The human adenovirus PI3K-Akt activator E4orf1 is targeted by the tumor suppressor p53.

Human adenoviruses (HAdV) are classified as DNA tumor viruses due to their potential to mediate oncogenic transformation in non-permissive mammalian cells and certain human stem cells. To achieve transformation, the viral early proteins of the E1 and E4 regions must block apoptosis and activate proliferation: the former predominantly through modulating the cellular tumor suppressor p53 and the latter by activating cellular pro-survival and pro-metabolism protein cascades, such as the phosphoinositide 3-kinase (PI3K-Akt) pathway, which is activated by HAdV E4orf1. Focusing on HAdV-C5, we show that E4orf1 is necessary and sufficient to stimulate Akt activation through phosphorylation in H1299 cells, which is not only hindered but repressed during HAdV-C5 infection with a loss of E4orf1 function in p53-positive A549 cells. Contrary to other research, E4orf1 localized not only in the common, cytoplasmic PI3K-Akt-containing compartment, but also in distinct nuclear aggregates. We identified a novel inhibitory mechanism, where p53 selectively targeted E4orf1 to destabilize it, also stalling E4orf1-dependent Akt phosphorylation. Co-IP and immunofluorescence studies showed that p53 and E4orf1 interact, and since p53 is bound by the HAdV-C5 E3 ubiquitin ligase complex, we also identified E4orf1 as a novel factor interacting with E1B-55K and E4orf6 during infection; overexpression of E4orf1 led to less-efficient E3 ubiquitin ligase-mediated proteasomal degradation of p53. We hypothesize that p53 specifically subverts the pro-survival function of E4orf1-mediated PI3K-Akt activation to protect the cell from metabolic hyper-activation or even transformation.IMPORTANCEHuman adenoviruses (HAdV) are nearly ubiquitous pathogens comprising numerous subtypes that infect various tissues and organs. Among many encoded proteins that facilitate viral replication and subversion of host cellular processes, the viral E4orf1 protein has emerged as an intriguing yet under-investigated player in the complex interplay between the virus and its host. Nonetheless, E4orf1 has gained attention as a metabolism activator and oncogenic agent, while recent research is showing that E4orf1 may play a more important role in modulating the cellular pathways such as phosphoinositide 3-kinase-Akt-mTOR. Our study reveals a novel and general impact of E4orf1 on host mechanisms, providing a novel basis for innovative antiviral strategies in future therapeutic settings. Ongoing investigations of the cellular pathways modulated by HAdV are of great interest, particularly since adenovirus-based vectors actually serve as vaccine or gene vectors. HAdV constitute an ideal model system to analyze the underlying molecular principles of virus-induced tumorigenesis.

Identification of Adenovirus E1B-55K Interaction Partners through a Common Binding Motif.

The adenovirus C5 E1B-55K protein is crucial for viral replication and is expressed early during infection. It can interact with E4orf6 to form a complex that functions as a ubiquitin E3 ligase. This complex targets specific cellular proteins and marks them for ubiquitination and, predominantly, subsequent proteasomal degradation. E1B-55K interacts with various proteins, with p53 being the most extensively studied, although identifying binding sites has been challenging. To explain the diverse range of proteins associated with E1B-55K, we hypothesized that other binding partners might recognize the simple p53 binding motif (xWxxxPx). In silico analyses showed that many known E1B-55K binding proteins possess this amino acid sequence; therefore, we investigated whether other xWxxxPx-containing proteins also bind to E1B-55K. Our findings revealed that many cellular proteins, including ATR, CHK1, USP9, and USP34, co-immunoprecipitate with E1B-55K. During adenovirus infection, several well-characterized E1B-55K binding proteins and newly identified interactors, including CSB, CHK1, and USP9, are degraded in a cullin-dependent manner. Notably, certain binding proteins, such as ATR and USP34, remain undegraded during infection. Structural predictions indicate no conservation of structure around the proposed binding motif, suggesting that the interaction relies on the correct arrangement of tryptophan and proline residues.

Mouse Adenovirus Type 1 E4orf6 Induces PKR Degradation.

