Analysis of HPV DNA replication using transient transfection and cell-free assays.

The genomes of human and animal papillomaviruses amplify in keratinocytes undergoing terminal squamous differentiation. Two approaches have been developed to facilitate the investigation into the requirement for viral DNA replication and its regulation outside the context of the host tissues. Under these conditions, the investigation can be conducted independently of viral genes required to re-establish the S-phase milieu in the differentiated keratinocytes to support viral DNA replication. The first method is transient replication in cell lines after transfection of a plasmid containing the viral origin of replication together with expression vectors of the necessary viral proteins that direct specific initiation from the origin. The second is cell-free replication in which purified viral replication proteins are complemented by cell extracts to initiate origin-specific replication. The two methods have identified the origin, the viral E1 and E2 proteins necessary for initiation, their functions, and the host factors that are required to support and regulate the viral DNA replication. These two methods are complementary in providing answers and insights that either one alone may not be able to achieve. This chapter provides a practical guide to these two replication assays.