The mechanism by which transcription factors stimulate DNA replication in eukaryotes is unknown. Bovine papillomavirus DNA synthesis requires the products of the viral E1 gene and the transcriptional activator protein encoded by the E2 gene. Experimental data showed that the 68-kilodalton (kD) E1 protein formed a complex with the 48-kD E2 transcription factor. This complex bound specifically to the viral origin of replication, which contains multiple binding sites for E2. Repressor proteins encoded by the E2 open reading frame failed to complex with E1 suggesting that the 162-amino acid region of E2 that participates in transactivation contained critical determinants for interaction with E1. The physical association between a replication protein and a transcription factor suggests that transcriptional activator proteins may function in targeting replication initiator proteins to their respective origins of replication.

No abstract

El is the largest open reading frame (ORF) of bovine papillomavirus type 1 (BPV-1) and is highly conserved among all papillomaviruses, maintaining its size, amino acid composition, and location in the viral genome with respect to other early genes. Multiple viral replication functions have been mapped to the El ORF of BPV-1, and evidence suggested that more than one protein was encoded by this ORF. We previously identified a small protein (M) whose gene consists of two exons, one encoded by the 5' end of the El ORF. We show here that a 68-kilodalton (kDa) phosphoprotein made from the El ORF can be detected in BPV-1-transformed cells, and we present evidence that this protein is encoded by sequences colinear with the entire El ORF. The full-length El protein immunoprecipitated from virally transformed cells and identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis comigrates with a protein expressed from a recombinant DNA construct capable of producing only the complete El protein. In addition, two different antisera directed against polypeptides encoded from either the 3' or the 5' end of the El ORF both recognize the full-length El product. A mutation converting the first methionine codon in the ORF to an isoleucine codon abolishes BPV-1 plasmid replication and El protein production. Consistent with the notion that this methionine codon is the start site for El, a mutant with a termination codon placed after the splice donor at nucleotide 1235 in El produces a truncated protein with the molecular mass predicted from the primary sequence as well as the previously identified M protein. When visualized by immunostaining, the El protein expressed in COS cells is localized to the cell nucleus. A high degree of similarity exists between the BPV-1 El protein and polyomavirus and simian virus 40 large-T antigens in regions of the T antigens that bind ATP. We show by ATP affinity labeling that the El protein produced in COS cells binds ATP and that this activity is abolished by a point mutation which converts the codon for proline 434 to serine. Furthermore, this mutation renders the viral genome defective for DNA replication, suggesting that the ATP-binding activity of El is necessary for its putative role in viral DNA replication. We conclude that the El ORF encodes at least two bona fide viral polypeptides in BPV-1-transformed cells: a small protein with an apparent molecular mass of 23 kDa translated from the 5' portion of El and a large protein with an apparent molecular mass of 68 kDa.

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