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...

The 55-kDa gene product from subgroup C adenovirus type 5 (Ad5) early region 1 (E1B-55kDa) plays a central role in the oncogenic transformation of primary rodent cells primarily by inactivating transcriptional and presumably other functional properties of the tumor suppressor protein p53. We have previously shown that Ad5 E1B-55kDa possesses a leucine-rich nuclear export signal (NES), which confers rapid nucleocytoplasmic shuttling via the CRM1-dependent export pathway. In this study we report that an export-deficient mutant of the viral protein (E1B-NES) substantially enhances focus formation of primary baby rat kidney cells in combination with Ad E1A. Transformed rat cells stably expressing the E1B-NES protein exhibited increased tumorigenicity and accelerated tumor growth in nude mice compared to transformants containing the wild-type E1B product. This 'gain of function' correlated with enhanced inhibition of p53 transactivation in transient reporter assays and the accumulation of the mutant protein and p53 in several dotlike subnuclear aggregates. Interestingly, these structures also contained a large fraction of cellular promyelocytic leukemia protein (PML), a known regulator of p53. These data indicate that E1B-NES promotes oncogenic transformation by combinatorial mechanisms that involve modulation of p53 in the context of PML nuclear bodies. In sum, these results extend our previous observation that inhibition of PML activities by E1B-55kDa is required for efficient focus formation and provide further support for the view that blocking p53 transcriptional functions is the principal mechanism by which the Ad protein contributes to complete cell transformation in conjunction with Ad E1A.

The Bacillus subtilis lacA gene, coding for ␤-galactosidase, has been explored as a new site able to accept DNA sequences from nonreplicating delivery vectors. Two such delivery expression vectors have been constructed and shown to be useful in obtaining regulated expression from the chromosomal location. In another experiment, it was shown that the integration of a regulatory gene at the lacA locus was able to control the expression of a transcriptional fusion at the amyE locus. These experiments demonstrate that both integration sites can be used simultaneously to obtain regulated expression of desired genes.Several different plasmids have been used as cloning vectors in Bacillus subtilis, but many of them suffer from the disadvantage that they replicate via a single-stranded DNA intermediate (2). Several steps in the replication cycle render these plasmids highly susceptible to structural rearrangements, and these effects are often dramatically enhanced in recombinant plasmids (5). An alternative method to avoid the problem of the instability of recombinant plasmids in B. subtilis is to use integrative plasmids. Such plasmids are usually based on an Escherichia coli replicon (mostly pBR322 or one of its derivatives) and carry an antibiotic resistance marker gene that can be selected in B. subtilis and DNA sequences homologous to the B. subtilis chromosome. The most prominent and widely used systems are delivery plasmids which allow the insertion of any kind of genetic information into the bacterial chromosome. The amyE locus, coding for a nonessential ␣-amylase, is used in most cases for ectopic integration. This system has been developed by Shimotsu and Henner (12) and contains in its simplest form an antibiotic resistance marker and a multiple cloning site sandwiched between the two halves of the amyE gene, designated amyE-front and amyE-back. Upon transformation of B. subtilis cells, both amyE sequences will recombine at their homologous sites, thereby stably inserting the DNA sequences in between amyE-front and amyE-back into the B. subtilis chromosome via a double-crossover event (12).In some cases, it is appropriate to have two different integration sites available, e.g., to use two different expression systems to allow the study of gene regulation. Therefore, the lacA locus has been explored as an additional site for ectopic integration of DNA sequences. The lacA gene codes for ␤-galactosidase (␤-Gal) and is weakly expressed, if at all, in B. subtilis (1). In addition, two different expression cassettes allowing the regulatable transcription of cloned genes have been developed. Construction of the lacA delivery expression vector pAX01.The delivery expression vector pAX01 (Fig. 1A) was assembled from different plasmids and from chromosomal DNA of B. subtilis in the following way. From pBgaB (8), the ColE1-bla backbone (3,537 bp) was PCR amplified using primers ON1 and ON2 (Table 1), which are both flanked by EcoRV restriction sites, resulting in pK1. Next, 5Ј lacA (lacA-front; 500 bp; made with ON3 and...

Adenoviral replication depends on viral as well as cellular proteins. However, little is known about cellular proteins promoting adenoviral replication. In our screens to identify such proteins, we discovered a cellular component of the ubiquitin proteasome pathway interacting with the central regulator of adenoviral replication. Our binding assays mapped a specific interaction between the N-terminal domains of both viral E1B-55K and USP7, a deubiquitinating enzyme. RNA interference-mediated downregulation of USP7 severely reduced E1B-55K protein levels, but more importantly negatively affected adenoviral replication. We also succeeded in resynthesizing an inhibitor of USP7, which like the knockdown background reduced adenoviral replication. Further assays revealed that not only adenoviral growth, but also adenoviral oncogene-driven cellular transformation relies on the functions of USP7. Our data provide insights into an intricate mechanistic pathway usurped by an adenovirus to promote its replication and oncogenic functions, and at the same time open up possibilities for new antiviral strategies.

Inhibition of p53-activated transcription is an integral part of the mechanism by which early region 1B 55K oncoprotein (E1B-55K) from adenovirus type 5 (Ad5) contributes to complete cell transformation in combination with Ad E1A. In addition, more recent data suggest that the mode of action of the Ad protein during transformation may involve additional functions and other protein interactions. In the present study, we performed a comprehensive mutational analysis to assign further transforming functions of Ad5 E1B-55K to distinct domains within the viral polypeptide. Results from these studies show that the functions required for transformation are encoded within several patches of the 55K primary sequence, including several clustered cysteine and histidine residues, some of which match the consensus for zinc fingers. In addition, two amino-acid substitutions (C454S/C456S) created a 55K mutant protein, which had substantially reduced transforming activity. Interestingly, the same mutations neither affected binding to p53 nor inhibition of p53-mediated transactivation. Therefore, an activity necessary for efficient transformation of primary rat cells can be separated from functions required for inhibition of p53-stimulated transcription. Our data indicate that this activity is linked to the ability of the Ad5 protein to bind to components of the Mre11/Rad50/NBS1 DNA doublestrand break repair complex, and/or its ability to assemble multiprotein aggregates in the cytoplasm and nucleus of transformed rat cells. These results introduce a new function for Ad5 E1B-55K and suggest that the viral protein contributes to cell transformation through p53 transcription-dependent and -independent pathways.