Virginia Commonwealth Universitygrid.224260.0 (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA.
VCU Massey Cancer Center, Richmond, Virginia, USA.
J Virol. 2022 Aug 24;96(16):e0083022. doi: 10.1128/jvi.00830-22. Epub 2022 Jul 26.
Human papillomavirus 16 (HPV16) E2 is a DNA-binding protein that regulates transcription, replication and potentially, segregation of the HPV16 genome during the viral life cycle. In the segregation model, E2 simultaneously binds to viral and host chromatin, acting as a bridge to ensure that viral genomes reside in daughter nuclei following cell division. The host chromatin receptor for E2 mediating this function is unknown. Recently, we demonstrated that CK2 phosphorylation of E2 on serine 23 (S23) is required for interaction with TopBP1, and that this interaction promotes E2 and TopBP1 recruitment to mitotic chromatin. Here, we demonstrate that in U2OS cells expressing wild-type E2 and a non-TopBP1-binding mutant (S23A, serine 23 mutated to alanine), interaction with TopBP1 is essential for E2 recruitment of plasmids to mitotic chromatin. Using novel quantitative segregation assays, we demonstrate that interaction with TopBP1 is required for E2 plasmid segregation function in U2OS and N/Tert-1 cells. Small interfering RNA (siRNA) knockdown of TopBP1 or CK2 enzyme components disrupts E2 segregation/retention function. The interaction of E2 with TopBP1 promotes increased levels of E2 protein during mitosis in U2OS and N/Tert-1 cells, as well as in human foreskin keratinocytes (HFK) immortalized by the HPV16 genome. Overall, our results demonstrate that E2 has plasmid segregation activity, and that the E2-TopBP1 interaction is essential for this E2 function. HPV16 causes 3% to 4% of all human cancers. It is proposed that during the viral life cycle, the viral genome is actively segregated into daughter nuclei, ensuring viral replication in the subsequent S phase. The E2 protein potentially bridges the viral and host genomes during mitosis to mediate segregation of the circular viral plasmid. Here, we demonstrate that E2 has the ability to mediate plasmid segregation, and that this function is dependent upon interaction with the host protein TopBP1. Additionally, we demonstrate that the E2-TopBP1 interaction promotes enhanced E2 expression during mitosis, which likely promotes the plasmid segregation function of E2. Overall, our results present a mechanism of how HPV16 can segregate its viral genome during an active infection, a critical aspect of the viral life cycle.
人乳头瘤病毒 16（HPV16）E2 是一种 DNA 结合蛋白，在病毒生命周期中调节转录、复制，并可能调节 HPV16 基因组的分离。在分离模型中，E2 同时与病毒和宿主染色质结合，充当桥梁以确保病毒基因组在细胞分裂后存在于子核中。介导 E2 这种功能的宿主染色质受体尚不清楚。最近，我们证明 CK2 磷酸化 E2 丝氨酸 23（S23）对于与 TopBP1 的相互作用是必需的，并且这种相互作用促进 E2 和 TopBP1 募集到有丝分裂染色质。在这里，我们证明在表达野生型 E2 和非 TopBP1 结合突变体（S23A，丝氨酸 23 突变为丙氨酸）的 U2OS 细胞中，与 TopBP1 的相互作用对于 E2 将质粒募集到有丝分裂染色质是必需的。使用新型定量分离测定法，我们证明在 U2OS 和 N/Tert-1 细胞中，与 TopBP1 的相互作用对于 E2 质粒分离功能是必需的。TopBP1 或 CK2 酶成分的小干扰 RNA（siRNA）敲低会破坏 E2 的分离/保留功能。E2 与 TopBP1 的相互作用促进 U2OS 和 N/Tert-1 细胞以及 HPV16 基因组永生化的人包皮角质形成细胞（HFK）中 E2 蛋白在有丝分裂期间的水平升高。总的来说，我们的结果表明 E2 具有质粒分离活性，并且 E2-TopBP1 相互作用对于这种 E2 功能是必需的。HPV16 导致 3%至 4%的人类癌症。据推测，在病毒生命周期中，病毒基因组被主动分离到子核中，以确保随后 S 期的病毒复制。E2 蛋白可能在有丝分裂期间桥接病毒和宿主基因组，以介导环状病毒质粒的分离。在这里，我们证明 E2 具有介导质粒分离的能力，并且该功能取决于与宿主蛋白 TopBP1 的相互作用。此外，我们证明 E2-TopBP1 相互作用促进有丝分裂期间 E2 表达的增强，这可能促进 E2 的质粒分离功能。总的来说，我们的结果提出了一种 HPV16 在活跃感染期间如何分离其病毒基因组的机制，这是病毒生命周期的一个关键方面。
