Cell-free replication of the human papillomavirus DNA with homologous viral E1 and E2 proteins and human cell extracts.
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.
Kuo
S R
SR
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
Liu
J S
JS
Broker
T R
TR
Chow
L T
LT
D000906:Antibodies; D001483:Base Sequence; D052688:Bovine papillomavirus 1 / Q000235:genetics; D002461:Cell Line, Transformed; D002474:Cell-Free System; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000502:physiology*; D000939:Epitopes; D006801:Humans; D008969:Molecular Sequence Data; D027383:Papillomaviridae / Q000235:genetics*; D018741:Replication Origin; D013698:Templates, Genetic; D014764:Viral Proteins / Q000502:physiology*
D000906:Antibodies; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C084304:E1 protein, Human papillomavirus type 11; D000939:Epitopes; D014764:Viral Proteins