Human papillomaviruses (HPVs) are small DNA viruses which are maintained stably in human cells. Many HPVs are also involved in the pathogenesis of anogenital cancer. The long term goals of this research are to understand the DNA-protein and protein-protein interactions involved in the regulated replication of HPV DNA. We intend to identify the cis-acting sequences that are important for the initiation and regulation of replication. We also propose to study the role of the viral E1 and E2 proteins in HPV DNA replication. Recent studies have shown that the E1 and E2 proteins are required for the replication of plasmids containing the HPV origin of replication. Using a transient replication assay, we will determine the sequence requirements for the HPV-1a origin function. The HPV-1a ori plasmids can replicate in the presence of E1 alone in a transient assay. Nucleotides that are important for E1 dependent replication and binding by E1 will be identified by site-directed mutagenesis. We will test the hypothesis that the HPV-1a E1 protein has higher binding affinity for the DNA than HPV-18 E1. The E1 and E2 proteins will be purified and their affinity for the DNA will be measured by determining the equilibrium constants. The effect of E2 on the DNA binding activity of E1 will also be studied. The mechanism of inhibition of replication by a 174-bp region of HPV-1a DNA will also be studied. The hypothesis that HPV-18 E1 can support DNA replication if it is targeted to the DNA will be tested using LexA-E1 fusions and DNA binding mutants of E1 and testing their ability to replicate plasmids containing the LexA binding sites, or the HPV-18 origin, respectively. We will identify the replication specificity, DNA binding and E2 complexing domains of the HPV-1a and 18 E1 proteins. This will involve the generation of truncated E1 derivatives, as well as small deletions and amino acid substitutions in E1. The various activities of these mutants will be analyzed in vivo and in vitro. We will also generate hybrids between HPV 1a and 18 E1 proteins to identify their replication specificity domains. A successful completion of this project should significantly enhance our understanding of the replication of these viruses and may provide important insights into the mechanism of replication and persistence of these viruses in human cells. These studies could also lead to a better understanding of the events that inhibit HPV replication, and may facilitate their integration into the chromosome that is thought to promote malignancy.