Protein kinase R (PKR) is a cellular kinase involved in the antiviral response. The inactivation or inhibition of this protein is a conserved activity in DNA and RNA virus infections. In contrast to human adenovirus type 5, mouse adenovirus type 1 (MAV-1) inhibits PKR activity through proteasome-dependent degradation. However, the molecular mechanism by which this process takes place is not fully understood. We investigated whether ubiquitination, MAV-1 early region 1B 55k (E1B 55k), and early region 4 orf6 (E4orf6) play a role in PKR degradation in MAV-1 infection, because the enzyme 3 (E3) ubiquitin ligase activity with these viral proteins is conserved among the Adenoviridae family. We provide evidence that E4orf6 is sufficient to induce mouse PKR degradation and that proteasome pathway inhibition blocks PKR degradation. Inhibition of neddylation of cullin, a component of E3 ubiquitin ligase complex, blocked efficient PKR degradation in MAV-1-infected cells. Finally, we demonstrated that MAV-1 degradation of PKR is specific for mouse PKR. These results indicate that counteracting PKR is mechanistically different in two species of adenoviruses. IMPORTANCE Viruses have evolved to counteract the immune system to successfully replicate in the host. Downregulation of several antiviral proteins is important for productive viral infection. Protein kinase R (PKR) is an antiviral protein that belongs to the first line of defense of the host. Because PKR senses dsRNA and blocks the cellular translation process during viral infections, it is not surprising that many viruses counteract this antiviral activity. We previously reported PKR degradation during mouse adenovirus type 1 (MAV-1) infection; however, the molecular mechanism of this activity was not fully known. This work provides evidence about the MAV-1 protein that induces PKR degradation and expands knowledge about involvement of the proteasome pathway.

Disruption of NBS1/MRN Complex Formation by E4orf3 Supports NF-κB That Licenses E1B55K-Deleted Adenovirus-Infected Cells to Accumulate DNA>4n.

Cells increase their DNA content greater than the G2/M (DNA > 4n) phases along the path to cancer. The signals that support this increase in DNA content remain poorly understood. Cells infected with adenovirus (Ad) similarly develop DNA > 4n and share a need to bypass the DNA damage response (DDR) signals that trigger cell cycle arrest, and/or cell death. Ads with deletion in early region 1B55K (ΔE1B Ad) are oncolytic agents that are currently being explored for use in vaccine delivery. Interestingly, they promote higher levels of DNA > 4n than Ads that contain E1B55K. Existing in these and almost all Ads that are being explored for clinical use, is early region 4 (E4). The Ad E4 open reading frame 3 (E4orf3) is a viral oncogene that interferes with the ability of cells to respond to DNA damage by disrupting MRN complex formation. Our study reveals that E4orf3 is required for the enhanced fraction of ΔE1B Ad-infected cells with DNA > 4n. For that reason, we explored signaling events mediated by E4orf3. We found that in ΔE1B Ad-infected cells, E4orf3, as reported by others, isolates NBS1 in nuclear dots and tracks. This allows for elevated levels of phosphorylated ATM that is linked to transcriptionally active NF-κB. Pharmacological inhibition of NF-κB reduced the fraction of ΔE1B Ad-infected cells with DNA > 4n while pharmacological inhibition of ATM reduced the levels of nuclear NF-κB and the fraction of ΔE1B Ad-infected cells with DNA > 4n and increased the fraction of dead or dying cells with fragmented DNA. This ability of E4orf3 to disrupt MRN complex formation that allows cells to bypass the cell cycle, evade death, and accumulate DNA > 4n, may be linked to its oncogenic potential. IMPORTANCE Genome instability, a hallmark of cancer, exists as part of a cycle that leads to DNA damage and DNA > 4n that further enhances genome instability. Ad E4orf3 is a viral oncogene. Here, we describe E4orf3 mediated signaling events that support DNA > 4n in ΔE1B Ad-infected cells. These signaling events may be linked to the oncogenic potential of E4orf3 and may provide a basis for how some cells survive with DNA > 4n.

E4orf1-induced reduction in endogenous insulin level is independent of pancreas endocrine function.

