The E3 ubiquitin ligase activity associated with the adenoviral E1B-55K-E4orf6 complex does not require CRM1-dependent export

The adenovirus type 5 (Ad5) E1B-55K and E4orf6 (E1B-55K/E4orf6) proteins are multifunctional regulators of Ad5 replication, participating in many processes required for virus growth. A complex containing the two proteins mediates the degradation of cellular proteins through assembly of an E3 ubiquitin ligase and induces shutoff of host cell protein synthesis through selective nucleocytoplasmic viral late mRNA export. Both proteins shuttle between the nuclear and cytoplasmic compartments via leucine-rich nuclear export signals (NES). However, the role of their NES-dependent export in viral replication has not been established. It was initially shown that mutations in the E4orf6 NES negatively affect viral late gene expression in transfection/infection complementation assays, suggesting that E1B-55K/E4orf6-dependent viral late mRNA export involves a CRM1 export pathway. However, a different conclusion was drawn from similar studies showing that E1B-55K/E4orf6 promote late gene expression without active CRM1 or functional NES. To evaluate the role of the E1B-55K/E4orf6 NES in viral replication in the context of Ad-infected cells and in the presence of functional CRM1, we generated virus mutants carrying amino acid exchanges in the NES of either or both proteins. Phenotypic analyses revealed that mutations in the NES of E1B-55K and/or E4orf6 had no or only moderate effects on viral DNA replication, viral late protein synthesis, or viral late mRNA export. Significantly, such mutations also did not interfere with the degradation of cellular substrates, indicating that the NES of E1B-55K or E4orf6 is dispensable both for late gene expression and for the activity associated with the E3 ubiquitin ligase.     

Adenovirus type 5 E4orf3 protein targets the Mre11 complex to cytoplasmic aggresomes

Virus infections have dramatic effects on structural and morphological characteristics of the host cell. The gene product of open reading frame 3 in the early region 4 (E4orf3) of adenovirus serotype 5 (Ad5) is involved in efficient replication and late protein synthesis. During infection with adenovirus mutants lacking the E4 region, the viral genomic DNA is joined into concatemers by cellular DNA repair factors, and this requires the Mre11/Rad50/Nbs1 complex. Concatemer formation can be prevented by the E4orf3 protein, which causes the cellular redistribution of the Mre11 complex. Here we show that E4orf3 colocalizes with components of the Mre11 complex in nuclear tracks and also in large cytoplasmic accumulations. Rearrangement of Mre11 and Rad50 by Ad5 E4orf3 is not dependent on interactions with Nbs1 or promyelocytic leukemia protein nuclear bodies. Late in infection the cytoplasmic inclusions appear as a distinct juxtanuclear accumulation at the centrosome and this requires an intact microtubule cytoskeleton. The large cytoplasmic accumulations meet the criteria defined for aggresomes, including gamma-tubulin colocalization and formation of a surrounding vimentin cage. E4orf3 also appears to alter the solubility of the cellular Mre11 complex. These data suggest that E4orf3 can target the Mre11 complex to an aggresome and may explain how the cellular repair complex is inactivated during adenovirus infection.     

Roles for the E4 orf6, orf3, and E1B 55-Kilodalton Proteins in Cell Cycle-Independent Adenovirus Replication

Adenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G(1) and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548-561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G(1) cells infected with the mutant viruses compared to that in G(1) cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G(1). However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G(1) than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G(1). Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth.     

