Functional Interaction of a Novel Cellular Protein with the Papillomavirus E2 Transactivation Domain

Abstract

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 ...

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