Virginia Commonwealth Universitygrid.224260.0 (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA.
Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussexgrid.12082.39, Brighton, United Kingdom.
mBio. 2021 Oct 26;12(5):e0116321. doi: 10.1128/mBio.01163-21. Epub 2021 Sep 21.
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 and 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 (E2 with serine 23 mutated to alanine) activates and represses transcription identically to E2-WT (wild-type E2), and E2-S23A is as efficient as E2-WT in transient replication assays. However, E2-S23A has compromised interaction with mitotic chromatin compared with E2-WT. In E2-WT cells, both E2 and TopBP1 levels increase during mitosis compared with vector control cells. In E2-S23A cells, neither E2 nor TopBP1 levels increase during mitosis. 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. Remarkably, S23A cells had a disrupted viral life cycle in organotypic raft cultures, with a loss of E2 expression and a failure of viral replication. Overall, our results demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 and that this interaction is critical for the viral life cycle. Human papillomaviruses are causative agents in around 5% of all cancers, with no specific antiviral therapeutics available for treating infections or resultant cancers. In this report, we demonstrate that phosphorylation of HPV16 E2 by CK2 promotes formation of a complex with the cellular protein TopBP1 and . This complex results in stabilization of E2 during mitosis. We demonstrate that CK2 phosphorylates E2 on serine 23 and that CK2 inhibitors disrupt the E2-TopBP1 complex. Mutation of E2 serine 23 to alanine disrupts the HPV16 life cycle, hindering immortalization and disrupting the viral life cycle, demonstrating a critical function for this residue.
在人类乳头瘤病毒 16（HPV16）生命周期中，E2 蛋白与宿主因子相互作用，调节病毒转录、复制和基因组分离/保留。我们对宿主伴侣蛋白及其在 E2 功能中的作用的理解仍然不完整。在这里，我们证明 CK2 对 E2 丝氨酸 23 的磷酸化促进了与 TopBP1 的相互作用，并且在 HPV16 生命周期中 E2 在此残基上被磷酸化。我们研究了突变 E2 丝氨酸 23 对 E2 功能的影响。E2-S23A（E2 中丝氨酸 23 突变为丙氨酸）与 E2-WT（野生型 E2）具有相同的激活和抑制转录的作用，并且在瞬时复制测定中，E2-S23A 与 E2-WT 一样有效。然而，与 E2-WT 相比，E2-S23A 与有丝分裂染色质的相互作用受损。在 E2-WT 细胞中，与载体对照细胞相比，E2 和 TopBP1 的水平在有丝分裂期间增加。在 E2-S23A 细胞中，E2 和 TopBP1 的水平在有丝分裂期间均不增加。将 S23A 突变引入 HPV16 基因组导致人包皮角朊细胞（HFK）的永生化延迟，并且在产生的已建立的 HFK 中，游离病毒基因组拷贝数更高。值得注意的是，S23A 细胞在器官样筏培养物中的病毒生命周期中断，E2 表达丧失，病毒复制失败。总的来说，我们的结果表明，E2 丝氨酸 23 上的 CK2 磷酸化促进了与 TopBP1 的相互作用，并且这种相互作用对于病毒生命周期至关重要。人类乳头瘤病毒是约 5%所有癌症的致病因子，目前尚无针对感染或相关癌症的特定抗病毒治疗方法。在本报告中，我们证明 CK2 对 HPV16 E2 的磷酸化促进了与细胞蛋白 TopBP1 的复合物的形成。该复合物导致 E2 在有丝分裂期间稳定。我们证明 CK2 磷酸化 E2 丝氨酸 23，并且 CK2 抑制剂破坏 E2-TopBP1 复合物。E2 丝氨酸 23 突变为丙氨酸会破坏 HPV16 生命周期，阻碍永生化并破坏病毒生命周期，表明该残基具有关键功能。
