Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes

Abstract

Using Cre-loxP-mediated recombination, we established a highly efficient and reproducible system that generates autonomous HPV-18 genomes in primary human keratinocytes (PHKs), the organotypic raft cultures of which recapitulated a robust productive program. While E7 promoted S-phase re-entry in numerous suprabasal differentiated cells, HPV DNA unexpectedly amplified following a prolonged G2 arrest in mid-and upper spinous cells. As viral DNA levels intensified, E7 activity diminished and then extinguished. These cells then exited the cell cycle to undergo virion morphogenesis. High titers of progeny virus generated an indistinguishable productive infection in naïve PHK raft cultures as before, never before achieved until now. An immortalization-defective HPV-18 E6 mutant genome was also characterized for the first time. Numerous cells accumulated p53 protein, without inducing apoptosis, but the productive program was severely curtailed. Complementation of mutant genomes by E6-expressing retrovirus restored proper degradation of p53 as well as viral DNA amplification and L1 production. This system will be invaluable for HPV genetic dissection and serves as a faithful ex vivo model for investigating infections and interventions.[Keywords: Human papillomavirus infection program; organotypic cultures of primary human keratinocytes; Cre-loxP-mediated recombination in vivo; cell cycle regulation; immortalization-and replication-defective E6 mutant; p53 protein stabilization] Supplemental material is available at http://www.genesdev.org.

Biology of Human Papillomavirus Infections in Head and Neck Carcinogenesis

“…HPV cannot be cultured but only recently, an efficient virus propagation in vitro has been introduced [6]. HPVs infect undifferentiated basal cells of epithelia after trauma or erosion, and are maintained there at a low copy number.…”

Bovine papillomavirus: opening new trends for comparative pathology

“…Papillomavirus are highly species-specific, making animal models for HPV difficult to obtain, except by using animal papillomavirus. Even though these difficulties in finding experimental models of HPV infection and carcinogenesis have been largely overcome during the first years of the new century, with the development of HPV-transfected organotypic cell cultures [2], research on animal papillomavirus, and particularly on BPV, does not seem to have diminished since then. This review will deal with selected recent literature on animal papillomavirus, especially with those papers published over the last two years.…”

A highly efficient system to produce infectious human papillomavirus: Elucidation of natural virus-host interactions

“…Using tissue sections from time course experiments, we have determined a temporal order of the HPV productive program by fluorescence in situ hybridization detection of viral DNA amplification (DNA-FISH), concurrently with antibody detection of BrdU incorporation and E7-induced cellular proteins or viral proteins, as well as by DAPI staining of nuclei. 8 These then stably accumulated in the cornified envelopes (Fig. 1B and data not shown).…”

“…In Wang et al 2009, 8 we reported a simple yet efficient and reproducible approach which overcame the limitations of the methods heretofore used by other investigators in the HPV field. We have recapitulated an exceptionally productive program in PHK raft cultures.…”

“…In agreement, we did not detect apoptotic cells based on tissue histology, nor did we detect cleaved caspase 3 in these raft cultures. 8 The various properties attributed to the E1^E4 protein are likely due to its overexpression in cycling, undifferentiated cells that express low molecular weight keratins as opposed to the high molecular weight forms in differentiated keratinocytes. Our Cre-loxP system will be ideal to elucidate the function of E1^E4 protein during the productive phase of the viral life cycle.…”