein (E4-ORF3; UniProt ID P04489) is the most highly conserved of all adenovirus-encoded gene products at the amino acid level. A conserved attribute of the E4-ORF3 proteins of different human adenoviruses is the ability to disrupt PML nuclear bodies from their normally punctate appearance into heterogeneous filamentous structures. This E4-ORF3 activity correlates with the inhibition of PML-mediated antiviral activity. The mechanism of E4-ORF3-mediated reorganization of PML nuclear bodies is unknown. Biophysical analysis of the purified WT E4-ORF3 protein revealed an ordered secondary/tertiary structure and the ability to form heterogeneous higher-order multimers in solution. Importantly, a nonfunctional E4-ORF3 mutant protein, L103A, forms a stable dimer with WT secondary structure content. Because the L103A mutant is incapable of PML reorganization, this result suggests that higher-order multimerization of E4-ORF3 may be required for the activity of the protein. In support of this hypothesis, we demonstrate that the E4-ORF3 L103A mutant protein acts as a dominant-negative effector when coexpressed with the WT E4-ORF3 in mammalian cells. It prevents WT E4-ORF3-mediated PML track formation presumably by binding to the WT protein and inhibiting the formation of higher-order multimers. In vitro protein binding studies support this conclusion as demonstrated by copurification of coexpressed WT and L103A proteins in Escherichia coli and coimmunoprecipitation of WT·L103A E4-ORF3 complexes in mammalian cells. These results provide new insight into the properties of the Ad E4-ORF3 protein and suggest that higher-order protein multimerization is essential for E4-ORF3 activity.

Development and validation of a novel reporter assay for human papillomavirus type 16 late gene expression

To facilitate the investigations of HPV-16 late gene expression HPV-16 reporter plasmids were generated using previously described sub-genomic HPV-16 plasmids, named pBEL and pBELM, that, similar to the full viral genome, produce primarily HPV-16 early mRNAs and very little, if any, late mRNAs in cervical cancer cells. The HPV-16 late L1 gene was replaced by the chloramphenicol acetyltransferase (CAT) reporter gene, or green fluorescent protein (GFP), preceded by the poliovirus internal ribosome entry site (IRES). Results show that the reporter genes mimic the expression of L1 from these plasmids. For example, overexpression of adenovirus E4orf4 protein (E4orf4), polypyrimidine tract binding protein (PTB), arginine/serine-rich SRp30c protein (SRp30c) or alternative splicing factor/splicing factor 2 (ASF/SF2) induced an increased expression of CAT or GFP. Stable cell lines with reporter plasmids pBELCAT and pBELMCAT were also generated. An induction of CAT was observed in HPV-16 reporter cell lines in the presence of the small molecule phorbol 12-myristate 13-acetate (TPA). Further experiments identified the TPA-inducible, hnRNP A2/B1 protein as a regulator of HPV-16 late gene expression. In conclusion, the HPV-16 reporter plasmids and reporter cell lines described herein can be used to identify small molecules and cellular factors that regulate HPV-16 gene expression.

Adenovirus E4orf3 Targets Transcriptional Intermediary Factor 1γ for Proteasome-Dependent Degradation during Infection

The ability of adenovirus early region proteins, E1B-55K and E4orf6, to usurp control of cellular ubiquitin ligases and target proteins for proteasome-dependent degradation during infection is well established. Here we show that the E4 gene product, E4orf3 can, independently of E1B-55K and E4orf6, target the transcriptional corepressor transcriptional intermediary factor 1γ (TIF1γ) for proteasome-mediated degradation during infection. Initial mass spectrometric studies identified TIF1 family members-TIF1α, TIF1β, and TIF1γ-as E1B-55K-binding proteins in both transformed and infected cells, but analyses revealed that, akin to TIF1α, TIF1γ is reorganized in an E4orf3-dependent manner to promyelocytic leukemia protein-containing nuclear tracks during infection. The use of a number of different adenovirus early region mutants identified the specific and sole requirement for E4orf3 in mediating TIF1γ degradation. Further analyses revealed that TIF1γ is targeted for degradation by a number of divergent human adenoviruses, suggesting that the ability of E4orf3 to regulate TIF1γ expression is evolutionarily conserved. We also determined that E4orf3 does not utilize the Cullin-based ubiquitin ligases, CRL2 and CRL5, or the TIF1α ubiquitin ligase in order to promote TIF1γ degradation. Further studies suggested that TIF1γ possesses antiviral activity and limits adenovirus early and late gene product expression during infection. Indeed, TIF1γ knockdown accelerates the adenovirus-mediated degradation of MRE11, while TIF1γ overexpression delays the adenovirus-mediated degradation of MRE11. Taken together, these studies have identified novel adenovirus targets and have established a new role for the E4orf3 protein during infection.

Adenovirus E4-ORF3-dependent relocalization of TIF1α and TIF1γ relies on access to the Coiled-Coil motif

The adenovirus E4-ORF3 protein promotes viral replication by relocalizing cellular proteins into nuclear track structures, interfering with potential anti-viral activities. E4-ORF3 targets transcriptional intermediary factor 1 alpha (TIF1α), but not homologous TIF1β. Here, we introduce TIF1γ as a novel E4-ORF3-interacting partner. E4-ORF3 relocalizes endogenous TIF1γ in virus-infected cells in vivo and binds to TIF1γ in vitro. We used the homologous nature, yet differing binding capabilities, of these proteins to study how E4-ORF3 targets proteins for track localization. We mapped the ability of E4-ORF3 to interact with specific TIF1 subdomains, demonstrating that E4-ORF3 interacts with the Coiled-Coil domains of TIF1α, TIF1β, and TIF1γ, and that the C-terminal half of TIF1β interferes with this interaction. The results of E4-ORF3-directed TIF1 protein relocalization assays performed in vivo were verified using coimmunoprecipitation assays in vitro. These results suggest that E4-ORF3 targets proteins for relocalization through a loosely homologous sequence dependent on accessibility.

