CK2 phosphorylation of human papillomavirus 16 E2 on serine 23 promotes interaction with TopBP1 and is critical for E2 plasmid retention function

Abstract

During the human papillomavirus 16 (HPV16) life cycle, the E2 protein interacts with host factors to regulate viral transcription, replication and genome segregation/retention. Our understanding of host partner proteins and their roles in E2 functions remains incomplete. Here, 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 investigated the consequences of mutating serine 23 on E2 functions. E2-S23A activates and represses transcription identically to E2-WT (wild-type), and E2-S23A is as efficient as E2-WT in transient replication assays. 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 next 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 resulted in delayed immortalization of human foreskin keratinocytes (HFK) and higher episomal viral genome copy number in resulting established HFK. Overall, our results demonstrate 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.

Regulation of the Human Papillomavirus Lifecyle through Post-Translational Modifications of the Viral E2 Protein

“…Most recently, HPV16 E2 was found to be phosphorylated at S23 and confirmed by employing specific phosphoantibodies [ 24 ]. This residue resides in the alpha-helix of the TAD.…”

“…This residue resides in the alpha-helix of the TAD. Interestingly, this phosphorylation, which is also mediated by CK2, promoted the interaction of E2 with topoisomerase binding protein 1, TopBP1, and was predicted to modulate partitioning of the viral genomes during mitosis [ 24 ]. Unpublished mass spectrometry data from our lab also identified S23 phosphorylation in BPV-1 E2, which further emphasizes the role of conserved phosphorylation sites across PVs.…”