E4orf1 Limits the Oncolytic Potential of the E1B-55K Deletion Mutant Adenovirus

Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G₁ phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G₁ restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3′-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function.Conditionally replicating adenoviruses are a novel class of biological agents used to treat cancer (57). The E1B-55K deletion mutant virus ONYX-015, originally known as dl1520 (4), is one of the first of such agents (7). H101 is another E1B-55K deletion mutant adenovirus that is being used for tumor therapy in China (30, 78). We previously reported that cells infected during the G₁ phase of the cell cycle with E1B-55K deletion mutant adenoviruses exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less effectively killed than cells infected during S phase (34, 35, 66). These observations indicated that the E1B-55K deletion mutant virus ONYX-015 is restricted in cells infected in G₁. This restriction is significant because a large fraction of cells within a tumor exist in the G₁ phase of the cell cycle (71). Here we show that the G₁ restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1).The E4orf1-encoded protein is a small adapter molecule that associates with PDZ domain-containing proteins including MUPP1, PATJ, MAGI-1, ZO-2, and Dlg1 (46). PDZ domain-containing proteins often serve as scaffolds for the assembly of signaling complexes at the plasma membrane (64). Through its association with PDZ domain-containing proteins, the E4orf1-encoded protein promotes signaling through the phosphatidylinositol 3′-kinase (PI3-kinase) pathway to effectors such as protein kinase B (Akt), the mammalian target of rapamycin (mTOR), and the S6 ribosomal protein kinase (p70 S6K) (27, 54). Through these effectors, PI3-kinase alters protein synthesis and cell survival (21, 28). E4orf1 is the principal oncogenic determinant of species D adenovirus type 9 (42). The transforming ability of E4orf1 can be blocked by the PI3-kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY249002","term_id":"1257710161","term_text":"LY249002"}}LY249002 (27). However, phosphorylation of p70 S6K can also proceed by pathways that are independent of PI3-kinase or Akt. For example, the Rho-like GTPase Rac1 can activate p70 S6K (17). Rac1 is itself regulated by cellular factors to which it binds, including the Rac1-specific guanine nucleotide exchange factor T-cell lymphoma invasion and metastasis 1 protein (Tiam1). Tiam1 and the neural tissue-associated F-actin-binding protein neurabin II or spinophilin recruit p70 S6K into a complex containing Rac1, resulting in increased phosphorylation of p70 S6K (12, 36, 50). Interestingly, both Tiam1 and neurabin II are PDZ-containing proteins. These observations provided a potential basis by which E4orf1 may modulate protein synthesis and cell survival.In this report, we show for the first time that E4orf1 restricts the abilities of the E1B-55K deletion mutant virus to produce viral progeny, to direct viral late protein synthesis, and to kill tumor cells. Drugs that are reported to prevent phosphorylation of p70 S6K or to disrupt the interaction between Tiam1 and Rac1 increase the cell-killing ability of the E1B-55K deletion mutant virus to nearly the same level observed for an E1B-55K/E4orf1 double mutant and the wild-type virus. By uncovering a role for E4orf1 in the course of a lytic adenovirus infection, this study presents novel genetic and pharmacological means by which the effectiveness of replicating oncolytic adenoviruses can be improved.     

Cytoplasmic Death Signal Triggered by Src-Mediated Phosphorylation of the Adenovirus E4orf4 Protein

In transformed cells, the adenovirus E4orf4 death factor works in part by inducing a Src-mediated cytoplasmic apoptotic signal leading to caspase-independent membrane blebbing and cell death. Here we show that Src-family kinases modulate E4orf4 phosphorylation on tyrosine residues. Mutation of tyrosines 26, 42, and 59 to phenylalanines inhibited Src-induced phosphorylation of E4orf4 in vivo and in vitro but had no effect on the molecular association of E4orf4 with Src. However, in contrast to wild-type E4orf4, the nonphosphorylatable E4orf4 mutant was unable to modulate Src-dependent phosphorylation and was deficient in recruiting a subset of tyrosine-phosphorylated proteins. Indeed, the Src substrates cortactin and p62dok were found to associate with wild-type E4orf4 but not with the nonphosphorylatable E4orf4. Importantly, the nonphosphorylatable mutant E4orf4 was preferentially distributed in the cell nucleus, was unable to induce membrane blebbing, and had a highly impaired killing activity. Conversely, an activated form of E4orf4 was obtained by mutation of tyrosine 42 to glutamic acid. This pseudophosphorylated mutant E4orf4 was enriched in the cytoplasm and plasma membrane, showed increased binding to phosphotyrosine-containing proteins, and induced a dramatic blebbing phenotype associated with increased cell death. Altogether, our findings strongly suggest that Src-mediated phosphorylation of adenovirus type 2 E4orf4 is critical to promoting its cytoplasmic and membrane localization and is required for the transduction of E4orf4-Src-dependent induction of membrane blebbing. We propose that E4orf4 acts in part by uncoupling Src-dependent signals to drive the formation of a signaling complex that triggers a cytoplasmic death signal.     

