A splice donor in E6 influences keratinocyte immortalization by beta-HPV49

Human papillomaviruses (HPV) from the genus beta have been implicated in the development of cutaneous squamous cell cancer in epidermodysplasia verruciformis and organ transplant patients. In contrast to alpha-high-risk HPV, which cause ano-genital and oropharyngeal cancers, beta-HPV replication is not well understood. The beta-HPV49 transcriptome was analyzed by RNA sequencing using stable keratinocyte cell lines maintaining high levels of extrachromosomally replicating E8- genomes, which can be established due to a lack of the viral E8^E2 repressor protein. This analysis indicated the presence of four transcription start sites, two polyadenylation signals, and splice donor (SD) and acceptor sites consistent with the conserved gene expression patterns of animal and human PV. Surprisingly, a novel SD in the E6 oncogene (SD217) was identified resembling the SD in E6 of carcinogenic alpha-HPV. Mutation of SD217 enhanced E6 protein expression but had no influence on the growth of keratinocytes transduced with retroviral HPV49 E6 and E7 expression vectors. Inactivation of SD217 in the context of the HPV49 wild-type genome did not enable immortalization and prevented immortalization in the context of the E8- genome. The analysis of SD217 mutant genomes revealed a strong down-regulation of SD217 usage, but only weak effects on other viral transcripts. This suggests that SD217 does not contribute to immortalization by modulating viral gene expression. Usage of SD217 is increased in immortalized E8- cell lines compared with transiently transfected cells, which may indicate that long-term extrachromosomal maintenance requires reduced E6 protein levels.IMPORTANCEHigh-risk (hr) human papillomaviruses (HPV) from the genus alpha cause ano-genital and oropharyngeal cancers, whereas beta-HPV have been implicated to cause skin cancer in epidermodysplasia verruciformis and organ transplant patients. In contrast to alpha hr-HPV, the replication cycle of beta-HPV is not very well understood. Transcriptional profiling of beta-HPV49 by RNA sequencing reveals transcription start sites and splice sites conserved among HPV. Surprisingly, a splice donor site in the E6 oncogene (SD217), previously only described for hr-HPV, was identified that controls E6 oncoprotein levels and is required for immortalization of keratinocytes by the HPV49 genome.