Institute for Virology and Immunobiology, Julius-Maximilians-University Würzburg, Würzburg, Germany.
Hannover Medical School, Institute of Virology, Hannover, Germany.
J Virol. 2024 Oct 22;98(10):e0117824. doi: 10.1128/jvi.01178-24. Epub 2024 Sep 24.
Transcriptional activity of RNA polymerase II (Pol II) is influenced by post-translational modifications of the C-terminal domain (CTD) of the largest Pol II subunit, RPB1. Herpes simplex virus type 1 (HSV-1) usurps the cellular transcriptional machinery during lytic infection to efficiently express viral mRNA and shut down host gene expression. The viral immediate-early protein ICP22 interferes with serine 2 phosphorylation (pS2) by targeting CDK9 and other CDKs, but the full functional implications of this are not well understood. Using Western blotting, we report that HSV-1 also induces a loss of serine 7 phosphorylation (pS7) of the CTD during lytic infection, requiring expression of the two immediate-early proteins ICP22 and ICP27. ICP27 has also been proposed to target RPB1 for degradation, but we show that pS2/S7 loss precedes the drop in total protein levels. Cells with the RPB1 polyubiquitination site mutation K1268R, preventing proteasomal degradation during transcription-coupled DNA repair, displayed loss of pS2/S7 but retained higher overall RPB1 protein levels later in infection, indicating this pathway is not involved in early CTD dysregulation but may mediate bulk protein loss later. Using α-amanitin-resistant CTD mutants, we observed differential requirements for Ser2 and Ser7 for the production of viral proteins, with Ser2 facilitating viral immediate-early genes and Ser7 appearing dispensable. Despite dysregulation of CTD phosphorylation and different requirements for Ser2/7, all CTD modifications tested could be visualized in viral replication compartments with immunofluorescence. These data expand the known means that HSV employs to create pro-viral transcriptional environments at the expense of host responses.IMPORTANCECells rapidly induce changes in the transcription of RNA in response to stress and pathogens. Herpes simplex virus (HSV) disrupts many processes of host mRNA transcription, and it is necessary to separate the actions of viral proteins from cellular responses. Here, we demonstrate that viral proteins inhibit two key phosphorylation patterns on the C-terminal domain (CTD) of cellular RNA polymerase II and that this is separate from the degradation of polymerases later in infection. Furthermore, we show that viral genes do not require the full "CTD code." Together, these data distinguish multiple steps in the remodeling of RNA polymerase during infection and suggest that shared transcriptional phenotypes during stress responses do not revolve around a core disruption of CTD modifications.
RNA 聚合酶 II（Pol II）的转录活性受最大 Pol II 亚基 RPB1 的 C 末端结构域（CTD）的翻译后修饰影响。单纯疱疹病毒 1 型（HSV-1）在裂解感染过程中篡夺细胞转录机制，以有效地表达病毒 mRNA 并关闭宿主基因表达。病毒早期蛋白 ICP22 通过靶向 CDK9 和其他 CDK 干扰丝氨酸 2 磷酸化（pS2），但人们对其完整的功能意义还了解甚少。通过 Western blot 分析，我们报告说 HSV-1 在裂解感染过程中还诱导 CTD 上丝氨酸 7 磷酸化（pS7）的丧失，这需要两种早期蛋白 ICP22 和 ICP27 的表达。ICP27 也被提议针对 RPB1 进行降解，但我们表明 pS2/S7 的丧失先于总蛋白水平下降。具有 RPB1 多泛素化位点突变 K1268R 的细胞（该突变可防止转录偶联的 DNA 修复过程中的蛋白酶体降解）显示出 pS2/S7 的丧失，但在感染后期仍保留更高的总 RPB1 蛋白水平，表明该途径不参与早期 CTD 失调，但可能介导后期的大量蛋白质丢失。使用α-鹅膏蕈碱抗性 CTD 突变体，我们观察到 Ser2 和 Ser7 对病毒蛋白产生的不同需求，Ser2 促进病毒早期基因，而 Ser7 似乎不需要。尽管 CTD 磷酸化失调和 Ser2/7 的不同需求，但免疫荧光检测显示所有测试的 CTD 修饰都可以在病毒复制隔间中观察到。这些数据扩展了 HSV 用于在宿主反应的代价上创造有利于病毒转录的环境的已知手段。
重要提示：细胞会迅速响应应激和病原体改变 RNA 的转录。单纯疱疹病毒（HSV）破坏了宿主 mRNA 转录的许多过程，有必要将病毒蛋白的作用与细胞反应区分开来。在这里，我们证明病毒蛋白抑制细胞 RNA 聚合酶 II 的 C 末端结构域（CTD）上的两个关键磷酸化模式，并且这与感染后期聚合酶的降解分开。此外，我们表明病毒基因不需要完整的“CTD 密码”。总的来说，这些数据区分了感染过程中 RNA 聚合酶重塑的多个步骤，并表明应激反应中的共享转录表型并非围绕 CTD 修饰的核心破坏展开。
