The adenovirus E4 17-kilodalton protein complexes with the cellular transcription factor E2F, altering its DNA-binding properties and stimulating E1A-independent accumulation of E2 mRNA

Abstract

E2F is a cellular DNA-binding factor. Its binding activity is changed within adenovirus-infected cells so that it binds cooperatively to pairs of properly spaced and oriented E2F recognition sites. In the work described in this report, the conversion to cooperative binding was shown to require the adenovirus E4 17-kilodalton (kDa) polypeptide. Mutant viruses carrying alterations within the E4 17-kDa coding region failed to generate the infection-specific, cooperatively binding form of E2F. It was possible to alter E2F from uninfected cells so that it bound cooperatively by incubation with a partially purified fraction obtained from infected cells. The E4 17-kDa protein copurified with this activity and was also found to be present in a complex containing E2F. Consistent with its ability to alter the binding of E2F to its recognition sites within the E2 promoter, the E4 17-kDa polypeptide contributed to maximal expression of E2 mRNAs in some cell types. Its ability to enhance E2 transcription did not require expression of the ElA transactivator protein. These results are consistent with a model which proposes that the E4 17-kDa polypeptide binds to the cellular E2F factor, altering its binding behavior and thereby enhancing its ability to stimulate transcription. Three different early adenovirus gene products have been shown to activate the expression of other viral genes in trans. The best-studied activator is the ElA 289R protein. This ElA product is required for efficient activation of all remaining early viral genes (4, 28), and it functions to enhance the rate of transcription (36). The other activating products are the E1B 21-kilodalton (kDa) polypeptide (23, 51, 54), which has not been shown to act at the level of transcription, and an E4 gene product (13, 49). The mechanism of transcriptional activation by the ElA protein is not yet clear (for reviews, see references 3, 11, and 40). It has been proposed that ElA-mediated activation of the adenovirus E2 early promoter involves the induction or "activation" of a cellular factor termed E2F (37, 38, 40). E2F is a sequence-specific DNA-binding activity that recognizes the sequence 5'-TTTC(G/C)CGC-3' (30). There are 2345

Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells

“…Immunoprecipitations were performed as described previously (62). The E1B-55 kDa-specific antibody, 2A6, was described by Sarnow et al (63), and the E4-specific monoclonal antibody, MAb 3, was described by Marton et al (42). Immune complexes were separated by SDS-polyacrylamide gel electrophoresis (PAGE).…”

“…Indirect immunofluorescence and photomicroscopy of whole cells was performed as previously described (51). Simultaneous double-label immunofluorescence was achieved with the rat monoclonal antibody 9C10 (Oncogene Science, Uniondale, N.Y.) (73), specific for the E1B-55 kDa protein, and the mouse monoclonal antibody MAb 3 (42), specific for the E4-34 kDa protein. The secondary antibodies (Jackson ImmunoResearch) were multiple-label-qualified goat antibodies conjugated to dichlorotriazinylamino fluorescein and lissamine rhodamine sulfonyl chloride.…”

“…cDNA expression vectors that direct expression of the E4-34 kDa and E1B-55 kDa proteins under control of the CMV immediate-early promoter were prepared and used to transfect HeLa cells. After transfection, the cells were processed for indirect immunofluorescence, and the localization of the E1B-55 kDa and E4-34 kDa proteins was determined by double-label immunofluorescence with a rat monoclonal antibody specific for the E1B-55 kDa protein, 9C10 (73), and a mouse monoclonal antibody specific for the E4-34 kDa protein, MAb 3 (42). The mouse antibody was visualized with a fluorescein-conjugated secondary antibody, and the rat antibody was visualized with a rhodamine-conjugated secondary antibody.…”

Two Distinct Activities Contribute to the Oncogenic Potential of the Adenovirus Type 5 E4orf6 Protein

“…The following primary antibodies were used in this study. RSA3 and M45 are monoclonal antibodies directed against the amino terminus of the E4orf6 protein (33,41). Monoclonal antibodies 12CA5 and 3F10 recognize the HA epitope and were obtained from Boehringer Mannheim.…”

A carboxy-terminal region required by the adenovirus type 9 E4 ORF1 oncoprotein for transformation mediates direct binding to cellular polypeptides

“…Nevertheless, the early gene products of DNA viruses generally act to establish conditions optimal for viral replication, often by targeting cellular proteins which control cell cycle progression (27,33). Aside from the E4 ORF1 protein, the E4 transcription unit codes for five additional polypeptides (4,8,9,26,39,40) and, interestingly, cellular targets for three of these early gene products, E4 ORF4, E4 ORF6, and E4 ORF6/7, have been identified as protein phosphatase 2A, p53, and E2F, respectively (10,18,26,29,32). Because adenovirus gene products with related functions frequently cluster within a single viral transcription unit (42), the cellular factors found to complex with the adenovirus E4 ORF1 proteins are similarly expected to represent important regulators of cellular proliferation.…”