Papillomaviruses induce benign epithelial tumors in a wide variety of vertebrate hosts with strict species- and tissue-specificity. Some have been strongly implicated in the occurrence of malignant lesions. Bovine papillomavirus type-1(BPV-1) is also able to induce fibropapillomas in its natural host, and it readily transforms established rodent cells in culture. It serves as the prototype papillomavirus and has provided the basis for most of the insights into papillomavirus molecular biology. BPV- 1 is a fascinating model system for studying eukaryotic DNA replication, transcriptional control, and cellular transformation. BPV-1 DNA exists as a stable multicopy extrachromosomal element in dermal fibroblasts, in the basal cells of the epidermis within fibropapillomas, and in BPV-1 transformed rodent cells. Within transformed cells, copy number appears to be controlled from one generation to the next. Data from studies of stable BPV-1 replication and transient replication assays implicate several cis- and trans-acting factors in viral DNA replication in stably transformed cells. However, their precise role in the replication reaction, its regulation, and the maintenance of copy number have yet to be elucidated. This proposal is to study the molecular mechanism of BPV-1 replication and its regulation by establishing an accurate in vitro BPV-1 replication system. First, the BPV-1 minimal origin will be localized in vivo utilizing the DNA replication terminus site (tau of the prokaryotic plasmid R6K, where, upon binding of the tau sequence-specific protein, Ter, movement of the DNA replication fork is either arrested or severely impeded. Initial experiments will be performed using the well- characterized SV40 replication system in vitro and in vivo to confirm the applicability of the method. Alternative approaches also will be considered. Then, the sequence-specific origin binding protein(s) will be identified as viral or cellular, and purified. Identification of the origin binding protein gene will help establish whether this protein is also the initiator protein for BPV-1 replication in transformed cells. Its properties will be characterized in vitro to help shed light on its biological function; for example, specific DNA-protein interactions, enzymatic properties, and interaction with other viral and cellular proteins. If the origin binding protein is not the initiator, the latter will be identified systematically. With the two key components of BPV-1 replication in hand, an accurate, efficient in vitro replication system will kinetic analysis, mutagenic analysis, and reconstitution experiments. The results will be correlated with in vivo effects. This in vitro system will greatly enhance the functional characterization of BPV-1 proteins implicated in viral DNA replication. This work should yield valuable information regarding the mechanism and regulation of BPV-1 replication in transformed cells which in turn should provide new insight into the mechanisms of cellular DNA replication.
