CTCF association with episomal HPV16 genomes regulates viral oncogene transcription and splicing

Human papillomavirus 16 (HPV16) is a high-risk alphapapillomavirus that is associated with cancers of mucosal epithelia. The virus genome exists in cells as an episome but can integrate and overexpress the E6 and E7 viral oncogenes. In related high-risk family members HPV18 and HPV31, host proteins including CTCF, an insulator, and SMC1A, a component of the cohesion complex, are known to interact with the viral genome and alter transcriptional activity, splicing patterns and episome amplification. However, the roles of these two proteins during HPV16 infection has not yet been fully examined. Here, we show during differentiation of the episomal HPV16-containing W12 cell line that CTCF association increases with the virus genome at the known E2 binding site, whilst additional CTCF binding now occurs at the putative L2 binding site, with SMC1A association occurring unchanged here. While expression of virus late transcripts (E4^L1, L2, L1) is stimulated, early transcript levels decrease by 48 hours, with the exception of the E6*IV spliced transcript. Conversely, in undifferentiated, monolayer W12 cells, CTCF knockdown increases the level of all early transcripts, whereas E6*IV level increases. Additionally, CTCF ablation as well as SMC1A knockdown results in decreases to HPV16 genome copy number. Taken together, this supports the model that while CTCF and SMC1A have a role in HPV16 genome maintenance, CTCF plays a greater part in regulating HPV16 oncogene splicing and expression during the natural lifecycle of the virus, and may be involved in a reduced risk of cancer development during episomal HPV16 infections.