Human Papillomaviruses Modulate Expression of MicroRNA 203 upon Epithelial Differentiation to Control Levels of p63 Proteins

Human papillomaviruses (HPV) link their life cycles to epithelial differentiation and induce productive replication of viral DNA in suprabasal cells. Viral-DNA amplification requires cells to remain active in the cell cycle upon differentiation. This is in contrast to normal cells, which lose proliferative capability upon differentiation. One factor that negatively regulates proliferative capability upon differentiation is microRNA 203 (miR-203), which is expressed primarily in suprabasal epithelial cells. Although HPVs do not encode their own microRNAs (miRNAs), they modulate expression of cellular miRNAs to regulate the activities of cellular proteins. We show that the HPV E7 protein downregulates miR-203 expression upon differentiation, which may occur through the mitogen-activated protein (MAP) kinase/protein kinase C (PKC) pathway. One target of miR-203 is the p63 family of transcription factors, and we demonstrate that HPV-positive cells maintain significantly higher levels of these factors upon differentiation than do normal keratinocytes. Several downstream targets of p63, CARM-1, p21, and Bax, were also increased in E7-expressing cells, and their levels were inversely correlated with amounts of miR-203. Introduction of expression vectors for miR-203 into keratinocytes that stably maintain HPV episomes resulted in short-term elevation of HPV genome copy numbers, but these were rapidly lost upon subsequent passage. When HPV-positive cells expressing high levels of miR-203 were induced to differentiate in methylcellulose, impaired genome amplification was observed. We conclude that high levels of miR-203 are inhibitory to HPV amplification and that HPV proteins act to suppress expression of this microRNA to allow productive replication in differentiating cells.