BACKGROUND: Obesity is often associated with hyperinsulinemia due to insulin resistance. In mice models of hyperinsulinemia, adenovirus-derived E4orf1 protein promotes glucose disposal via insulin-independent pathway, and reduces insulin response to glucose load, described as its "Insulin Sparing Action". This is likely because less insulin is needed for disposing glucose in presence of E4orf1, however, there are other potential possibilities. This study determined if E4orf1 reduces insulin response to glucose load because it a) suppresses the ability of pancreatic ß-cells to secret insulin, or b) upregulates glucagon production by the pancreas. METHODS: C57BL/6J wild type (control) and transgenic C57BL/6J (E4orf1) mice that express E4orf1 protein in adipose tissue upon doxycycline feeding, were used. Post-doxycycline feeding, insulin and glucagon secretion in response to glibenclamide or phenylephrine were compared between the two groups. The pancreases were examined for histological changes. RESULTS: In response to glibenclamide, E4orf1 mice secreted more insulin and exhibited lower blood glucose compared to control (47.4 ± 4.4 vs 27.4 ± 3.7 mg/dl, p < 0.003), but showed no difference in glucagon secretion. Post-phenylephrine injection, no differences were observed between the two groups for glucagon or insulin, except E4orf1 mice had a lower blood glucose rise after 10-min of injection compared to the control (39.7 ± 4.7 vs. 58.3 ± 7.5 mg/dl, p < 0.05). E4orf1 mice had significantly larger pancreatic islets and higher number of islets per mm2 tissue area. Neither the size nor the number of islets met the criteria of hypertrophy or hyperplasia. CONCLUSIONS/INTERPRETATION: E4orf1 retains and may enhance the ability of the pancreases to secret insulin in response to insulin secretagogue. Glucagon does not seem to play a role in the Insulin Sparing Action of E4orf1. Overall, the histology studies support better pancreatic islet health in presence of E4orf1, compared to that in control mice. The "insulin-independent" role of E4orf1 has potential therapeutic implications in addressing hyperinsulinemia in obesity.

Enhanced oncolytic activity of E4orf6-deficient adenovirus by facilitating nuclear export of HuR.

An AU-rich element (ARE) is RNA element that enhances the rapid decay of mRNA. The RNA binding protein HuR stabilizes ARE-mRNA by exporting it to the cytoplasm. In most of cancer cells, HuR is exported to the cytoplasm and ARE-mRNA is stabilized. In addition, the viral gene product E4orf6 exports HuR to stabilize ARE-mRNA in adenovirus-infected cells and the stabilization is required for full virus replication. Previously we showed the oncolytic activity of E4orf6-deleted adenovirus dl355, which can replicate in cancer cells where ARE-mRNA is stabilized. In this study, we examined whether the further enhancement of HuR export can stimulate the replication and the oncolytic activity of dl355. We found that ethanol treatment promoted the cytoplasmic relocalization of HuR in cancer cells. In addition, the replication efficiency of dl355 increased in ethanol-treated cells, and in response, the cytolytic activity of the virus also increased in vitro and in vivo. Upregulation of a cleaved-PARP level in infected cells mediated by ethanol is suggesting that ethanol activated the apoptosis induced by dl355. IVa2 mRNA, the only ARE-mRNA among transcripts of adenovirus was augmented by ethanol treatment. These data indicate that the enhancement of ARE-mRNA stabilization as a result of ethanol treatment upregulates the oncolytic activity of dl355 and suggests that the combined use of an oncolytic adenovirus and ethanol treatment may be a good strategy for cancer therapy.

Reducing endogenous insulin is linked with protection against hepatic steatosis in mice.

