An activity associated with human chromosome 21 permits nuclear colocalization of the adenovirus E1B-55K and E4orf6 proteins and promotes viral late gene expression

The adenovirus E1B-55K and E4orf6 proteins cooperate during virus infection while performing several tasks that contribute to a productive infection, including the selective nucleocytoplasmic transport of late viral mRNA. Previous studies have shown that the E4orf6 protein retains the E1B-55K protein in the nucleus of human and monkey cells, but not in those of rodents, suggesting that primate-specific cellular factors contribute to the E4orf6-mediated retention of the E1B-55K protein in the nucleus. In an effort to identify these proposed primate-specific cellular factors, the interaction of the E1B-55K and E4orf6 proteins was studied in a panel of stable human-rodent monochromosomal somatic cell hybrids. Analysis of this panel of cell lines has demonstrated the existence of an activity associated with human chromosome 21 that permits the E1B-55K and E4orf6 proteins to colocalize in the nucleus of a rodent cell. Additional hybrid cells bearing portions of human chromosome 21 were used to map this activity to a 10-megabase-pair segment of the chromosome, extending from 21q22.12 to a region near the q terminus. Strikingly, this region also facilitates the expression of adenovirus late genes in a rodent cell background while having little impact on the expression of early viral genes.     

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.     

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.     

Diverse roles for E4orf3 at late times of infection revealed in an E1B 55-kilodalton protein mutant background

Species C human adenovirus mutants that fail to express open reading frame 3 of early region 4 (E4orf3) are phenotypically indistinguishable from the wild-type virus when evaluated in cells cultured in vitro. However, E4orf3 gene function has been productively studied in the context of additional viral mutations. This study identifies diverse roles for the E4orf3 protein that are evident in the absence of early region 1B 55-kDa protein (E1B-55K) function. In an E1B-55K-deficient background, the E4orf3 protein promotes viral replication by increasing both the burst size and the probability that an infected cell will produce virus. Early viral gene expression is not impaired in E1B-55K/E4orf3 double mutant virus-infected cells. Cells infected with the double mutant virus accumulated concatemers of viral DNA. However, the E1B-55K/E4orf3 double mutant virus did not replicate any better in MO59J cells, in which viral DNA concatemers did not accumulate, than in MO59K cells, in which viral DNA concatemers were produced, suggesting that viral DNA concatenation is not the primary growth defect of the E1B-55K/E4orf3 double mutant virus. Accumulation of viral mRNA in the nucleus and cytoplasm of E1B-55K/E4orf3 double mutant virus-infected cells was severely reduced compared to that on wild-type virus-infected cells. Thus, in an E1B-55K mutant background, the E4orf3 protein promotes the accumulation of late viral RNA and enhances late gene expression. Finally, within the context of an E1B-55K mutant virus, the E4orf3 protein acts to suppress host cell translation and preserve the viability of cells at moderately late times of infection.     

Adenovirus ubiquitin-protein ligase stimulates viral late mRNA nuclear export

Theadenovirus type 5 (Ad5) E1B-55K and E4orf6 proteins are required together to stimulate viral late nuclear mRNA export to the cytoplasm and to restrict host cell nuclear mRNA export during the late phase of infection. Previous studies have shown that these two viral proteins interact with the cellular proteins elongins B and C, cullin 5, RBX1, and additional cellular proteins to form an E3 ubiquitin-protein ligase that polyubiquitinates p53 and probably one or more subunits of the MRE11-RAD50-NBS1 (MRN) complex, directing their proteasomal degradation. The MRN complex is required for cellular DNA double-strand break repair and induction of the DNA damage response by adenovirus infection. To determine if the ability of E1B-55K and E4orf6 to stimulate viral late mRNA nuclear export requires the ubiquitin-protein ligase activity of this viral ubiquitin-protein ligase complex, we designed and expressed a dominant-negative mutant form of cullin 5 in HeLa cells before infection with wild-type Ad5 or the E1B-55K null mutant dl1520. The dominant-negative cullin 5 protein stabilized p53 and the MRN complex, indicating that it inhibited the viral ubiquitin-protein ligase but had no effect on viral early mRNA synthesis, early protein synthesis, or viral DNA replication. However, expression of the dominant-negative cullin 5 protein caused a decrease in viral late protein synthesis and viral nuclear mRNA export similar to the phenotype produced by mutations in E1B-55K. We conclude that the stimulation of adenovirus late mRNA nuclear export by E1B-55K and E4orf6 results from the ubiquitin-protein ligase activity of the adenovirus ubiquitin-protein ligase complex.     

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.     