The adenovirus type 5 E1B-55K oncoprotein actively shuttles in virus-infected cells, whereas transport of E4orf6 is mediated by a CRM1-independent mechanism

The E1B-55K and E4orf6 proteins of adenovirus type 5 are involved in viral mRNA export. Here we demonstrate that adenovirus infection does not inhibit the function of the E1B-55K nuclear export signal and that E1B-55K also shuttles in infected cells. Even during virus infection, E1B-55K was exported by the leptomycin B-sensitive CRM1 pathway, whereas E4orf6 transport appeared to be mediated by an alternative mechanism. Our results strengthen the potential role of E1B-55K as the "driving force" for adenoviral late mRNA export.     

Transforming Potential of the Adenovirus Type 5 E4orf3 Protein

Previous observations that the adenovirus type 5 (Ad5) E4orf6 and E4orf3 gene products have redundant effects in viral lytic infection together with the recent findings that E4orf6 possesses transforming potential prompted us to investigate the effect of E4orf3 expression on the transformation of primary rat cells in combination with adenovirus E1 oncogene products. Our results demonstrate for the first time that E4orf3 can cooperate with adenovirus E1A and E1A plus E1B proteins to transform primary baby rat kidney cells, acting synergistically with E4orf6 in the presence of E1B gene products. Transformed rat cells expressing E4orf3 exhibit morphological alterations, higher growth rates and saturation densities, and increased tumorigenicity compared with transformants expressing E1 proteins only. Consistent with previous results for adenovirus-infected cells, the E4orf3 protein is immunologically restricted to discrete nuclear structures known as PML oncogenic domains (PODs) in transformed rat cells. As opposed to E4orf6, the ability of E4orf3 to promote oncogenic cell growth is probably not linked to a modulation of p53 functions and stability. Instead, our results indicate that the transforming activities of E4orf3 are due to combinatorial effects that involve the binding to the adenovirus 55-kDa E1B protein and the colocalization with PODs independent from interactions with the PML gene product. These data fit well with a model in which the reorganization of PODs may trigger a cascade of processes that cause uncontrolled cell proliferation and neoplastic growth. In sum, our results provide strong evidence for the idea that interactions with PODs by viral proteins are linked to oncogenic transformation.     

Control of mRNA export by adenovirus E4orf6 and E1B55K proteins during productive infection requires E4orf6 ubiquitin ligase activity

During the adenovirus infectious cycle, the early proteins E4orf6 and E1B55K are known to perform several functions. These include nuclear export of late viral mRNAs, a block of nuclear export of the bulk of cellular mRNAs, and the ubiquitin-mediated degradation of selected proteins, including p53 and Mre11. Degradation of these proteins occurs via a cellular E3 ubiquitin ligase complex that is assembled through interactions between elongins B and C and BC boxes present in E4orf6 to form a cullin 5-based ligase complex. E1B55K, which has been known for some time to associate with the E4orf6 protein, is thought to bind to specific substrate proteins to bring them to the complex for ubiquitination. Earlier studies with E4orf6 mutants indicated that the interaction between the E4orf6 and E1B55K proteins is optimal only when E4orf6 is able to form the ligase complex. These and other observations suggested that most if not all of the functions ascribed to E4orf6 and E1B55K during infection, including the control of mRNA export, are achieved through the degradation of specific substrates by the E4orf6 ubiquitin ligase activity. We have tested this hypothesis through the generation of a virus mutant in which the E4orf6 product is unable to form a ligase complex and indeed have found that this mutant behaves identically to an E4orf6⁻ virus in production of late viral proteins, growth, and export of the late viral L5 mRNA.     

Localization and Importance of the Adenovirus E4orf4 Protein during Lytic Infection

