CK2 phosphorylation of human papillomavirus 16 E2 on serine 23 promotes interaction with TopBP1 and is critical for E2 plasmid retention function
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Doctor of Philosophy
Oral Health Research
Human papillomaviruses (HPVs) are the causative agents in most cervical cancers and have been implicated in a rising number of oropharyngeal cancers. HPV positive oropharyngeal cancers have increased significantly over the last two decades with no specific anti-viral therapeutic options for the treatment of HPV diseases. During its lifecycle, HPV16 encoded E2 protein interacts with host cellular factors to regulate viral transcription, replication and genome segregation/retention. Our understanding of the cellular partner proteins that E2 uses to mediate its functions remains incomplete.
Previously, an interaction between E2 and the host protein TopBP1 was identified. In earlier studies, it was also demonstrated that E2 and TopBP1 co-locate to late telophase chromatin, and that TopBP1 regulates the interaction of E2 with interphase chromatin, suggesting that TopBP1 may be the chromatin receptor protein mediating E2 segregation function.
In this study, we sought a more mechanistic understanding of the E2-TopBP1 interaction. We demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 in vitro and in vivo, and that E2 is phosphorylated on this residue during the HPV16 life cycle. We further investigated the consequences of mutating serine 23 on E2 functions. E2-S23A activates and represses transcription identically to E2-WT (wild-type), and in transient replication assays, E2-S23A is as efficient as E2-WT. However, E2-S23A has compromised interaction with mitotic chromatin when compared with E2-WT. In E2-WT cells, both E2 and TopBP1 levels increase during mitosis when compared with vector control cells. In E2-S23A cells, neither E2 nor TopBP1 levels increase during mitosis. We additionally tested whether this difference in E2-S23A levels during mitosis disrupts E2 plasmid retention function. We developed a novel plasmid retention assay and demonstrate that E2-S23A is deficient in plasmid retention when compared with E2-WT. siRNA targeted knockdown of TopBP1 abrogates E2-WT plasmid retention function.
Introduction of the S23A mutation into the HPV16 genome ensued delayed immortalization of human foreskin keratinocytes (HFK) and higher episomal viral genome copy number in resulting established HFK. Furthermore, S23A mutation results in an aberrant life cycle; there is an increase in koilocytes (indicative of a more transformed keratinocyte), a failure of E2 to be stabilized in differentiating epithelium, and a failure to amplify the viral genome in the epithelium. Overall, our results reveal that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1, which is critical for E2 plasmid retention function and in HPV16 immortalization of keratinocytes. Moreover, we demonstrate that TopBP1 regulation of E2 function is required for an optimum HPV life cycle. These results will enhance the molecular understanding of the HPV16 life cycle in order to identify potential anti-viral targets against HPV.
© Apurva Tadimari Prabhakar
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