Department of Urology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany.
Department of Oral and Maxillofacial Surgery, Medical University of Innsbruck, Innsbruck, Austria.
J Virol. 2023 Jun 29;97(6):e0037023. doi: 10.1128/jvi.00370-23. Epub 2023 May 23.
DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus. Our analyses of this issue revealed that in particular inhibition of cyclin-dependent kinases 4/6 (CDK4/6i) increased E1-independent adenovirus E2-expression and viral DNA replication. Detailed analysis of the E2-expression in dl70-3 infected cells by RT-qPCR showed that the increase in E2-expression originated from the E2-early promoter. Mutations of the two E2F-binding sites in the E2-early promoter (pE2early-LucM) caused a significant reduction in E2-early promoter activity in -activation assays. Accordingly, mutations of the E2F-binding sites in the E2-early promoter in a virus named dl70-3/E2Fm completely abolished CDK4/6i induced viral DNA replication. Thus, our data show that E2F-binding sites in the E2-early promoter are crucial for E1A independent adenoviral DNA replication of E1-deleted vectors in cancer cells. E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells. Here, we report, that the two E2F-binding sites in the adenoviral E2-early promoter contribute substantially to the so-called E1A-like activity in tumor cells. With this finding, on the one hand, the safety profile of viral vaccine vectors can be increased and, on the other hand, the oncolytic property for cancer therapy might be improved through targeted manipulation of the host cell.
E1 缺失的第一代腺病毒（AdV）在培养的癌细胞中的 DNA 复制已被反复报道，并且有人提出某些细胞蛋白可以在功能上补偿 E1A，导致早期区域 2（E2）编码蛋白的表达，随后是病毒复制。因此，将这种现象称为 E1A 样活性。在这项研究中，我们研究了不同的细胞周期抑制剂，以了解它们增加 E1 缺失腺病毒 dl70-3 的病毒 DNA 复制的能力。我们对这个问题的分析表明，特别是细胞周期蛋白依赖性激酶 4/6（CDK4/6i）的抑制显著增加了 E1 非依赖性腺病毒 E2 表达和病毒 DNA 复制。通过 RT-qPCR 对 dl70-3 感染细胞中的 E2 表达进行详细分析表明，E2 表达的增加源于 E2 早期启动子。E2 早期启动子中两个 E2F 结合位点的突变（pE2early-LucM）在失活测定中导致 E2 早期启动子活性显著降低。因此，在一种名为 dl70-3/E2Fm 的病毒中，E2 早期启动子中 E2F 结合位点的突变完全消除了 CDK4/6i 诱导的病毒 DNA 复制。因此，我们的数据表明，E2 早期启动子中的 E2F 结合位点对于 E1 缺失载体在癌细胞中 E1A 非依赖性腺病毒 DNA 复制至关重要。E1 缺失的 AdV 载体被认为是复制缺陷的，是病毒生物学、基因治疗和大规模疫苗开发研究的重要工具。然而，E1 基因的缺失并不能完全消除癌细胞中的病毒 DNA 复制。在这里，我们报告称，腺病毒 E2 早期启动子中的两个 E2F 结合位点对肿瘤细胞中所谓的 E1A 样活性有很大贡献。有了这一发现，一方面可以提高病毒疫苗载体的安全性，另一方面可以通过靶向宿主细胞的操纵来提高癌症治疗的溶瘤特性。
