Reconstruction of Human Papillomavirus Type 16-Mediated Early-Stage Neoplasia Implicates E6/E7 Deregulation and the Loss of Contact Inhibition in Neoplastic Progression

Abstract

H igh-risk human papillomaviruses (HPV), such as HPV type 16 (HPV-16), are associated with a spectrum of precancerous neoplastic changes which occur within the locally infected epithelium. HPV infections that exhibit mild neoplastic changes are classified as low-grade squamous intraepithelial lesions (LSIL), whereas infections showing more severe neoplastic changes are classified as high-grade squamous intraepithelial lesions (HSIL). LSIL and HSIL occur in mucosal epithelia, such as the cervix, and equivalent lesion grades can occur in cutaneous epithelia, such as the vulva (3).How the virus influences the pathological progression from LSIL to HSIL is not completely understood. Recent studies of both cutaneous and mucosal epithelial lesions have shown that the numbers of cells expressing cell cycle proteins, such as the E7 surrogate marker, minichromosome maintenance protein 7 (MCM-7), are increased in HSIL (2,15,22). Furthermore, the prolonged expression of E7 and MCM-7 in cells of the upper epithelial layers coincides with a delay in HPV-16 late gene expression, including that of the genes coding for the E4 and L1 capsid proteins (15). Regions of HSIL often do not support a productive virus life cycle, even though the majority of cells within the lesion still maintain intact viral episomes (8, 11). These observations suggest a model in which the deregulated expression of the HPV-16 early E7 and/or E6 oncogene from intact viral episomes allows infected cells to remain in cycle throughout the upper epithelial layers, thus resulting in an HSIL and an abortive virus life cycle.While studying viral gene expression patterns in organotypic raft cultures of an HPV-16 episome-containing normal immortalized human keratinocyte line (NIKS), we noticed that in marked contrast to what is seen with cell populations, individual HPV-16 cell line clones of the same early passage number were heterogeneous when propagated in raft culture and could mimic either LSIL or HSIL phenotypes with respect to viral gene expression patterns (i.e., E7/MCM, E4, and L1) and cellular pathology. In both the LSIL-like and HSIL-like clones, the viral oncogenes were expressed exclusively from intact viral episomes rather than from integrated sequences, with expression at confluence correlating closely with both the phenotype in raft culture and the extent of suprabasal E7/MCM-7 expression. This work supports the recent vaccine trial results showing that LSIL and HSIL may sometimes arise within a similar time frame and that, in some instances, a cervical intraepithelial neoplasia grade of 2ϩ (CIN2ϩ) can be detected within months or even weeks of first infection (16,17,20). The use of episomal cell lines provides a novel model of early-stage cervical disease and has revealed a correlation between the extent of expression of viral oncogenes and the severity of neoplasia prior to the acquisition of the cancer phenotype.Characterization of HPV16 LSIL-and HSIL-like phenotypes following life cycle reconstruction in organotypic raft culture. To study episo...

Human Papillomavirus E2 Regulates SRSF3 (SRp20) To Promote Capsid Protein Expression in Infected Differentiated Keratinocytes

“…4B, lane 2) but not in undifferentiated (Fig. 4B, lane 1) NIKS16 cells, as expected from data from raft cultures of this line (27). Differentiation was assessed by increased expression of the epithelial differentiation marker involucrin (Fig.…”

“…If used at low passage, NIKS16 clone 2L stably maintains episomal genomes. Like W12E and CIN612 9E cells, they can differentiate in monolayer culture and display a CIN1-like phenotype in organotypic raft culture (27). Differentiation of W12E, NIKS16, and the parental NIKS cells can be induced by culturing in 1.88 mM Ca 2ϩ to high density (10,26).…”

“…For these experiments, we used both the HPV16-infected W12E life cycle model we had used previously (24) and a second model of the HPV16 infectious life cycle (NIKS16) to corroborate our data with W12 cells. NIKS16 cells are normal immortalized foreskin keratinocytes (NIKS) stably transfected with episomal HPV16 genomes (27). If used at low passage, NIKS16 clone 2L stably maintains episomal genomes.…”

“…NIKS16 cells are normal immortalized keratinocytes (NIKS) stably transfected with the HPV16 genome. Clone 2L maintains episomal HPV16 genomes (27). CIN612-9E cells are cervical keratinocytes containing episomal HPV31 genomes (28).…”

The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters

“…Linear genomes were gel purified, recircularized using a dilute ligation reaction, and introduced into NIKS cells by cotransfection with a blasticidin-resistant plasmid using Effectene transfection reagent (Qiagen Srl, Milan, Italy), as previously described (38). Following treatment with blasticidin (5 g/ml for 1 week), resistant colonies were pooled, expanded, and named NIKS-HPV18.…”