ATRX promotes transcription initiation of HSV-1 immediate early genes [HEP2 PRO-seq]

Herpes simplex virus 1 (HSV-1) transcribes its genome in a highly coordinated temporal cascade, utilizing the host RNA polymerase II (Pol II). Repression of transcription precedes the cascade's progression in a process requiring viral immediate early (IE) genes, in a phenomenon termed Transient Immediate Early gene Mediated Repression (TIEMR). Given that components of promyelocytic leukaemia nuclear bodies (PML-NBs) are known to rapidly engage incoming HSV-1 genomes, we investigated their potential role in TIEMR regulation. Using siRNA knockdown of PML-NB constituents (PML, DAXX, and ATRX), we unexpectedly observed that ATRX depletion resulted in a significant reduction in nascent viral transcription on viral IE promoters at 1.5 hours post-infection (hpi). ChIP-Seq analysis indicated ATRX is associated with both highly transcriptionally active and transcriptionally restricted regions, indicating that ATRX has diverse functions in the viral genome. We show here that ATRX association with active transcription on IE genes is correlated to the presence of G-quadruplexes (G4s). Drug stabilization of G4s mimicked the effects of ATRX knockdown, significantly reducing transcription initiation on IE genes. These findings suggest that ATRX promotes transcriptional initiation of HSV-1 IE genes by preventing G4 formation. In sum, our results reveal a previously unrecognized pro-transcriptional role for ATRX early in HSV-1 infection. Cells were infected with HSV-1F at an MOI of 5 and infection allowed to proceed for 1.5 hpi. Nuclei were extracted and subjected to PRO-Seq.

单纯疱疹病毒1型（Herpes simplex virus 1, HSV-1）借助宿主的RNA聚合酶II（RNA polymerase II, Pol II），以高度协调的时序级联方式完成其基因组的转录。该转录级联反应的推进需以转录抑制为前置步骤，此过程依赖病毒即刻早期（immediate early, IE）基因参与，这一现象被称为瞬时即刻早期基因介导的抑制（Transient Immediate Early gene Mediated Repression, TIEMR）。鉴于早幼粒细胞白血病核小体（promyelocytic leukaemia nuclear bodies, PML-NBs）的组成成分可快速结合侵入宿主的HSV-1基因组，本研究探讨了其在TIEMR调控中的潜在作用。通过小干扰RNA（small interfering RNA, siRNA）敲低PML-NB的组成蛋白（PML、DAXX及ATRX），我们意外发现，在感染后1.5小时（hpi），ATRX的缺失会导致病毒即刻早期启动子区域的新生病毒转录水平显著降低。染色质免疫共沉淀测序（ChIP-Seq）分析显示，ATRX既可结合转录高度活跃的区域，也可结合转录受限的区域，表明ATRX在病毒基因组中发挥多种功能。本研究证实，ATRX在即刻早期基因上与活跃转录的关联与G-四链体（G-quadruplexes, G4s）的存在相关。对G4s进行药物稳定化处理可模拟ATRX敲低的效应，显著降低即刻早期基因的转录起始水平。上述研究结果表明，ATRX通过抑制G4结构的形成，促进HSV-1即刻早期基因的转录起始。综上，本研究揭示了ATRX在HSV-1感染早期此前未被发现的促转录功能。本实验以感染复数（multiplicity of infection, MOI）为5的HSV-1F毒株感染细胞，感染持续至1.5 hpi时收集样本，提取细胞核后进行新生RNA测序（PRO-Seq）分析。