Replication of bovine papilloma virus (BPV) DNA requres two virus-encoded proteins, El and E2, while afl other proteins are supplied by the host cell. Here, we describe the isolation of the El protein and show that it is a mult nal protein. antigen, which plays a pivotal role in SV40 DNA replication. However, unlike the SV40 large tumor antigen, the helicase activity of El was stimulated 5-fold by the presence of a fork structure at the junction between single-stranded and doublestranded DNA and was supported efficiently by all eight nudeoside triphosphates. The El-catalyzed ATPase activity required the presence of single-stranded or double-stranded DNAs.In cells transformed with bovine papilloma virus (BPV), the viral DNA exists as a free replicating plasmid at a constant copy number (1). The replication of BPV DNA in vivo was shown to depend completely upon two BPV-encoded proteins, the 48-kDa E2 protein and the 68-kDa El protein (2). In addition, the minimal origin sequence required to support BPV DNA replication has been identified (nt 7911-22 of BPV DNA) (3,4). This sequence contains a binding site for the El protein and part of a sequence that acts as a binding site for the E2 protein.The in vitro replication of BPV origin-containing DNA (ori+ DNA), using extracts of a mouse mammary tumor cell line (FM3A), was established in Botchan's laboratory (4). They demonstrated that the in vitro synthesis of DNA absolutely required the BPV minimal origin and the viral El protein, whereas all other required proteins were supplied by the uninfected mouse cell extracts. At low concentrations of El protein, the replication reaction was stimulated markedly by the BPV E2 protein (4).We have isolated and characterized the two viral-encoded proteins required for this replication pathway, El and E2. In this report, we describe the biochemical properties of the purified BPV El protein. We show that this protein supports the replication of BPV oril DNA in vitro, as reported (4). We also demonstrate that this protein possesses a number of different activities required for the replication of the BPV viral genome. These include a DNA helicase activity that moves in the 3' -* 5' direction, a BPV ori+ DNA-binding activity that is stimulated by ATP and MgCl2, and the ability to unwind superhelical DNA leading to the production of highly unwound superhelical DNA (form U DNA). Thus the role of El in the BPV system is analogous to the role of large tumor antigen (T antigen) in the replication of simian virus 40 (SV40) DNA (5-7). MATERIALS AND METHODSCell Cultures and Recombinant Baculoviruses. Growth conditions of the mouse FM3A cell line and the preparation of extracts were as described (4). Recombinant baculoviruses containing wild-type BPV El and E2 genes have been described (8). The El protein used here contained the hemagglutinin epitope of influenza virus (9) followed by a thrombin cleavage site. An oligonucleotide encoding the amino acid residues MYPYDVPDYASLGGPLPRGS was ligated in the Nia III site (nt 848) at the first ...

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