BACKGROUND: Obesity and type 2 diabetes (T2D) are closely associated with hepatic steatosis (HS), which if untreated can advance to serious liver conditions. Since insulin promotes hepatic lipogenesis, reducing hyperinsulinemia may help in treating HS. E4orf1 is an adenovirus-derived protein that improves glucose clearance independent of insulin, lowers insulin amount required for glucose disposal, and reduces HS. As a next step, we evaluated the mechanism for E4orf1-induced reduction in HS and tested that E4orf1 does not induce hypoglycemia, an important attribute for its application as a potential anti-diabetic agent. METHODS: C57Bl/6J mice that transgenically express E4orf1 in adipose tissue (E4orf-Tg) and wild-type (WT) mice received a chow diet for 6 weeks, followed by a high-fat (HF) diet for additional 10 weeks. Body composition, blood glucose, and serum insulin levels upon glucose load were measured at 0, 6, 7, and 16 weeks. Serum free fatty acid (FFA), triglyceride (TG), and hepatic TG were measured at study termination. We compared histology and the mRNA/protein markers of hepatic and adipose tissue lipid metabolism between the two groups of mice. RESULTS: On chow diet, both groups remained normoglycemic, but E4orf1 expression reduced insulin response. On HF diet, glycemic control in WT deteriorated, whereas E4orf1 significantly enhanced glycemic control, lowered insulin response, reduced hepatic triglycerides, and serum FFA. Overall, a comparison of hepatic mRNA and/or protein expression suggested that E4orf1 expression significantly decreased de novo lipogenesis (DNL) and intracellular lipid transport and increased fat oxidation and TG export. Adipose tissue mRNA and protein markers suggested that E4orf1 expression lowered DNL and increased lipolysis. CONCLUSION: Considering that E4orf1 is not secreted in circulation, we postulate that reduced endogenous insulin in E4orf1 mice indirectly contributes to reduce HS by altering hepatic lipid metabolism, including lipogenesis. This study underscores the possibility of indirectly impacting HS by manipulating adipose tissue metabolism.

Cell transformation by the adenovirus oncogenes E1 and E4.

Extensive studies on viral-mediated oncogenic transformation by human adenoviruses have revealed much of our current understanding on the molecular mechanisms that are involved in the process. To date, these studies have shown that cell transformation is a multistep process regulated by the cooperation of several adenoviral gene products encoded in the early regions 1 (E1) and 4 (E4). Early region 1A immortalizes primary rodent cells, whereas co-expression of early region protein 1B induces full manifestation of the transformed phenotype. Beside E1 proteins, also some E4 proteins have partial transforming activities through regulating many cellular pathways. Here, we summarize recent data of how adenoviral oncoproteins may contribute to viral transformation and discuss the challenge of pinpointing the underlying mechanisms.

Epigenetics and the dynamics of chromatin during adenovirus infections.

The DNA genome of eukaryotic cells is compacted by histone proteins within the nucleus to form chromatin. Nuclear-replicating viruses such as adenovirus have evolved mechanisms of chromatin manipulation to promote infection and subvert host defenses. Epigenetic factors may also regulate persistent adenovirus infection and reactivation in lymphoid tissues. In this review, we discuss the viral proteins E1A and protein VII that interact with and alter host chromatin, as well as E4orf3, which separates host chromatin from sites of viral replication. We also highlight recent advances in chromatin technologies that offer new insights into virus-directed chromatin manipulation. Beyond the role of chromatin in the viral replication cycle, we discuss the nature of persistent viral genomes in lymphoid tissue and cell lines, and the potential contribution of epigenetic signals in maintaining adenovirus in a quiescent state. By understanding the mechanisms through which adenovirus manipulates host chromatin, we will understand new aspects of this ubiquitous virus and shed light on previously unknown aspects of chromatin biology.

Adenovirus oncoprotein E4orf6 triggers Cullin5 neddylation to activate the CLR5 E3 ligase for p53 degradation.

The human adenovirus oncoprotein E4orf6 hijacks intracellular Cullin 5-based E3 ubiquitin ligases (CRL5s) to induce the degradation of host proteins, including p53, that impede efficient viral replication. The complex also relies on another viral protein, E1B55K, to recruit substrates for ubiquitination. However, the determinants of adenoviral E4orf6-CRL5 E3 ligase-mediated p53 degradation in the scaffolding protein Cullin5 remain rarely investigated. Here, we demonstrated that the viral protein E4orf6 triggered relocalization of the Cullin5 protein from the cytoplasm to the nucleus and induced activation of the CRL5 E3 ligase via facilitating neddylation. The expression of the deneddylase SENP8/Den1 was significantly downregulated by E4orf6. We then identified SENP8 as a natural restriction factor for E4orf6-induced p53 degradation. Furthermore, our results indicated that the NEDD8-conjugating E2 enzyme UBE2M was essential for E4orf6-mediated p53 degradation and that its dominant negative mutant UBE2M C111S dramatically blocked E4orf6 functions. The Nedd8-activating enzyme inhibitor MLN4924 decreased E4orf6-induced neddylation of the cullin5 protein and subsequently suppressed p53 degradation. Collectively, our findings illuminate the strategy by which this viral oncoprotein specifically utilizes the neddylation pathway to activate host CRL E3 ligases to degrade host restriction factors. Disrupting this post-translational modification is an attractive pharmacological intervention against human adenoviruses.

