Abstract
Human papillomaviruses (HPV) are the causative agents of cervical cancers. The infectious HPV life cycle is closely linked to the differentiation state of the host epithelia, with viral genome amplification, late gene expression and virion production restricted to suprabasal cells. The E6 and E7 proteins provide an environment conducive to DNA synthesis upon differentiation, but little is known concerning the mechanisms that regulate productive viral genome amplification. Using keratinocytes that stably maintain HPV-31 episomes, and chemical inhibitors, we demonstrate that viral proteins activate the ATM DNA damage response in differentiating cells, as indicated by phosphorylation of CHK2, BRCA1 and NBS1. This activation is necessary for viral genome amplification, as well as for formation of viral replication foci. In contrast, inhibition of ATM kinase activity in undifferentiated keratinocytes had no effect on the stable maintenance of viral genomes. Previous studies have shown that HPVs induce low levels of caspase 3/7 activation upon differentiation and that this is important for cleavage of the E1 replication protein and genome amplification. Our studies demonstrate that caspase cleavage is induced upon differentiation of HPV positive cells through the action of the DNA damage protein kinase CHK2, which may be activated as a result of E7 binding to the ATM kinase. These findings identify a major regulatory mechanism responsible for productive HPV replication in differentiating cells. Our results have potential implications for the development of anti-viral therapies to treat HPV infections.
Highlights
Human papillomaviruses (HPV) are the etiological agents of most anogenital cancers and their productive life cycle is dependent upon epithelial differentiation [1,2]
The infectious life cycle of HPV is dependent on differentiation of the host epithelial cell, with viral genome amplification and virion production restricted to differentiated suprabasal cells
We demonstrate that HPV induces an ATM-dependent DNA damage response that is essential for viral genome amplification in differentiating cells
Summary
Human papillomaviruses (HPV) are the etiological agents of most anogenital cancers and their productive life cycle is dependent upon epithelial differentiation [1,2]. HPVs infect cells in the basal layer of stratified epithelia, but restrict the productive phase of the life cycle to highly differentiated suprabasal cells [3]. Upon differentiation HPV genomes are replicated to thousands of copies per cell in a process referred to as amplification [4]. While normal epithelial cells exit the cell cycle upon differentiation, HPVinfected cells are able to over-ride normal checkpoint controls and remain active in the cell cycle, allowing for the synthesis of cellular proteins that are necessary for viral replication [5,6]. HPV proteins activate low levels of caspases belonging to the intrinsic pathway in differentiating cells, and this is necessary for viral replication [7]. The mechanisms regulating productive replication of HPVs upon differentiation, remain largely unknown
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