Post-Transcriptional Regulation of KLF4 by High-Risk Human Papillomaviruses Is Necessary for the Differentiation-Dependent Viral Life Cycle

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Abstract
Human papillomaviruses (HPVs) are epithelial tropic viruses that link their productive life cycles to the differentiation of infected host keratinocytes. A subset of the over 200 HPV types, referred to as high-risk, are the causative agents of most anogenital malignancies. HPVs infect cells in the basal layer, but restrict viral genome amplification, late gene expression, and capsid assembly to highly differentiated cells that are active in the cell cycle. In this study, we demonstrate that HPV proteins regulate the expression and activities of a critical cellular transcription factor, KLF4, through post-transcriptional and post-translational mechanisms. Our studies show that KLF4 regulates differentiation as well as cell cycle progression, and binds to sequences in the upstream regulatory region (URR) to regulate viral transcription in cooperation with Blimp1. KLF4 levels are increased in HPV-positive cells through a post-transcriptional mechanism involving E7-mediated suppression of cellular miR-145, as well as at the post-translational level by E6–directed inhibition of its sumoylation and phosphorylation. The alterations in KLF4 levels and functions results in activation and suppression of a subset of KLF4 target genes, including TCHHL1, VIM, ACTN1, and POT1, that is distinct from that seen in normal keratinocytes. Knockdown of KLF4 with shRNAs in cells that maintain HPV episomes blocked genome amplification and abolished late gene expression upon differentiation. While KLF4 is indispensable for the proliferation and differentiation of normal keratinocytes, it is necessary only for differentiation-associated functions of HPV-positive keratinocytes. Increases in KLF4 levels alone do not appear to be sufficient to explain the effects on proliferation and differentiation of HPV-positive cells indicating that additional modifications are important. KLF4 has also been shown to be a critical regulator of lytic Epstein Barr virus (EBV) replication underscoring the importance of this cellular transcription factor in the life cycles of multiple human cancer viruses. Viruses that induce persistent infections often alter the expression and activities of cellular transcription factors to regulate their productive life cycles. Human papillomaviruses (HPVs) are epithelial tropic viruses that link their productive life cycles to the differentiation of infected host keratinocytes. Our studies show that KLF-4, originally characterized as a pluripotency factor, binds HPV-31 promoters activating viral transcription as well as modulates host cell differentiation and cell cycle progression. KLF4 levels and activity are enhanced in HPV-positive cells by E6 and E7 mediated post-transcriptional and post-translational mechanisms resulting in altered target gene expression and biological functions from that seen in normal keratinocytes. Importantly, silencing KLF4 hinders viral genome amplification and late gene expression. Along with its recently identified role in Epstein Barr Virus reactivation during differentiation, our studies demonstrate the importance of KLF4 in the life cycles of multiple human cancer viruses.
Funding Information
  • National Cancer Institute (RO1CA059655)

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