CELL-FREE REPLICATION OF THE HUMAN PAPILLOMAVIRUS DNA WITH HOMOLOGOUSVIRAL E1 AND E2 PROTEINS AND HUMAN CELL-EXTRACTS

We have established the first homologous cell-free DNA replication sys tem 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 c ells infected with recombinant baculoviruses. Efficient replication de pends on the HPV-11 origin, the HPV-11 E1 and E2 proteins, as well as human DNA polymerase alpha, delta, replication protein A, topoisomeras e I, and topoisomerase II. High concentrations of E1 protein also prom oted a low level of origin-independent replication which was suppresse d by the addition of the E2 protein, whereas E2 protein stimulated ori gin-dependent replication. We also show that an intact E2 protein bind ing site was absolutely necessary for origin activity, as a strong HPV -11 origin was rendered inactive when one half-site of each of the thr ee E2 binding sites was mutated. In contrast, there was only a relativ ely small reduction in this mutant origin activity when the cell extra cts were supplemented with the bovine papillomavirus type 1 (BPV-1) pr oteins. These results suggest that the HPV-11 E2 protein plays a prima ry role in HPV origin recognition. Furthermore, unlike transient repli cation in which HPV-11 and BPV-1 viral proteins promote efficient repl ication of homologous and heterologous origins, efficient cell-free re plication took place only with the homologous combinations.