E4orf1 protein reduces the need for endogenous insulin.

BACKGROUND: E4orf1 protein derived from adenovirus-36 reduces glucose excursion in mice, and lowers endogenous insulin response, suggesting a reduced need for insulin. We tested if the E4orf1-mediated lowering of insulin response is due to increased tissue sensitivity to insulin, reduced ability to produce or release insulin, or a reduced need for insulin release. METHODS: Experiment 1: hyperinsulinemic-euglycemic clamps (HEC) and glucose tolerance test (GTT) were performed in high fat fed transgenic mice expressing E4orf1 or non-transgenic littermates (n = 12 each), for 4 weeks. Experiments 2, 3, and 4: E4orf1 or null vectors were expressed in rat-pancreatic ß-cell line (INS-1) for 72 h, and cells were exposed to varying levels of glucose. Cell lysates and media were collected. Experiment 5: 3T3L1-preadipocytes that express E4orf1 upon doxycycline induction, or null vector were induced with doxycycline and then exposed to protein transport inhibitor. Supernatant and cell lysate were collected. Experiment 6: 3T3L1-preadipocytes that express E4orf1 upon doxycycline induction, or null vector were co-cultured with INS-1 cells for 24 h. Media was collected. RESULTS: Experiment 1: E4orf1 transgenic mice cleared glucose faster compared to non-transgenic mice during GTT. HEC showed that E4orf1 did not alter tissue sensitivity to exogenous insulin in mice. Experiments 2, 3, and 4: in INS1 cells, E4orf1 did not alter Glut2 abundance or Akt activation, suggesting no reduction in glucose sensing or insulin synthesis, respectively. E4orf1 did not influence glucose-stimulated insulin secretion in media by INS1 cells. Experiment 5: E4orf1 was present in cell lysate, but not in media, indicating it is not a secretory protein. Experiment 6: INS1 cells released less insulin in media when co-cultured in the presence of E4orf1-expressing 3T3-L1 cells. CONCLUSIONS: Our studies support the working hypothesis that the E4orf1-mediated lowering of insulin response is not due to increased tissue sensitivity to insulin, or reduced ability to produce or release insulin, but likely to be due to a reduced need for insulin release.

Biphasic Functional Interaction between the Adenovirus E4orf4 Protein and DNA-PK.

The adenovirus (Ad) E4orf4 protein contributes to virus-induced inhibition of the DNA damage response (DDR) by reducing ATM and ATR signaling. Consequently, E4orf4 inhibits DNA repair and sensitizes transformed cells to killing by DNA-damaging drugs. Inhibition of ATM and ATR signaling contributes to the efficiency of virus replication and may provide one explanation for the cancer selectivity of cell death induced by the expression of E4orf4 alone. In this report, we investigate a direct interaction of E4orf4 with the DDR. We show that E4orf4 physically associates with the DNA-dependent protein kinase (DNA-PK), and we demonstrate a biphasic functional interaction between these proteins, wherein DNA-PK is required for ATM and ATR inhibition by E4orf4 earlier during infection but is inhibited by E4orf4 as infection progresses. This biphasic process is accompanied by initial augmentation and a later inhibition of DNA-PK autophosphorylation as well as by colocalization of DNA-PK with early Ad replication centers and distancing of DNA-PK from late replication centers. Moreover, inhibition of DNA-PK improves Ad replication more effectively when a DNA-PK inhibitor is added later rather than earlier during infection. When expressed alone, E4orf4 is recruited to DNA damage sites in a DNA-PK-dependent manner. DNA-PK inhibition reduces the ability of E4orf4 to induce cancer cell death, likely because E4orf4 is prevented from arriving at the damage sites and from inhibiting the DDR. Our results support an important role for the E4orf4-DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.IMPORTANCE Several DNA viruses evolved mechanisms to inhibit the cellular DNA damage response (DDR), which acts as an antiviral defense system. We present a novel mechanism by which the adenovirus (Ad) E4orf4 protein inhibits the DDR. E4orf4 interacts with the DNA damage sensor DNA-PK in a biphasic manner. Early during infection, E4orf4 requires DNA-PK activity to inhibit various branches of the DDR, whereas it later inhibits DNA-PK itself. Furthermore, although both E4orf4 and DNA-PK are recruited to virus replication centers (RCs), DNA-PK is later distanced from late-phase RCs. Delayed DNA-PK inhibition greatly contributes to Ad replication efficiency. When E4orf4 is expressed alone, it is recruited to DNA damage sites. Inhibition of DNA-PK prevents both recruitment and the previously reported ability of E4orf4 to kill cancer cells. Our results support an important role for the E4orf4-DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.