The human adenovirus type 5 (Ad5) E4orf4 product has been studied extensively although in most cases as expressed from vectors in the absence of other viral products. Thus, relatively little is known about its role in the context of an adenovirus infection. Although considerable earlier work had indicated that the E4orf4 protein is not essential for replication, a recent study using dl359, an Ad5 mutant believed to produce a nonfunctional E4orf4 protein, suggested that E4orf4 is essential for virus growth in primary small-airway epithelial cells (C. O'Shea, et al., EMBO J. 24:1211-1221, 2005). Hence, to examine further the role of E4orf4 during virus infection, we generated for the first time a set of E4orf4 virus mutants in a common Ad5 genetic background. Such mutant viruses included those that express E4orf4 proteins containing various individual point mutations, those defective entirely in E4orf4 expression, and a mutant expressing wild-type E4orf4 fused to the green fluorescent protein. E4orf4 protein was found to localize primarily in nuclear structures shown to be viral replication centers, in nucleoli, and in perinuclear bodies. Importantly, E4orf4 was shown not to be essential for virus growth in either human tumor or primary cells, at least in tissue culture. Unlike E4orf4-null virus, mutant dl359 appeared to exhibit a gain-of-function phenotype that impairs virus growth. The dl359 E4orf4 protein, which contains a large in-frame internal deletion, clustered in aggregates enriched in Hsp70 and proteasome components. In addition, the late viral mRNAs produced by dl359 accumulated abnormally in a nuclear punctate pattern. Altogether, our results indicate that E4orf4 protein is not essential for virus growth in culture and that expression of the dl359 E4orf4 product interferes with viral replication, presumably through interactions with structures in the nucleus.     

E4orf6 Variants with Separate Abilities To Augment Adenovirus Replication and Direct Nuclear Localization of the E1B 55-Kilodalton Protein

The E4orf6 protein of group C adenovirus is an oncoprotein that, in association with the E1B 55-kDa protein and by E1B-independent means, promotes virus replication. An arginine-faced amphipathic alpha-helix in the E4orf6 protein is required for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein and to enhance replication of an E4 deletion virus. In this study, E4orf6 protein variants containing arginine substitutions in the amphipathic alpha-helix were analyzed. Two of the six arginine residues within the alpha-helix, arginine-241 and arginine-243, were critical for directing nuclear localization of the E1B 55-kDa protein. The four remaining arginine residues appear to provide a net positive charge for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein. The molecular determinants of the arginine-faced amphipathic alpha-helix that were required for the functional interaction between the E4orf6 and E1B 55-kDa proteins seen in the transfected cell differed from those required to support a productive infection. Several E4orf6 protein variants with arginine-to-glutamic acid substitutions that failed to direct nuclear localization of the E1B 55-kDa protein restored replication of an E4 deletion virus. Additionally, a variant containing an arginine-to-alanine substitution at position 243 that directed nuclear localization of the E1B 55-kDa protein failed to enhance virus replication. These results indicate that the ability of the E4orf6 protein to relocalize the E1B 55-kDa protein to the nucleus can be separated from the ability of the E4orf6 protein to support a productive infection.     

腺病毒E4orf4蛋白诱导肿瘤特异性细胞死亡及其在癌症治疗中的应用

腺病毒E4orf4蛋白由腺病毒早期第4转录区第4开放读码框编码,为一种多功能调节蛋白质,其活性包括下调早期病毒基因表达和下调影响病毒复制的细胞基因表达,调控病毒基因的选择性转录后剪切以影响病毒感染进程等。当E4orf4脱离病毒环境单独表达时,可诱导不依赖p53和胱天蛋白酶途径的癌细胞特异性细胞死亡,而不影响原代细胞的正常生长。这表明E4orf4对癌细胞有特异性的杀伤作用。本文主要介绍了：（1）E4orf4主要蛋白质伴侣（蛋白磷酸酶2A和Src家族激酶）对E4orf4细胞杀伤作用的贡献;（2）E4orf4诱导的细胞死亡独特模式的基本机制及其特点;（3）近年来利用E4orf4治疗癌症的研究。     

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

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

The adenovirus E1b55K/E4orf6 complex induces degradation of the Bloom helicase during infection

The adenovirus (Ad) E1b55K and E4orf6 gene products assemble an E3 ubiquitin ligase complex that promotes degradation of cellular proteins. Among the known substrates are p53 and the Mre11-Rad50-Nbs1 (MRN) complex. Since members of the RecQ helicase family function together with MRN in genome maintenance, we investigated whether adenovirus affects RecQ proteins. We show that Bloom helicase (BLM) is degraded during adenovirus type 5 (Ad5) infection. BLM degradation is mediated by E1b55K/E4orf6 but is independent of MRN. We detected BLM localized at discrete foci around viral replication centers. These studies identify BLM as a new substrate for degradation by the adenovirus E1b55K/E4orf6 complex.