Rolling circle replication of human papillomavirus type 16 DNA in epithelial cell extracts.
Replication of human papillomavirus (HPV) genomes requires an origin of replication and two viral proteins: the DNA helicase E1 and the auxiliary factor E2. To dissect the profile of HPV replication in the epithelium, we analyzed replication of an HPV16 origin-containing plasmid in human epithelial cell extracts supplemented with purified E1 and E2. We found that in addition to well-defined circular replication products, high-molecular-weight DNA was synthesized in a manner that depended on the origin, E1 and E2. The high-molecular-weight DNA was converted to a unit-length linear DNA by treatment with restriction enzymes that cleave the plasmid once, implying that a concatemeric DNA was generated by rolling circle replication. Nicking or relaxing the template plasmid enhanced the level of HPV rolling circle replication. In contrast, the addition of an extract from non-epithelial cells diminished the generation of the rolling circle replication product in the epithelial cell extract, indicating factors that counteract HPV rolling circle replication. These results suggest a rolling circle replication mechanism for the HPV genome in cervical epithelial cells, which may have physiological implications for generation of the tandem-repeated HPV genomes occasionally found integrated into the chromosome of cervical cancer cells.
Pathogen Genomics Center, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-murayama, Tokyo 208-0011, Japan.
Rika
R
Kusumoto-Matsuo
Tadahito
T
Kanda
Iwao
I
Kukimoto
D002457:Cell Extracts; D004261:DNA Replication; D004270:DNA, Circular / Q000096:biosynthesis*; D004279:DNA, Viral / Q000096:biosynthesis*; D004847:Epithelial Cells / Q000821:virology; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D010957:Plasmids; D018741:Replication Origin; D014764:Viral Proteins / Q000235:genetics
D002457:Cell Extracts; D004270:DNA, Circular; D004279:DNA, Viral; D014764:Viral Proteins