Adenovirus 5 E1A Interacts with E4orf3 To Regulate Viral Chromatin Organization.

Human adenovirus expresses several early proteins that control various aspects of the viral replication program, including an orchestrated expression of viral genes. Two of the earliest viral transcriptional units activated after viral genome entry into the host cell nucleus are the E1 and E4 units, which each express a variety of proteins. Chief among these are the E1A proteins that function to reprogram the host cell and activate transcription of all other viral genes. The E4 gene encodes multiple proteins, including E4orf3, which functions to disrupt cellular antiviral defenses, including the DNA damage response pathway and activation of antiviral genes. Here we report that E1A directly interacts with E4orf3 via the conserved N terminus of E1A to regulate the expression of viral genes. We show that E4orf3 indiscriminately drives high nucleosomal density of viral genomes, which is restrictive to viral gene expression and which E1A overcomes via a direct interaction with E4orf3. We also show that during infection E1A colocalizes with E4orf3 to nuclear tracks that are associated with heterochromatin formation. The inability of E1A to interact with E4orf3 has a significant negative impact on overall viral replication, the ability of the virus to reprogram the host cell, and the levels of viral gene expression. Together these results show that E1A and E4orf3 work together to fine-tune the viral replication program during the course of infection and highlight a novel mechanism that regulates viral gene expression.IMPORTANCE To successfully replicate, human adenovirus needs to carry out a rapid yet ordered transcriptional program that executes and drives viral replication. Early in infection, the viral E1A proteins are the key activators and regulators of viral transcription. Here we report, for the first time, that E1A works together with E4orf3 to perfect the viral transcriptional program and identify a novel mechanism by which the virus can adjust viral gene expression by modifying its genome's nucleosomal organization via cooperation between E1A and E4orf3.

Mechanism of Adenovirus E4-ORF3-Mediated SUMO Modifications.

Regulation of a variety of different cellular processes, including posttranslational modifications, is critical for the ability of many viruses to replicate efficiently within host cells. The adenovirus (Ad) E4-ORF3 protein assembles into polymers and forms a unique nuclear scaffold that leads to the relocalization and sequestration of cellular proteins, including small ubiquitin-like modifiers (SUMOs). Previously, we showed that E4-ORF3 functions as a SUMO E3 ligase of transcriptional intermediary factor-1 gamma (TIF-1γ) and promotes poly-SUMO chain formation. Here, we present cellular and biochemical data to further understand E4-ORF3 SUMO ligase activity. E4-ORF3 proteins from five different Ad species were found to possess SUMO E3 ligase activities in vitro In infected cells, SUMO modifications of target proteins occurred only when the proteins were recruited into E4-ORF3 polymeric structures. By analyzing SUMO-deficient TIF-1γ, we demonstrated that SUMO conjugations are not required for E4-ORF3-mediated relocalization of target proteins in infected cells, implying that sequestration is followed by SUMO modification. In vitro SUMO conjugation assays revealed the Ad E1B-55K oncoprotein as a new viral target of E4-ORF3-mediated SUMOylation. We also verified a direct function of E4-ORF3 as a SUMO ligase for multiple cellular proteins, including transcription factor II-I (TFII-I), Nbs1, and Mre11. Moreover, we discovered that E4-ORF3 associates with SUMO-bound UBC9, and E4-ORF3 polymerization is crucial for this ternary interaction. Together, our findings characterize E4-ORF3 as a novel polymer-type SUMO E3 ligase and provide mechanistic insights into the role of E4-ORF3 in SUMO conjugation.IMPORTANCE Viruses interplay with the host SUMOylation system to manipulate diverse cellular responses. The Ad E4-ORF3 protein forms a dynamic nuclear network to interfere with and exploit different host processes, including the DNA damage and interferon responses. We previously reported that E4-ORF3 is a SUMO E3 ligase. Here, we demonstrate that this activity is a conserved function of evolutionarily diverse human Ad E4-ORF3 proteins and that E4-ORF3 functions directly to promote SUMO conjugations to multiple cellular proteins. Recruitment of cellular substrates into E4-ORF3 nuclear inclusions is required for SUMO conjugation to occur in vivo We probed the mechanism by which E4-ORF3 functions as a SUMO E3 ligase. Only multimeric, but not dimeric, E4-ORF3 binds to the SUMO E2 conjugation enzyme UBC9 in vitro only in a trimeric complex with SUMO. These results reveal a novel mechanism by which a conserved viral protein usurps the cellular SUMO conjugation machinery.

