Adenovirus E1B 55-Kilodalton Protein Is a p53-SUMO1 E3 Ligase That Represses p53 and Stimulates Its Nuclear Export through Interactions with Promyelocytic Leukemia Nuclear Bodies

Abstract

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...

The Repression Domain of the E1B 55-Kilodalton Protein Participates in Countering Interferon-Induced Inhibition of Adenovirus Replication

“…On the other hand, mutations that prevent binding by the only motif in the protein implicated in interaction with nucleic acids, the RNP motif (79), did not reduce the resistance of viral replication to IFN (Table 1), nor did analysis of the clusters of genes differentially expressed in cells infected by Ad5 and an E1B 55-kDa protein null mutant (44) by using FIRE (Finding Informative Regulatory Elements) (80) identify any sequence motif(s) common to or overrepresented among the promoters of E1B-repressed genes (data not shown). Furthermore, the E1B 55-kDa protein has more recently been demonstrated to function as a Sumol E3 ligase (33,34) and could therefore regulate transcription indirectly via this activity.…”

“…For example, it is also a Sumol E3 ligase (33,34) that modifies p53 to induce association of this cellular protein with nuclear Pml bodies and its subsequent export from the nucleus (34). This mechanism of blocking regulation of transcription by p53 is thought to contribute to the ability of the E1B 55-kDa protein to cooperate with viral E1A proteins to transform rodent cells in culture (33,34), as does a second E4 Orf6 protein-independent activity, inhibition of p53-dependent transcription. Early studies using transient expression assays established that the E1B 55-kDa protein is sufficient to repress expression of p53-dependent reporter genes (35).…”

DNA Virus Replication Compartments

“…Interestingly, the E1B-55K and E4orf3 proteins, are known to participate in SUMOylation of cellular substrates (197,198), and both associate with PML-NBs (199).…”

The adenovirus E4-ORF3 protein functions as a SUMO E3 ligase for TIF-1γ sumoylation and poly-SUMO chain elongation

“…SUMO E1 and E2 enzymes are sufficient to conjugate SUMO proteins at consensus conjugation sites (Ψ-K-X-E/D; Ψ is a large hydrophobic amino acid, X is any amino acid), whereas SUMO E3 ligases are thought to be important for conjugating SUMO at nonconsensus sites. Some viral proteins function as a SUMO E3 ligase, including the Ad5 E1B-55K protein (16,17) for p53 sumoylation and Kaposi's sarcoma-associated herpesvirus (KSHV) KbZIP (18) for sumoylation of p53 and the retinoblastoma protein. Recent studies have demonstrated that poly-SUMO2/3 chains serve as a ubiquitination signal for SUMO-targeted ubiquitin ligases (STUbLs) and proteasomal degradation (19).…”