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The adenovirus 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 dominantnegative cullin 5 protein stabilized p53 and the MRN complex, indicating that it inhibited the viral ubiquitinprotein 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.Two adenovirus type 5 (Ad5) early proteins, E1B-55K and E4orf6, function to stimulate nuclear export and translation of viral late mRNAs, inhibit host cell mRNA nuclear export, and inhibit the functions of p53 and the MRE11-RAD50-NBS1 (MRN) DNA double-strand break repair complex during wildtype (WT) Ad5 infection (3,4,6,11,18,30,35,47,50,64,83). Initial insight into how these two proteins are involved in these diverse processes came from characterizing adenoviral mutant forms of E1B-55K and/or E4orf6. Mutants defective in expressing either E1B-55K or E4orf6 or with a mutation in genes for both show similar phenotypes: They all have a defect in viral late nuclear message export to the cytoplasm and cannot inhibit host cell mRNA nuclear export to the cytoplasm, and synthesis of viral late proteins is reduced compared to that of WT Ad5 (3,4,18,29,30,47,64). These mutants are also unable to induce the degradation of p53 or subunits of the MRN complex (56,65,73,78).Much information has accumulated that is relevant to the functions of E1B-55K and E4orf6. The two viral proteins associate in vivo (68) and colocalize in Ad5-infected cell nuclei at viral DNA replication-transcription centers, as well as other regions of the nucleoplasm and cytoplasm during the late phase of infection (59). Both E1B-55K and E4orf6 have nuclear export signals and nuclear localization signals req...

Oncogenic transformation by adenovirus E1A and E1B-55K requires E1B-55K inhibition of p53 activity to prevent E1A-induced apoptosis. During viral infection, E1B-55K and E4orf6 substitute for the substratebinding subunits of the host cell cullin 5 class of ubiquitin ligases, resulting in p53 polyubiquitinylation and proteasomal degradation. Here we show that E1B-55K alone also functions as an E3 SUMO1-p53 ligase. Fluorescence microscopy studies showed that E1B-55K alone, in the absence of other viral proteins, causes p53 to colocalize with E1B-55K in promyelocytic leukemia (PML) nuclear bodies, nuclear domains with a high concentration of sumoylated proteins. Photobleaching experiments with live cells revealed that E1B-55K tethering of p53 in PML nuclear bodies decreases the in vivo nuclear mobility of p53 nearly 2 orders of magnitude. E1B-55K-induced p53 sumoylation contributes to maximal inhibition of p53 function since mutation of the major p53 sumoylation site decreases E1B-55K-induced p53 sumoylation, tethering in PML nuclear bodies, and E1B-55K inhibition of p53 activity. Mutation of the E1B-55K sumoylation site greatly inhibits E1B-55K association with PML nuclear bodies and the p53 nuclear export to cytoplasmic aggresomes observed in E1A-E1B-transformed cells. Purified E1B-55K and p53 form high-molecular-weight complexes potentially through the formation of a network of E1B-55K dimers bound to the N termini of p53 tetramers. In support of this model, a p53 mutation that prevents tetramer formation greatly reduces E1B-55K-induced tethering in PML nuclear bodies and p53 nuclear export. These data indicate that E1B-55K's association with PML nuclear bodies inactivates p53 by first sequestering it in PML nuclear bodies and then greatly facilitating its nuclear export.During adenovirus type 5 (Ad5) infection and oncogenic transformation of primary cells by E1A and E1B, E1B-55K protein inhibits the activity of cellular p53 (17, 82), a major regulator of cell cycle arrest and apoptosis (11,25,76), through multiple mechanisms (4,7,78). Upon activation, p53 becomes localized in promyelocytic leukemia (PML) nuclear bodies (PML-nb), presumably to be activated by colocalized nuclear acetyltransferases (e.g., p300 and CBP) and kinases (e.g., HIPK2, ATR, and Chk2) through acetylation and phosphorylation, respectively (5, 14, 23, 46). PML-nb are dynamic, heterogeneous macromolecular multiprotein networks ϳ1 m in diameter found in the nuclei of mammalian cells. There are 1 to 30 PML-nb per nucleus (5, 46). They recruit and release a number of proteins to facilitate several different nuclear processes, such as apoptosis, senescence, tumor suppression, and antiviral defenses.Many of the proteins associated with PML-nb, including several isoforms of PML, a major component of PML-nb required for their formation, are reversibly modified posttranslationally by conjugation to lysine ε-amino groups of small ubiquitin-like modifiers (SUMOs) (34,75,85). This modification is important for their recruitment to PML-nb and consequentl...

In the pediatric population, B-acute lymphoblastic leukemia (B-ALL) is the most prevalent childhood hematological malignancy, as well as the leading cause of childhood cancer-related mortality. Advances in cytogenetics utilizing array-based technologies and next-generation sequencing (NGS) techniques have revealed exciting insights into the genetic basis of this disease, with the hopes of developing individualized treatment plans for affected children. In this comprehensive review, we discuss our current understanding of childhood (pediatric) B-ALL and highlight the most recent genetic advances and their therapeutic implications.