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. 1990 Oct;64(10):5093–5105. doi: 10.1128/jvi.64.10.5093-5105.1990

Identification of a 68-kilodalton nuclear ATP-binding phosphoprotein encoded by bovine papillomavirus type 1.

S Sun 1, L Thorner 1, M Lentz 1, P MacPherson 1, M Botchan 1
PMCID: PMC248001  PMID: 2168988

Abstract

E1 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 E1 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 E1 ORF. We show here that a 68-kilodalton (kDa) phosphoprotein made from the E1 ORF can be detected in BPV-1-transformed cells, and we present evidence that this protein is encoded by sequences colinear with the entire E1 ORF. The full-length E1 protein immunoprecipitated from virally transformed cells and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis comigrates with a protein expressed from a recombinant DNA construct capable of producing only the complete E1 protein. In addition, two different antisera directed against polypeptides encoded from either the 3' or the 5' end of the E1 ORF both recognize the full-length E1 product. A mutation converting the first methionine codon in the ORF to an isoleucine codon abolishes BPV-1 plasmid replication and E1 protein production. Consistent with the notion that this methionine codon is the start site for E1, a mutant with a termination codon placed after the splice donor at nucleotide 1235 in E1 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 E1 protein expressed in COS cells is localized to the cell nucleus. A high degree of similarity exists between the BPV-1 E1 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 E1 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 E1 is necessary for its putative role in viral DNA replication.(ABSTRACT TRUNCATED AT 400 WORDS)

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