Characterization of an HPV33 natural variant with enhanced transcriptional activity suggests a role for C/EBPβ in the regulation of the viral early promoter

The Long Control Region (LCR) of the human papillomavirus (HPV) genome encompasses the early promoter (EP) that drives expression of the viral oncogenes in infected cells and HPV-associated cancers. Here, we report on a natural variant of HPV33 that displays higher EP activity than the prototype in transfected C33A and HeLa cervical carcinoma cells, and in the osteosarcoma U2OS cell line which supports replication of HPV episomes. This increased promoter activity was ascribed to a single nucleotide variation in the LCR, T7791C, in a putative binding site for the transcription factor C/EBPβ. T7791C abrogated binding of recombinant C/EBPβ to this site in vitro and stimulated the EP in vivo, suggesting that it abrogates a negatively-acting regulatory element. A second C/EBPβ binding site was identified in vitro that activated the EP in vivo and whose function and location in the epithelial-specific enhancer is shown to be conserved in the highly prevalent HPV18. These results suggest that C/EBPβ is both an activator and a repressor of the HPV33 EP, acting via two distinct binding sites. Prediction of C/EBPβ sites in the LCR of 186 HPV types suggests that C/EBPβ regulation of the EP is common among high‐risk viruses from the α genus.