Bovine papillomavirus type 1 DNA replication: the transcriptional activator E2 acts in vitro as a specificity factor

We previously devised cell-free conditions supporting efficient replication of bovine papillomavirus type 1 (BPV1) DNA (C. Bonne-Andréa, S. Santucci, and P. Clertant, J. Virol. 69:3201-3205, 1995): the use of highly active preparations of viral initiator protein E1, together with extract from a particular cell source, allowed the synthesis of complete DNA circles through successive rounds of replication; this occurred in the absence of the viral transcriptional activator E2, required in vivo for the replication of viral genomes. We now report that adding E2 to cell-free assays produced only slight effects both on the yield of E1-dependent DNA synthesis and on the quality of newly made DNA molecules when a template carrying a wild-type BPV1 replication origin (ori) was used. The performance of mouse cell extracts, unable to sustain efficient BPV1 DNA replication in the presence of E1 only, was likewise not improved by the addition of E2. In a proper in vitro environment, E1 is thus fully capable of efficiently initiating viral DNA synthesis by itself, an activity which is not enhanced by interaction with E2. An important effect, however, was detected: E2 totally suppressed the nonspecific replication of ori-defective DNA templates, otherwise observed in high E1 concentrations. We examined the requirements for building a minimal DNA sequence behaving in vitro as a specific ori sequence under stringent recognition conditions, i.e., in the presence of both E1 and E2. Only two elements, the 18-bp E1 binding palindrome and an AT-rich sequence, were required in cis to allow specific cell-free DNA replication; there seemed to be no need for an E2 binding site to ensure discrimination between specific ori templates and other DNA molecules, even in the presence of E2. This suggests that during the initiation of BPV1 DNA replication, at least in vitro, E2 acts as a specificity factor restricting the action of E1 to a defined ori sequence; this function, likely not demanding the direct binding of E2 to cognate DNA sites, might primarily involve protein-protein interactions.