Virginia Commonwealth University (VCU), Philips Institute for Oral Health Research, School of Dentistry, Richmond, Virginia, USA.
VCU Massey Cancer Center, Richmond, Virginia, USA.
J Virol. 2023 Mar 30;97(3):e0006323. doi: 10.1128/jvi.00063-23. Epub 2023 Feb 22.
CK2 phosphorylation of HPV16 E2 at serine 23 promotes interaction with TopBP1, and this interaction is important for E2 plasmid segregation function. Here, we demonstrate that the E2-TopBP1 interaction is critical for E2 and viral genome stability during the viral life cycle. Introduction of the S23A mutation into the HPV16 genome results in a loss of E2 expression and viral genome integration during organotypic rafting. Coculture of N/Tert-1+E2-S23A cells with J2 fibroblasts results in E2-S23A degradation via the proteasome; wild-type E2 is not degraded. TopBP1 siRNA treatment of N/Tert-1+E2-WT cells results in E2 degradation only in the presence of J2 cells demonstrating the critical role for TopBP1 in maintaining E2 stability. The CK2 inhibitor CX4945 promotes E2-WT degradation in the presence of fibroblasts as it disrupts E2-TopBP1 interaction. siRNA targeting SIRT1 rescues E2-S23A stability in N/Tert-1 cells treated with J2 fibroblasts, with an increased E2-S23A acetylation. The results demonstrate that the E2-TopBP1 interaction is critical during the viral life cycle as it prevents fibroblast stimulated SIRT1 mediated deacetylation of E2 that promotes protein degradation. This means that the E2-TopBP1 complex maintains E2 and viral genome stability and that disruption of this complex can promote viral genome integration. Finally, we demonstrate that HPV11 E2 also interacts with TopBP1 and that this interaction is critical for HPV11 E2 stability in the presence of J2 cells. Treatment of N/Tert-1 + 11E2-WT cells with CX4945 results in 11E2 degradation. Therefore, CK2 inhibition is a therapeutic strategy for alleviating HPV11 diseases, including juvenile respiratory papillomatosis. Human papillomaviruses are pathogens that cause a host of diseases ranging from benign warts to cancers. There are no therapeutics available for combating these diseases that directly target viral proteins or processes; therefore, we must enhance our understanding of HPV life cycles to assist with identifying novel treatments. In this report, we demonstrate that HPV16 and HPV11 E2 protein expression is dependent upon TopBP1 interaction in keratinocytes interacting with fibroblasts, which recapitulate stromal interactions in culture. The degradation of 16E2 promotes HPV16 genome integration; therefore, the E2-TopBP1 interaction is critical during the viral life cycle. We demonstrate that the CK2 inhibitor CX4945 disrupts HPV11 interaction with TopBP1 and destabilizes HPV11 E2 protein in the presence of J2 fibroblasts; we propose that CX4945 could alleviate HPV11 disease burden.
HPV16 E2 的 CK2 磷酸化在丝氨酸 23 促进与 TopBP1 的相互作用，这种相互作用对于 E2 质粒分离功能很重要。在这里，我们证明 E2-TopBP1 相互作用对于病毒生命周期中 E2 和病毒基因组的稳定性至关重要。将 S23A 突变引入 HPV16 基因组会导致在器官样筏中 E2 表达和病毒基因组整合的丧失。将 N/Tert-1+E2-S23A 细胞与 J2 成纤维细胞共培养会导致 E2-S23A 通过蛋白酶体降解；野生型 E2 不会降解。TopBP1 siRNA 处理 N/Tert-1+E2-WT 细胞仅在存在 J2 细胞的情况下导致 E2 降解，表明 TopBP1 在维持 E2 稳定性方面起着关键作用。CK2 抑制剂 CX4945 在存在成纤维细胞的情况下促进 E2-WT 降解，因为它破坏了 E2-TopBP1 相互作用。靶向 SIRT1 的 siRNA 挽救了 N/Tert-1 细胞中 J2 成纤维细胞处理后的 E2-S23A 稳定性，E2-S23A 乙酰化增加。结果表明，E2-TopBP1 相互作用在病毒生命周期中至关重要，因为它可以防止成纤维细胞刺激的 SIRT1 介导的 E2 去乙酰化，从而促进蛋白质降解。这意味着 E2-TopBP1 复合物维持 E2 和病毒基因组的稳定性，破坏这种复合物可以促进病毒基因组整合。最后，我们证明 HPV11 E2 也与 TopBP1 相互作用，并且这种相互作用对于存在 J2 细胞时 HPV11 E2 的稳定性至关重要。用 CX4945 处理 N/Tert-1+11E2-WT 细胞会导致 11E2 降解。因此，CK2 抑制是一种缓解 HPV11 疾病的治疗策略，包括青少年呼吸道乳头状瘤病。人乳头瘤病毒是引起一系列疾病的病原体，从良性疣到癌症不等。目前尚无针对这些疾病的直接针对病毒蛋白或过程的治疗方法；因此，我们必须增强对 HPV 生命周期的理解，以协助确定新的治疗方法。在本报告中，我们证明 HPV16 和 HPV11 E2 蛋白表达依赖于角化细胞与成纤维细胞相互作用时的 TopBP1 相互作用，这再现了培养物中基质相互作用。16E2 的降解促进 HPV16 基因组整合；因此，E2-TopBP1 相互作用在病毒生命周期中至关重要。我们证明 CK2 抑制剂 CX4945 破坏了 HPV11 与 TopBP1 的相互作用，并在存在 J2 成纤维细胞的情况下使 HPV11 E2 蛋白不稳定；我们提出 CX4945 可以减轻 HPV11 疾病负担。