Temporal dynamics of adenovirus 5 gene expression in normal human cells.

Adenovirus executes a finely tuned transcriptional program upon infection of a cell. To better understand the temporal dynamics of the viral transcriptional program we performed highly sensitive digital PCR on samples extracted from arrested human lung fibroblasts infected with human adenovirus 5 strain dl309. We show that the first transcript made from viral genomes is the virus associated non-coding RNA, in particular we detected abundant levels of virus associated RNA II four hours after infection. Activation of E1 and E4 occurred nearly simultaneously later in infection, followed by other early genes as well as late genes. Our study determined that genomes begin to replicate between 29 and 30 hours after infection. This study provides a comprehensive view of viral mRNA steady-state kinetics in arrested human cells using digital PCR.

Localization of the kinase Ataxia Telangiectasia Mutated to Adenovirus E4 mutant DNA replication centers is important for its inhibitory effect on viral DNA accumulation.

Adenovirus (Ad) type 5 (Ad5) E4 deletion mutants including H5dl1007 (E4-) induce a DNA damage response (DDR) that activates the kinase ataxia-telangiectasia mutated (ATM), which can interfere with efficient viral DNA replication. We find that localization of active phosphorylated ATM (pATM) to E4- viral replication centers (VRCs) is important for its inhibitory effect. ATM is necessary for localization of RNF8 and 53BP1 to E4 mutant VRCs, while recruitment of DDR factors Mre11, Mdc1 and γH2AX is ATM-independent, raising the possibility that ATM may affect viral chromatin at VRCs. We assessed E4- and Ad5 chromatin organization by micrococcal nuclease (MN) digestion. A significant fraction of Ad5 DNA is somewhat resistant to MN digestion, whereas E4- DNA is more susceptible. ATM inhibition increases the fraction of E4- DNA that is resistant to MN digestion. Our results address possible mechanisms through which ATM inhibits E4- DNA replication.

Degradation of a Novel DNA Damage Response Protein, Tankyrase 1 Binding Protein 1, following Adenovirus Infection.