The nuclear export signal within the E4orf6 protein of adenovirus type 5 supports virus replication and cytoplasmic accumulation of viral mRNA

During the late phase of adenovirus infection, viral mRNA is efficiently transported from the nucleus to the cytoplasm while most cellular mRNA species are retained in the nucleus. Two viral proteins, E1B-55 kDa and E4orf6, are both necessary for these effects. The E4orf6 protein of adenovirus type 5 binds and relocalizes E1B-55 kDa, and the complex of the two proteins was previously shown to shuttle continuously between the nucleus and cytoplasm. Nucleocytoplasmic transport of the complex is achieved by a nuclear export signal (NES) within E4orf6. Mutation of this signal sequence severely reduces the ability of the E1B-55 kDa-E4orf6 complex to leave the nucleus. Here, we examined the role of functional domains within E4orf6 during virus infection. E4orf6 or mutants derived from it were transiently expressed, followed by infection with recombinant adenovirus lacking the E4 region and determination of virus yield. An arginine-rich putative alpha helix near the carboxy terminus of E4orf6 contributes to E1B-55 kDa binding and relocalization as well as to the synthesis of viral DNA, mRNA, and proteins. Further mutational analysis revealed that mutation of the NES within E4orf6 considerably reduces its ability to support virus production. The same effect was observed when nuclear export was blocked with a competitor. Further, a functional NES within E4orf6 contributed to the efficiency of late virus protein synthesis and viral DNA replication, as well as total and cytoplasmic accumulation of viral late mRNA. Our data support the view that NES-mediated nucleocytoplasmic shuttling strongly enhances most, if not all, intracellular activities of E4orf6 during the late phase of adenovirus infection.     

Distinct requirements of adenovirus E1b55K protein for degradation of cellular substrates

The E1b55K and E4orf6 proteins of adenovirus type 5 (Ad5) assemble into a complex together with cellular proteins including cullin 5, elongins B and C, and Rbx1. This complex possesses E3 ubiquitin ligase activity and targets cellular proteins for proteasome-mediated degradation. The ligase activity has been suggested to be responsible for all functions of E1b55K/E4orf6, including promoting efficient viral DNA replication, preventing a cellular DNA damage response, and stimulating late viral mRNA nuclear export and late protein synthesis. The known cellular substrates for degradation by E1b55K/E4orf6 are the Mre11/Rad50/Nbs1 DNA repair complex, the tumor suppressor p53, and DNA ligase IV. Here we show that the degradation of individual targets can occur independently of other substrates. Furthermore, we identify separation-of-function mutant forms of E1b55K that can distinguish substrates for binding and degradation. Our results identify distinct regions of E1b55K that are involved in substrate recognition but also imply that there are additional requirements beyond protein association. These mutant proteins will facilitate the determination of the relevance of specific substrates to the functions of E1b55K in promoting infection and inactivating host defenses.     

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.     

Analyses of single-amino-acid substitution mutants of adenovirus type 5 E1B-55K protein

The E1B-55K protein plays an important role during human adenovirus type 5 productive infection. In the early phase of the viral infection, E1B-55K binds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is required for efficient nucleocytoplasmic transport and translation of late viral mRNAs, as well as for host cell shutoff. In an effort to separate the p53 binding and inactivation function and the late functions of the E1B-55K protein, we have generated 26 single-amino-acid mutations in the E1B-55K protein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expression, and support virus replication in human cancer cells. Of the 26 mutants, 24 can mediate p53 degradation as efficiently as the wild-type protein. Two mutants, R240A (ONYX-051) and H260A (ONYX-053), failed to degrade p53 in the infected cells. In vitro binding assays indicated that R240A and H260A bound p53 poorly compared to the wild-type protein. When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its ability to bind E4orf6, while R240A was fully functional in this interaction. Another mutant, T255A, lost the ability to bind E4orf6, but unexpectedly, viral late-gene expression was not affected. This raised the possibility that the interaction between E1B-55K and E4orf6 was not required for efficient viral mRNA transport. Both R240A and H260A have retained, at least partially, the late functions of wild-type E1B-55K, as determined by the expression of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype. Virus expressing R240A (ONYX-051) replicated very efficiently in human cancer cells, while virus expressing H260A (ONYX-053) was attenuated compared to wild-type virus dl309 but was more active than ONYX-015. The ability to separate the p53-inactivation activity and the late functions of E1B-55K raises the possibility of generating adenovirus variants that retain the tumor selectivity of ONYX-015 but can replicate more efficiently than ONYX-015 in a broad spectrum of cell types.     

Adenovirus exploits the cellular aggresome response to accelerate inactivation of the MRN complex

Results reported here indicate that adenovirus 5 exploits the cellular aggresome response to accelerate inactivation of MRE11-RAD50-NBS1 (MRN) complexes that otherwise inhibit viral DNA replication and packaging. Aggresomes are cytoplasmic inclusion bodies, observed in many degenerative diseases, that are formed from aggregated proteins by dynein-dependent retrograde transport on microtubules to the microtubule organizing center. Viral E1B-55K protein forms aggresomes that sequester p53 and MRN in transformed cells and in cells transfected with an E1B-55K expression vector. During adenovirus infection, the viral protein E4orf3 associates with MRN in promyelocytic leukemia protein nuclear bodies before MRN is bound by E1B-55K. Either E4orf3 or E4orf6 is required in addition to E1B-55K for E1B-55K aggresome formation and MRE11 export to aggresomes in adenovirus-infected cells. Aggresome formation contributes to the protection of viral DNA from MRN activity by sequestering MRN in the cytoplasm and greatly accelerating its degradation by proteosomes following its ubiquitination by the E1B-55K/E4orf6/elongin BC/Cullin5/Rbx1 ubiquitin ligase. Our results show that aggresomes significantly accelerate protein degradation by the ubiquitin-proteosome system. The observation that a normal cellular protein is inactivated when sequestered into an aggresome through association with an aggresome-inducing protein has implications for the potential cytotoxicity of aggresome-like inclusion bodies in degenerative diseases.     

