Department of Viral Transformation, Leibniz Institute for Experimental Virology (HPI), Hamburg, Germany.
Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, México.
J Virol. 2022 Mar 9;96(5):e0206221. doi: 10.1128/jvi.02062-21. Epub 2022 Jan 12.
The multifunctional adenoviral E1B-55K phosphoprotein is a major regulator of viral replication and plays key roles in virus-mediated cell transformation. While much is known about its function in oncogenic cell transformation, the underlying features and exact mechanisms that implicate E1B-55K in the regulation of viral gene expression are less well understood. Therefore, this work aimed to unravel basic intranuclear principles of E1B-55K-regulated viral mRNA biogenesis using wild-type human adenovirus C5 (HAdV-C5) E1B-55K, a virus mutant with abrogated E1B-55K expression, and a mutant that expresses a phosphomimetic E1B-55K. By subnuclear fractionation, mRNA, DNA, and protein analyses as well as luciferase reporter assays, we show that (i) E1B-55K promotes the efficient release of viral late mRNAs from their site of synthesis in viral replication compartments (RCs) to the surrounding nucleoplasm, (ii) E1B-55K modulates the rate of viral gene transcription and splicing in RCs, (iii) E1B-55K participates in the temporal regulation of viral gene expression, (iv) E1B-55K can enhance or repress the expression of viral early and late promoters, and (v) the phosphorylation of E1B-55K regulates the temporal effect of the protein on each of these activities. Together, these data demonstrate that E1B-55K is a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes during HAdV-C5 infection. Human adenoviruses are useful models to study basic aspects of gene expression and splicing. Moreover, they are one of the most commonly used viral vectors for clinical applications. However, key aspects of the activities of essential viral proteins that are commonly modified in adenoviral vectors have not been fully described. A prominent example is the multifunctional adenoviral oncoprotein E1B-55K that is known to promote efficient viral genome replication and expression while simultaneously repressing host gene expression and antiviral host responses. Our study combined different quantitative methods to study how E1B-55K promotes viral mRNA biogenesis. The data presented here propose a novel role for E1B-55K as a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes.
多功能腺病毒 E1B-55K 磷酸蛋白是病毒复制的主要调节剂，在病毒介导的细胞转化中发挥关键作用。虽然人们对其在致癌性细胞转化中的功能有了很多了解，但涉及 E1B-55K 调节病毒基因表达的基本特征和确切机制还不太清楚。因此，这项工作旨在使用野生型人腺病毒 C5（HAdV-C5）E1B-55K、一种表达缺失 E1B-55K 的病毒突变体以及一种表达磷酸模拟 E1B-55K 的突变体，揭示 E1B-55K 调节病毒 mRNA 生物发生的基本核内原理。通过亚核部分分离、mRNA、DNA 和蛋白质分析以及荧光素酶报告基因测定，我们表明：（i）E1B-55K 促进病毒晚期 mRNA 从其在病毒复制区（RC）中的合成部位有效地释放到周围核质中，（ii）E1B-55K 调节 RC 中病毒基因转录和剪接的速率，（iii）E1B-55K 参与病毒基因表达的时间调节，（iv）E1B-55K 可以增强或抑制病毒早期和晚期启动子的表达，以及（v）E1B-55K 的磷酸化调节该蛋白对这些活性的时间效应。总之，这些数据表明，E1B-55K 是 HAdV-C5 感染过程中病毒基因的一种依赖于磷酸化的转录和转录后调节剂。人类腺病毒是研究基因表达和剪接基本方面的有用模型。此外，它们是临床应用中最常用的病毒载体之一。然而，腺病毒载体中常见修饰的重要病毒蛋白的关键活性方面尚未得到充分描述。一个突出的例子是多功能腺病毒致癌蛋白 E1B-55K，它已知可促进病毒基因组的有效复制和表达，同时抑制宿主基因表达和抗病毒宿主反应。我们的研究结合了不同的定量方法来研究 E1B-55K 如何促进病毒 mRNA 的生物发生。这里提出的数据为 E1B-55K 作为病毒基因的依赖于磷酸化的转录和转录后调节剂提供了一个新的作用机制。
