Cell-free replication of the human papillomavirus DNA with homologous viral E1 and E2 proteins and human cell extracts

Abstract

We have established the first homologous cell-free DNA replication system for a papillomavirus. The replication of the human papillomavirus type 11 (HPV-11) origin was achieved by using human 293 cell extracts supplemented with the HPV-11 E1 and E2 proteins purified from insect cells infected with recombinant baculoviruses. Efficient replication depends on the HPV-11 origin, the HPV-11 E1 and E2 proteins, as well as human DNA polymerase alpha, delta, replication protein A, topoisomerase I, and topoisomerase II. High concentrations of E1 protein also promoted a low level of origin-independent replication which was suppressed by the addition of the E2 protein, whereas E2 protein stimulated origin-dependent replication. We also show that an intact E2 protein binding site was absolutely necessary for origin activity, as a strong HPV-11 origin was rendered inactive when one half-site of each of the three E2 binding sites was mutated. In contrast, there was only a relatively small reduction in this mutant origin activity when the cell extracts were supplemented with the bovine papillomavirus type 1 (BPV-1) proteins. These results suggest that the HPV-11 E2 protein plays a primary role in HPV origin recognition. Furthermore, unlike transient replication in which HPV-11 and BPV-1 viral proteins promote efficient replication of homologous and heterologous origins, efficient cell-free replication took place only with the homologous combinations.