The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells.Components of the transcriptional apparatus that mediate the function of the transactivation domain (AD) of bovine papillomavirus type 1 (BPV1) E2 have not been characterized. The E2 AD encompasses its N-terminal 215 amino acids, and the C-terminal 125 amino acids bind the DNA sequence ACCGN 4 CGGT. Separating the AD and the DNA binding domain (DBD) is a region of approximately 100 amino acids termed the hinge. In addition to the full-length 410-amino-acid E2 protein (E2TA), the C-terminal 248 amino acids encoded by the E2 open reading frame (ORF) constitute a species (E2TR) lacking most of the AD, which retains DNA binding and represses transcription by E2TA.Within the E2 AD are two regions of net negative charge (14), a motif common in viral and cellular transcriptional activators. Specific amino acids in these regions are essential for transcriptional activation, although the distal portion of the E2 AD (amino acids 100 to 215) is also required for activity in yeast and mammalian cells (14,20,23,28). Whereas other ADs have been shown to interact with the basal factors TATA binding protein (TBP) and TFIIB, these associations have been reported not for the E2 AD but rather for its C-terminal DNA binding and dimerization domain (34, 39). These Cterminal interactions are dispensable for the function of E2, since it was observed that chimeric E2 proteins in which the E2 DBD was replaced with the GAL4 or LexA DBD activate transcription in vivo (7, 46). E2 cooperatively stimulates transcription with several cellular transcription factors, including Sp1 (27) and USF and CTF (8,19,42). Of these factors, only Sp1 has been reported to physically interact with E2 (27).E2TA also cooperates with the papillomavirus E1 protein to initiate papillomavirus DNA replication. E1 is a site-specific DNA binding protein that can unwind DNA and associate with DNA polymerase (5,36,41,48). The viral origin of replication contains binding sites for both E1 and E2 proteins (43). Although BPV1 ...

The cellular protein AMF-1 (Gps2) positively modulates gene expression by the papillomavirus E2 protein (D. E. Breiding et al., Mol. Cell. Biol. 17:7208-7219, 1997). We show here that AMF-1 also binds the transcriptional coactivator p300 in vitro and in vivo. E2 interacted weakly with p300. These observations led to a model in which AMF-1 recruits p300 into a complex with E2. Cotransfection of AMF-1 or p300 stimulated levels of E2-dependent transcription, while cotransfection of both AMF-1 and p300 showed an additive effect. The functional significance of p300 recruitment for E2 transactivation was evidenced by repression of E2-activated transcription by adenovirus E1A, which inhibits both coactivator and acetylase activities of p300. Antibodies to AMF-1 or E2 immunoprecipitated histone acetylase activity from cell lysates. Western blotting using antibody against acetyl-lysine failed to detect acetylation of AMF-1 or E2 in complex with p300. These results suggest that AMF-1 facilitates the recruitment of p300 and its histone acetylase activity into complexes with E2 and represents a novel mechanism of transcriptional activation.The bovine papillomavirus type 1 (BPV-1) E2 protein serves multiple functions for the virus. Binding of its carboxy-terminal sequence-specific DNA binding domain (DBD) to recognition elements in the papillomavirus genome regulates viral gene expression and replication of the viral genome (1,5,8,16,19,57). The amino-terminal activation domain (AD) of E2 mediates protein-protein interactions with the cellular transcription machinery. This region also binds the papillomavirus E1 protein, a DNA binding helicase essential for viral DNA replication (17, 27, 54). E2 targets E1 to the origin of replication. It is also believed that E2 plays an additional role in DNA replication, as several E2 mutants retained E1 and DNA binding were unable to stimulate replication (16). Because E2 is required for replication of viral nucleosomal DNA but not naked plasmid DNA in vitro (33,53), it has been proposed that E2 may relieve chromatin suppression and allowing entry of cellular replication and transcription factors to the origin and promoter, respectively.The cooperation of at least two functional units within the large 220-amino-acid E2 AD is essential for transcriptional activation. The crystal structure of the human papillomavirus (HPV) E2 AD reveals multiple surfaces available for proteinprotein interactions (2, 25). A transcription factor IIB (TFIIB)

Primary mouse cells transformed by adenovirus type 12 (Adl2) expressed negligible amounts of class I antigens H-2K, -D, and -L on the cell surface and were capable of forming tumors in syngeneic animals, whereas cells transformed by Ad5 continued to express class I antigens and were nontumorigenic.