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.     

E1B 55-Kilodalton-Associated Protein: a Cellular Protein with RNA-Binding Activity Implicated in Nucleocytoplasmic Transport of Adenovirus and Cellular mRNAs

The adenovirus type 5 (Ad5) early 1B 55-kDa protein (E1B-55kDa) is a multifunctional phosphoprotein that regulates viral DNA replication and nucleocytoplasmic RNA transport in lytically infected cells. In addition, E1B-55kDa provides functions required for complete oncogenic transformation of rodent cells in cooperation with the E1A proteins. Using the far-Western technique, we have isolated human genes encoding E1B-55kDa-associated proteins (E1B-APs). The E1B-AP5 gene encodes a novel nuclear RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that is highly related to hnRNP-U/SAF-A. Immunoprecipitation experiments indicate that two distinct segments in the 55-kDa polypeptide which partly overlap regions responsible for p53 binding are required for complex formation with E1B-AP5 in Ad-infected cells and that this protein interaction is modulated by the adenovirus E4orf6 protein. Expression of E1B-AP5 efficiently interferes with Ad5 E1A/E1B-mediated transformation of primary rat cells. Furthermore, stable expression of E1B-AP5 in Ad-infected cells overcomes the E1B-dependent inhibition of cytoplasmic host mRNA accumulation. These data suggest that E1B-AP5 might play a role in RNA transport and that this function is modulated by E1B-55kDa in Ad-infected cells.     

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 adenoviral E4orf6 protein induces atypical apoptosis in response to DNA damage

Adenoviral proteins interact with host-cell proteins to either exploit or inhibit cellular functions for the purpose of viral propagation. E4orf6, the 34-kDa gene product of the E4 gene, interacts with the double-strand break repair (DSBR) protein DNA-dependent protein kinase and cooperates with binding partner E1B-55K to degrade MRE11, preventing viral DNA concatemer formation. We previously demonstrated that E4orf6 radiosensitizes human tumor cells through the inhibition of DSBR, notably in the absence of E1B-55K. Here, we report that E4orf6 prolongs the signaling of DNA damage by inhibiting the activity of protein phosphatase 2A (PP2A), the phosphatase responsible for dephosphorylating gammaH2AX. The inhibition of PP2A occurs without significant disruption of the DNA re-ligation rate. Prolonged signaling of DNA damage in the presence of E4orf6 initiates caspase-dependent and independent cell death. This is accompanied by poly(ADP-ribose) polymerase (PARP) hyperactivation and the translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus. Knockdown of AIF by shRNA rescues the radiosensitization induced by E4orf6. Taken together, these data suggest that E4orf6 disrupts cellular DSBR signaling by inhibiting PP2A, leading to prolonged H2AX phosphorylation, hyperactivation of PARP, and AIF translocation to the nucleus. The function of E4orf6 as an inhibitor of PP2A and activator of PARP in the absence of other adenoviral gene products is of importance in delineating the adenovirus-host cell interplay.     

The adenovirus E4orf6 protein inhibits DNA double strand break repair and radiosensitizes human tumor cells in an E1B-55K-independent manner

The adenoviral protein E4orf6 has been shown to inhibit both in vitro V(D)J recombination and adenoviral DNA concatenation, two processes that rely on cellular DNA double strand break repair (DSBR) proteins. Most of the known activities of E4orf6 during adenoviral infection require its interaction with another adenoviral protein, E1B-55K. Here we report that E4orf6, stably expressed in RKO human colorectal carcinoma cells or transiently expressed by adenoviral vector in U251 human glioblastoma cells, inhibits DSBR and induces significant radiosensitization in the absence of E1B-55K. Expression of a mutant form of E4orf6 (L245P) failed to radiosensitize RKO cells. E4orf6 reduced DSBR capacity in transfected and infected cells, as measured by sublethal DNA damage repair assay and phosphorylated H2AX (gamma-H2AX) levels, respectively. Consistent with the inhibitory effect of E4orf6 on DSBR, expression of wild-type but not mutant E4orf6 reduced recovery of a transfected, replicating reporter plasmid (pSP189) in 293 cells but did not increase the mutation frequency measured in the reporter plasmid. The kinase activity of DNA-PKcs (the DNA-dependent protein kinase catalytic subunit) toward heterologous substrates was not affected by expression of E4orf6; however, autophosphorylation of DNA-PKcs at Thr-2609 following ionizing radiation was prolonged in the presence of E4orf6 when compared with control-infected cells. Our results demonstrate for the first time that E4orf6 expression hinders the cellular DNA repair process in mammalian cells in the absence of E1B-55K or other adenoviral genes and suggest that viral-mediated delivery of E4orf6, combined with localized external beam radiation, could be a useful approach for the treatment of radioresistant solid tumors such as glioblastomas.