Establishment of the human papillomavirus type 16 (HPV-16) life cycle in an immortalized human foreskin keratinocyte cell line.
The study of human papillomaviruses (HPVs) in cell culture has been hindered because of the difficulty in recreating the three-dimensional structure of the epithelium on which the virus depends to complete its life cycle. Additionally, the study of genetic mutations in the HPV genome and its effects on the viral life cycle are difficult using the current method of transfecting molecularly cloned HPV genomes into early-passage human foreskin keratinocytes (HFKs) because of the limited life span of these cells. Unless the HPV genome transfected into the early-passage HFK extends the life span of the cell, analysis of stable transfectants becomes difficult. In this study, we have used BC-1-Ep/SL cells, an immortalized human foreskin keratinocyte cell line, to recreate the HPV-16 life cycle. This cell line exhibits many characteristics of the early-passage HFKs including the ability to stratify and terminally differentiate in an organotypic raft culture system. Because of their similarity to early-passage HFKs, these cells were tested for their ability to support the HPV-16 life cycle. The BC-1-Ep/SL cells could stably maintain two HPV genotypes, HPV-16 and HPV-31b, episomally. Additionally, when the BC-1-Ep/SL cell line was stably transfected with HPV-16 and cultured using the organotypic raft culture system (rafts), it sustained the HPV-16 life cycle. Evidence for the productive stage of the HPV-16 life cycle was provided by: DNA in situ hybridization demonstrating HPV-16 DNA amplification in the suprabasal layers of the rafts, immunohistochemical staining for L1 showing the presence of capsid protein in the suprabasal layers of the rafts, and electron microscopy indicating the presence of virus like particles (VLPs) in nuclei from cells in the differentiated layers of the rafts.
Flores
E R
ER
McArdle Laboratory for Cancer Research, Department of Pathology, University of Wisconsin Medical School, 1400 University Avenue, Madison, Wisconsin, 53706, USA.
Allen-Hoffmann
B L
BL
Lee
D
D
Sattler
C A
CA
Lambert
P F
PF
D036022:Capsid Proteins; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000821:virology; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D000091344:Filaggrin Proteins; D016679:Genome, Viral; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007381:Intermediate Filament Proteins / Q000032:analysis; D053550:Keratin-10; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure / Q000821:virology*; D007633:Keratins / Q000032:analysis; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000032:analysis; D027383:Papillomaviridae / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification / Q000648:ultrastructure; D010957:Plasmids / Q000235:genetics*; D012692:Serial Passage; D014162:Transfection
D036022:Capsid Proteins; D004279:DNA, Viral; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; C507493:KRT10 protein, human; D009856:Oncogene Proteins, Viral; D053550:Keratin-10; D007633:Keratins; C063317:L1 protein, Human papillomavirus type 16