Infection by most DNA viruses activates a cellular DNA damage response (DDR), which may be to the detriment or advantage of the virus. In the case of adenoviruses, they neutralize antiviral effects of DDR activation by targeting a number of proteins for rapid proteasome-mediated degradation. We have now identified a novel DDR protein, tankyrase 1 binding protein 1 (TNKS1BP1) (also known as Tab182), which is degraded during infection by adenovirus serotype 5 and adenovirus serotype 12. In both cases, degradation requires the action of the early region 1B55K (E1B55K) and early region 4 open reading frame 6 (E4orf6) viral proteins and is mediated through the proteasome by the action of cullin-based cellular E3 ligases. The degradation of Tab182 appears to be serotype specific, as the protein remains relatively stable following infection with adenovirus serotypes 4, 7, 9, and 11. We have gone on to confirm that Tab182 is an integral component of the CNOT complex, which has transcriptional regulatory, deadenylation, and E3 ligase activities. The levels of at least 2 other members of the complex (CNOT3 and CNOT7) are also reduced during adenovirus infection, whereas the levels of CNOT4 and CNOT1 remain stable. The depletion of Tab182 with small interfering RNA (siRNA) enhances the expression of early region 1A proteins (E1As) to a limited extent during adenovirus infection, but the depletion of CNOT1 is particularly advantageous to the virus and results in a marked increase in the expression of adenovirus early proteins. In addition, the depletion of Tab182 and CNOT1 results in a limited increase in the viral DNA level during infection. We conclude that the cellular CNOT complex is a previously unidentified major target for adenoviruses during infection.IMPORTANCE Adenoviruses target a number of cellular proteins involved in the DNA damage response for rapid degradation. We have now shown that Tab182, which we have confirmed to be an integral component of the mammalian CNOT complex, is degraded following infection by adenovirus serotypes 5 and 12. This requires the viral E1B55K and E4orf6 proteins and is mediated by cullin-based E3 ligases and the proteasome. In addition to Tab182, the levels of other CNOT proteins are also reduced during adenovirus infection. Thus, CNOT3 and CNOT7, for example, are degraded, whereas CNOT4 and CNOT1 are not. The siRNA-mediated depletion of components of the complex enhances the expression of adenovirus early proteins and increases the concentration of viral DNA produced during infection. This study highlights a novel protein complex, CNOT, which is targeted for adenovirus-mediated protein degradation. To our knowledge, this is the first time that the CNOT complex has been identified as an adenoviral target.

Adenoviral E4 34K protein interacts with virus packaging components and may serve as the putative portal.

Studies on dsDNA bacteriophages have revealed that a DNA packaging complex assembles at a special vertex called the 'portal vertex' and consists of a portal, a DNA packaging ATPase and other components. AdV protein IVa2 is presumed to function as a DNA packaging ATPase. However, a protein that functions as a portal is not yet identified in AdVs. To identify the AdV portal, we performed secondary structure analysis on a set of AdV proteins and compared them with the clip region of the portal proteins of bacteriophages phi29, SPP1 and T4. Our analysis revealed that the E4 34K protein of HAdV-C5 contains a region of strong similarity with the clip region of the known portal proteins. E4 34K was found to be present in empty as well as mature AdV particles. In addition, E4 34K co-immunoprecipitates and colocalizes with AdV packaging proteins. Immunogold electron microscopy demonstrated that E4 34K is located at a single site on the virus surface. Finally, tertiary structure prediction of E4 34K and its comparison with that of single subunits of Phi29, SPP1 and T4 portal proteins revealed remarkable similarity. In conclusion, our results suggest that E4 34K is the putative AdV portal protein.

Insulin-sparing and fungible effects of E4orf1 combined with an adipocyte-targeting sequence in mouse models of type 1 and type 2 diabetes.

Obesity impairs glycemic control and causes insulin resistance and type 2 diabetes. Adenovirus 36 (Ad36) infection can increase the uptake of excess glucose from blood into adipocytes by increasing GLUT4 translocation through the Ras-Akt signaling pathway, which bypasses PI3K-Akt-mediated insulin receptor signaling. E4orf1, a viral gene expressed early during Ad36 infection, is responsible for this insulin-sparing effect and may be an alternative target for improving insulin resistance. To deliver the gene to adipocytes only, we connected the adipocyte-targeting sequence (ATS) to the 5' end of E4orf1 (ATS-E4orf1). In vitro transfection of ATS-E4orf1 into preadipocytes activated factors for GLUT4 translocation and adipogenesis to the same extent as did Hemagglutinin (HA)-E4orf1 transfection as positive reference. Moreover, the Transwell migration assay also showed that ATS-E4orf1 secreted by liver cells activated Akt in preadipocytes. We used a hydrodynamic gene delivery technique to deliver ATS-E4orf1 into high-fat diet-fed and streptozotocin-injected mice (disease models of type 2 and type 1 diabetes, respectively). ATS-E4orf1 improved the ability to eliminate excess glucose from the blood and ameliorated liver function in both disease models. These findings suggest that ATS-E4orf1 has insulin-sparing and fungible effects in type 2 and 1 diabetes independent of the presence of insulin.