We have investigated the requirements for CRM1-mediated nuclear export and SUMO1 conjugation of the adenovirus E1B-55K protein during productive infection. Our data show that CRM1 is the major export receptor for E1B-55K in infected cells. Functional inactivation of the E1B-55K CRM1-dependent nuclear export signal (NES) or leptomycin B treatment causes an almost complete redistribution of the viral protein from the cytoplasm to the nucleus and its accumulation at the periphery of the viral replication centers. Interestingly, however, this nuclear restriction imposed on the wild type and the NES mutant protein is fully compensated by concurrent inactivation of the adjacent SUMO1 conjugation site. Moreover, the same mutation fully reverses defects of the NES mutant in the nucleocytoplasmic transport of Mre11 and proteasomal degradation of p53. These results show that nuclear export of E1B-55K in infected cells occurs via CRM1-dependent and -independent pathways and suggest that SUMO1 conjugation and deconjugation provide a molecular switch that commits E1B-55K to a CRM1-independent export pathway.he 55K product from subgroup C adenovirus type 5 (Ad5) early region 1B (E1B-55K) belongs to a group of adenoviral regulatory proteins required for maximal virus production in a number of different normal human cell strains and human tumor cell lines (reviewed in ref. 1). In wild-type (WT) Ad5-infected cells, E1B-55K controls several processes, including selective nuclear export of viral late RNA transcripts, inhibition of cellular mRNA transport, and proteasomal degradation of the tumor suppressor protein p53 and Mre11, a subunit of the Mre11/ Rad50/Nbs1 (MRN) DNA double-strand break repair complex (reviewed in ref.2). Collectively available data suggest that these multiple lytic activities result from oligomerization, posttranslational modifications such as phosphorylation, continuous nucleocytoplasmic shuttling, and interactions with a variety of cellular and viral factors, most importantly the protein product from early region 4 ORF 6 (E4orf6) (reviewed in ref. 3 and references therein).Over the past years, it has been well established that complex formation with E4orf6 increases the multifunctionality of the E1B protein. Several studies have shown that E4orf6 alters the intracellular distribution of E1B-55K in virus-infected cells directing the E1B protein to the nuclear matrix compartment (4) and the sites of viral RNA transcription and processing (5, 6). In addition, a substantial amount of novel information demonstrates that E4orf6 connects E1B-55K to components of a cellular E3 ubiquitin ligase, thereby allowing the proteasomal degradation of p53, Mre11, and Rad50 (reviewed in ref. 7). It appears that the latter activity also involves active nuclear export and cytoplasmic deposition of MRN subunits into aggresomes (8). Finally, several lines of evidence suggest that the E1B-55K/ E4orf6 complex directly participates in the selective nuclear export of late viral mRNAs through active nucleocytoplasmic shuttling (3) a...

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.The late phase of an adenoviral infection, typified by human adenovirus type 5 (Ad5), is characterized by a massive production of progeny virions. To support this production, the virus takes control of the cellular machinery to produce abundant amounts of its own late proteins to the detriment of the synthesis of cellular proteins. This host cell shutoff is achieved via several mechanisms. The translation of cellular mRNAs is blocked by the action of the L4-100K protein on the ribosomal machinery (15, 16) so that only the late viral mRNAs containing the tripartite leader sequence can be translated (26). At the same time, export of cellular mRNAs to the cytoplasm is blocked by the action of the early viral proteins E4orf6 and E1B55K (3, 6, 23, 42). These same proteins were also shown to be required for the export of the late viral mRNAs (4,20,23,42,53). The E4orf6 and E1B55K proteins were shown to interact during infection (48) and to function in the same pathway, as a viral mutant with defects in both of these products was seen to have essentially the same phenotype as mutants affecting only one of these species (3,17,23,42). The role that E4orf6 and E1B55K proteins play in the control of mRNA export has yet to be defined.It has been known for some time that the E4orf6 and E1B55K proteins play a role in the degradation of the p53 tumor suppressor, and recently the mechanism for such degradation has been elucidated by our group. Expression of the virus E1A protein results in increased levels of p53 (10, 34); however, during infection, in the presence of both E4orf6 and E1B55K products, p53 is degraded ...

The adenovirus type 5 (Ad5) early region 1B 55-kDa (E1B-55K) protein is a multifunctional regulator of cell-cycle-independent virus replication that participates in many processes required for maximal virus production. As part of a study of E1B-55K function, we generated the Ad5 mutant H5pm4133, carrying stop codons after the second and seventh codons of the E1B reading frame, thereby eliminating synthesis of the full-length 55K product and its smaller derivatives. Unexpectedly, phenotypic studies revealed that H5pm4133 fully exhibits the characteristics of wild-type (wt) Ad5 in all assays tested. Immunoblot analyses demonstrated that H5pm4133 and wt Ad5 produce very low levels of two distinct polypeptides in the 48-to 49-kDa range, which lack the amino-terminal region but contain segments from the central and carboxy-terminal part of the 55K protein. Genetic and biochemical studies with different Ad5 mutants show that at least one of these isoforms consists of two closely migrating polypeptides of 433 amino acid residues (433R) and 422R, which are produced by translation initiation at two downstream AUG codons of the 55K reading frame. Significantly, a virus mutant producing low levels of the 433R isoform alone replicated to levels comparable to those of wt Ad5, demonstrating that this polypeptide provides essentially all functions of E1B-55K required to promote maximal virus growth in human tumor cells. Altogether, these results extend previous findings that the wt Ad5 E1B region encodes a series of smaller isoforms of E1B-55K and demonstrate that very low levels of at least one of these novel proteins (E1B-433R) are sufficient for a productive infection.Products of early region 1B (E1B) from subgroup C adenovirus type 5 (Ad5) are required for productive infection of human cells and for complete transformation of primary mammalian cells in cooperation with the E1A gene products (reviewed in reference 6). Most of these activities can be assigned to two different polypeptides of 176 amino acid residues (176R) (E1B 19-kDa protein [E1B-19K]) and 496R (E1B-55K). Both proteins are produced by translation of two overlapping reading frames starting from two separate initiation codons of the 22S transcript (28, 48) (Fig. 1). The 19-kDa product is also translated from three alternatively spliced transcripts of 13S, 14S, and 14.5S, which additionally direct the synthesis of three smaller 55K-related proteins (156R, 93R, and 84R) containing the amino-terminal 79 residues of E1B-55K (12, 45). E1B-156R also shares an identical carboxy terminus with 55K, while the 93R and 84R E1B polypeptides contain unique carboxy termini.It has been well established that E1B-19K and E1B-55K promote efficient viral replication via a number of different mechanisms. In the early phase of the infection, both E1B proteins counteract antiproliferative processes induced by the host cell, including activation of p53-dependent and -independent apoptosis, induction of cell cycle arrest, and stimulation of cellular DNA damage responses (reviewed in r...

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.