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.

The adenovirus E4orf4 protein targets PP2A to the ACF chromatin remodeling factor and controls cell death through different SNF2h‐containing complexes

The adenovirus 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 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 homologue, 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 vs. death decisions and contributes to E4orf4 functions during adenovirus infection.

Adenovirus Core Protein VII Protects the Viral Genome from a DNA Damage Response at Early Times after Infection

Adenovirus has a linear, double-stranded DNA genome that is perceived by the cellular Mre11-Rad50-Nbs1 (MRN) DNA repair complex as a double-strand break. If unabated, MRN elicits a double-strand break repair response that blocks viral DNA replication and ligates the viral genomes into concatemers. There are two sets of early viral proteins that inhibit the MRN complex. The E1B-55K/E4-ORF6 complex recruits an E3 ubiquitin ligase and targets MRN proteins for proteasome-dependent degradation. The E4-ORF3 protein inhibits MRN through sequestration. The mechanism that prevents MRN recognition of the viral genome prior to the expression of these early proteins was previously unknown. Here we show a temporal correlation between the loss of viral core protein VII from the adenovirus genome and a gain of checkpoint signaling due to the double-strand break repair response. While checkpoint signaling corresponds to the recognition of the viral genome, core protein VII binding to and checkpoint signaling at viral genomes are largely mutually exclusive. Transcription is known to release protein VII from the genome, and the inhibition of transcription shows a decrease in checkpoint signaling. Finally, we show that the nuclease activity of Mre11 is dispensable for the inhibition of viral DNA replication during a DNA damage response. These results support a model involving the protection of the incoming viral genome from checkpoint signaling by core protein VII and suggest that the induction of an MRN-dependent DNA damage response may inhibit adenovirus replication by physically masking the origins of DNA replication rather than altering their integrity.

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 E4orf6/E1B55K E3 Ubiquitin Ligase Complexes of Human Adenoviruses Exhibit Heterogeneity in Composition and Substrate Specificity

Although human adenovirus type 5 (Ad5) has been widely studied, relatively little work has been done with other human adenovirus serotypes. The Ad5 E4orf6 and E1B55K proteins form Cul5-based E3 ubiquitin ligase complexes to degrade p53, Mre11, DNA ligase IV, integrin α3, and almost certainly other targets, presumably to optimize the cellular environment for viral replication and perhaps to facilitate persistence or latency. As this complex is essential for the efficient replication of Ad5, we undertook a systematic analysis of the structure and function of corresponding E4orf6/E1B55K complexes from other serotypes to determine the importance of this E3 ligase throughout adenovirus evolution. E4orf6 and E1B55K coding sequences from serotypes representing all subgroups were cloned, and each pair was expressed and analyzed for their capacity to assemble the Cullin-based ligase complex and to degrade substrates following plasmid DNA transfection. The results indicated that all formed Cullin-based E3 ligase complexes but that heterogeneity in both structure and function existed. Whereas Cul5 was present in the complexes of some serotypes, others recruited primarily Cul2, and the Ad16 complex clearly bound both Cul2 and Cul5. There was also heterogeneity in substrate specificity. Whereas all serotypes tested appeared to degrade DNA ligase IV, complexes from some serotypes failed to degrade Mre11, p53, or integrin α3. Thus, a major evolutionary pressure for formation of the adenovirus ligase complex may lie in the degradation of DNA ligase IV; however, it seems possible that the degradation of as-yet-unidentified critical targets or, perhaps even more likely, appropriate combinations of substrates plays a central role for these adenoviruses.

Genetic Analysis of B55α/Cdc55 Protein Phosphatase 2A Subunits: Association with the Adenovirus E4orf4 Protein

The human adenovirus E4orf4 protein is toxic in both human tumor cells and Saccharomyces cerevisiae. Previous studies indicated that most of this toxicity is dependent on an interaction of E4orf4 protein with the B55 class of regulatory subunits of protein phosphatase 2A (PP2A) and in yeast with the B55 homolog Cdc55. We have found previously that E4orf4 inhibits PP2A activity against at least some substrates. In an attempt to understand the mechanism of this inhibition, we used a genetic approach to identify residues in the seven-bladed β-propeller proteins B55α and Cdc55 required for E4orf4 binding. In both cases, amino-terminal polypeptides composed only of blade 1 and at least part of blade 2 were found to bind E4orf4 and overexpression blocked E4orf4 toxicity in yeast. Furthermore, certain amino acid substitutions in blades 1 and 2 within full-length B55α and Cdc55 resulted in loss of E4orf4 binding. Recent mutational analysis has suggested that segments of blades 1 and 2 present on the top face of B55α form part of the "substrate-binding groove." Additionally, these segments are in close proximity to the catalytic C subunit of the PP2A holoenzyme. Thus, our results are consistent with the hypothesis that E4orf4 binding could affect the access of substrates, resulting in the failure to dephosphorylate some PP2A substrates.