Restriction of viral gene expression and replication prevents immortalization of human keratinocytes by a beta-human papillomavirus

Beta-human papillomaviruses (HPV) have been implicated in the development of cutaneous squamous cell cancer (cSCC) in epidermodysplasia verruciformis (EV) patients and organ transplant recipients. In contrast to high-risk (HR) HPV, which cause anogenital and oropharyngeal cancers, immortalizing activity of complete beta-HPV genomes in normal human keratinocytes (NHK), the natural target cells for HPV, has not been reported. We now demonstrate that the beta-HPV49 wild-type genome is transcriptionally active in NHK but lacks immortalizing activity unless the E8 gene, which encodes the E8^E2 repressor, is inactivated. HPV49 E8− immortalized keratinocytes maintain high levels of viral gene expression and very high copy numbers of extrachromosomal viral genomes during long-term cultivation. Not only disruption of the viral E6 and E7 oncogenes but also of the E1 or E2 replication genes renders E8− genomes incapable of immortalization. E8−/E1− and E8−/E2− genomes display greatly reduced E6 and E7 RNA levels in short-term assays. This strongly suggests that high-level expression of E6 and E7 from extrachromosomal templates is necessary for immortalization. The requirement for an inactivation of E8 while maintaining E1 and E2 expression highlights important differences in the carcinogenic properties of HR-HPV and beta-HPV. These findings strengthen the notion that beta-HPV have carcinogenic potential that warrants further investigations into the distribution of beta-HPV in human cancers.