The role of CTCF in the life cycle of human papillomavirus

Abstract

Papillomaviruses (PV) are epithelium specific DNA viruses that can cause health problems ranging from harmless warts to invasive cancer. Papillomavirus induced tumours most often arise in the cervix where human papillomavirus (HPV) infections were shown to cause 99.7 % of all malignancies. This study aims to map binding sites of the multifunctional host protein CCCTC binding factor (CTCF) to the papillomavirus genome, validate them and determine the function of CTCF in the papillomavirus life cycle. Computer predictions of CTCF binding sites in the sequence of 8 different PV revealed a CTCF binding pattern including a conserved high-affinity binding site around nucleotide 3000 in high risk HPV and around nucleotide 5400 in low risk HPV. This binding pattern was experimentally confirmed using electrophoretic mobility shift assays (EMSA). The binding site around nucleotide 3000 in HPV18 was mutated and human foreskin keratinocytes (HFK) were transfected with mutant and wild type HPV18 to analyse the effect of the mutation on viral gene expression and life cycle. Western blotting of methylcellulose differentiated HFK revealed earlier expression of E2 and decreased expression of E1^E4 in the mutant compared to the wild type. Immunostaining of organotypic raft cultures grown from the mutant maintaining cells showed a significant increase in proliferating cells compared to the HFK maintaining the wild type. This was accompanied by pseudo-differentiation of keratinocytes since the cells of the granular layer of the raft expressed the terminal differentiation marker loricrin but maintained the morphology of undifferentiated cells. Thus CTCF was shown to have a major impact on the HPV life cycle and it may play a role in HPV induced carcinogenesis. Furthermore a function of CTCF in long term maintenance of the viral episome was revealed as cells maintaining the CTCF mutant were shown to lose episomes more quickly compared to wild type maintaining cells.