The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression

The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Cellular differentiation, which is necessary for HPV life cycle completion disrupts CTCF-dependent chromatin looping of HPV18 episomes inducing enhanced activity of the HPV18 early promoter P105 and increased viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species in these cultures, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells (HPV18-{Delta}CTCF) identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF of HPV18 promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate importance of CTCF-dependent transcription regulation at multiple stages of the HPV life cycle. IMPORTANCEOncogenic human papillomavirus (HPV) infection is the cause of a subset of epithelial cancers of the uterine cervix, other anogenital areas and the oropharynx. HPV infection is established in the basal cells of epithelia where a restricted programme of viral gene expression is required for replication and maintenance of the viral episome. Completion of the HPV life cycle is dependent on the maturation (differentiation) of infected cells which induces enhanced viral gene expression and induction of capsid production. We previously reported that the host cell transcriptional regulator, CTCF, is hijacked by HPV to control viral gene expression. In this study, we use long-read mRNA sequencing to quantitatively map the variety and abundance of HPV transcripts produced in early and late stages of the HPV life cycle and to dissect the function of CTCF in controlling HPV gene expression and transcript processing.