Intracellular expression patterns of the human papillomavirus type 59 E1/E4 protein in COS cells, keratinocytes, and genital epithelium.

OBJECTIVE: To compare and contrast the intracellular distribution pattern of the human papillomavirus type 59 (HPV 59) E1/E4 protein in COS cells, human keratinocytes, and naturally infected genital epithelium. METHODS: The HPV 59 E1/E4 protein was expressed in COS cells and NIKS cells (immortalized human keratinocytes). A subset of NIKS cells was induced to differentiate. The intracellular distribution pattern of E1/E4 and the effects of E1/E4 expression on the cytoskeleton network were compared for COS and NIKS cells. Expression of E1/E4 was examined in HPV 59-infected foreskin xenografts grown in athymic mice and in a natural HPV 59-infected genital lesion. RESULTS: The HPV 59 E1/E4 protein formed dense perinuclear inclusions in COS cells, similar to those reported for the HPV 16 E1/E4 protein. In contrast, the E1/E4 protein was diffusely cytoplasmic in undifferentiated NIKS cells, co-localizing with an intact cytokeratin filament network. The E1/E4 protein was concentrated in the region of the cornified cell envelope (CCE) of differentiated NIKS cells, co-localizing with involucrin, a CCE component. A similar distribution in the region of the CCE was observed for E1/E4 protein in HPV 59-infected human epithelial tissues. CONCLUSIONS: The HPV 59 E1/E4 protein is cytoplasmic and co-localizes with an intact cytokeratin filament network in undifferentiated keratinocytes. The E1/E4 protein is distributed in the region of the CCE and co-localizes with involucrin in differentiated human keratinocytes, consistent with the intracellular distribution pattern observed in HPV 59-infected epithelium.

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

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.