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

• Published in: The Journal of biological chemistry Vol. 269; no. 39; pp. 24058 - 24065
• Main Authors: Kuo, S R, Liu, J S, Broker, T R, Chow, L T
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 30.09.1994
• Subjects: Antibodies; Base Sequence; Bovine papillomavirus 1 - genetics; Cell Line, Transformed; Cell-Free System; DNA Replication; DNA, Viral - biosynthesis; DNA-Binding Proteins - physiology; Epitopes; Humans; Molecular Sequence Data; Papillomaviridae - genetics; Replication Origin; Templates, Genetic; Viral Proteins - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)51047-X

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.