Human papillomavirus type 59 immortalized keratinocytes express late viral proteins and infectious virus after calcium stimulation

Elizabeth E. Lehr, Brahim Qadadri, Calla R. Brown, Darron Brown

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Human papillomavirus type 59 (HPV 59) is an oncogenic type related to HPV 18. HPV 59 was recently propagated in the athymic mouse xenograft system. A continuous keratinocyte cell line infected with HPV 59 was created from a foreskin xenograft grown in an athymic mouse. Cells were cultured beyond passage 50. The cells were highly pleomorphic, containing numerous abnormally shaped nuclei and mitotic figures. HPV 59 sequences were detected in the cells by DNA in situ hybridization in a diffuse nuclear distribution. Southern blots were consistent with an episomal state of HPV 59 DNA at approximately 50 copies per cell. Analysis of the cells using a PCR/reverse blot strip assay, which amplifies a portion of the L1 open reading frame, was strongly positive. Differentiation of cells in monolayers was induced by growth in F medium containing 2 mM calcium chloride for 10 days. Cells were harvested as a single tissue-like sheet, and histologic analysis revealed a four-to-six cell-thick layer. Transcripts encoding involucrin, a cornified envelope protein, and the E1∧E4 and E1∧E4∧L1 viral transcripts were detected after several days of growth in F medium containing 2 mM calcium chloride. The E1∧E4 and L1 proteins were detected by immunohistochemical analysis, and virus particles were seen in electron micrographs in a subset of differentiated cells. An extract of differentiated cells was prepared by vigorous sonication and was used to infect foreskin fragments. These fragments were implanted into athymic mice. HPV 59 was detected in the foreskin xenografts removed 4 months later by DNA in situ hybridization and PCR/reverse blot assay. Thus, the complete viral growth cycle, including production on infectious virus, was demonstrated in the HPV 59 immortalized cells grown in a simple culture system.

Original languageEnglish
Pages (from-to)562-571
Number of pages10
JournalVirology
Volume314
Issue number2
DOIs
StatePublished - Sep 30 2003

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Viral Proteins
Keratinocytes
Viruses
Calcium
Foreskin
Heterografts
Nude Mice
Calcium Chloride
In Situ Hybridization
DNA
Growth
Human papillomavirus 18
Polymerase Chain Reaction
Sonication
Southern Blotting
Cell Extracts
Virion
Open Reading Frames
Cell Differentiation
Cultured Cells

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

Human papillomavirus type 59 immortalized keratinocytes express late viral proteins and infectious virus after calcium stimulation. / Lehr, Elizabeth E.; Qadadri, Brahim; Brown, Calla R.; Brown, Darron.

In: Virology, Vol. 314, No. 2, 30.09.2003, p. 562-571.

Research output: Contribution to journalArticle

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