Regulation and Herpes simplex virus 1 counter-regulation of transcriptional bursting kinetics in the early type I interferon response

I型干扰素诱导数百个基因的表达，增强对入侵病毒的控制。有效的逃避对于有效的病毒感染和长期的持续至关重要。我们发现，在巨细胞病毒感染的头两个小时内，干扰素刺激基因(ISGs)的诱导主要是由于转录爆发的可能性增加。然而，与未感染细胞中ISGs的基础表达相比，爆发的幅度没有变化。最近，DTX3L/PARP9泛素连接酶复合体被证明与STAT1/STAT2/IRF9(ISGF3)复合体结合，并泛素化ISG启动子内的组蛋白，从而增强了ISG的大亚群的表达。有趣的是，我们发现大的HSV 1被膜蛋白pUL36通过其N端去泛素化(DUB)结构域靶向DTX3L/PARP9，并抑制DTX3L/PARP9所归属的两种泛素介导的功能，即诱导ISGs和将p53结合蛋白1(53BP1)募集到DNA损伤部位。我们假设(I)通过ISGF3复合体被招募到ISG启动子中的DTX3L/PARP9有助于将ISG启动子从非允许状态切换到允许状态，从而诱导更频繁的转录突发，(Ii)HSV-1 pUL36 Dub抵消了这一作用，从而增强了生产性感染。本项目的主要目的是阐明pUL36 Dub在DXT3L/PARP9-ISGF3相互作用中调控ISGs基因特异性转录输出的机制。所获得的数据将阐明一种令人兴奋的新的细胞机制，通过这种机制，ISGs在转录爆发的水平上被调节，以及HSV-1是如何干扰它的。最后，我们将使用我们的SCSLAM-SEQ方法并开发所需的计算框架来研究单细胞水平的ISG爆发动力学。综上所述，我们将研究病毒对ISG诱导的操纵，以阐明在染色质水平上控制ISG爆发动力学的新的细胞机制。

Type I interferons (IFN) induce the expression of hundreds of genes that augment control of invading viruses. Efficient evasion thereof is crucial for productive virus infection and long-term persistence. We discovered that the induction of interferon-stimulated genes (ISGs) in the first two hours of cytomegalovirus infection predominantly results from an increased likelihood of transcriptional bursts. The magnitude of bursts, however, was unchanged compared to basal expression of ISGs in uninfected cells. Recently, the DTX3L/PARP9 ubiquitin ligase complex was shown to bind to the STAT1/STAT2/IRF9 (ISGF3) complex and ubiquitinate histones within ISG promoters, thereby enhancing the expression of a large subset of ISGs. Interestingly, we found that the large HSV 1 tegument protein pUL36 targets DTX3L/PARP9 with its N terminal deubiquitinating (DUB) domain and inhibits two DTX3L/PARP9-attributed ubiquitin-mediated functions, namely the induction of ISGs, and the recruitment of p53 binding protein 1 (53BP1) to sites of DNA damage. We hypothesize that (i) DTX3L/PARP9, which is recruited to ISG promoters via the ISGF3 complex, facilitates switching ISG promoters from a non-permissive to a permissive state and thereby induces more frequent transcriptional bursts, and (ii) that the HSV-1 pUL36 DUB counteracts this to augment productive infection. The main objective of this project is to elucidate the mechanistic role of the pUL36 DUB on the DXT3L/PARP9-ISGF3 interaction in governing the gene-specific transcriptional output of ISGs. The obtained data will illuminate an exciting new cellular mechanism by which ISGs are regulated at the level of transcriptional bursting and how HSV-1 interferes with it. Finally, we will employ our scSLAM-Seq approach and develop the required computational framework to study ISG bursting kinetics at single cell level. In summary, we will study viral manipulation of ISG induction to elucidate novel cellular mechanisms that govern ISG bursting kinetics at chromatin level.