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

I型干扰素（IFN）诱导数百个基因的表达，这些基因增强对入侵病毒的控制。其有效逃避对于生产性病毒感染和长期持久性至关重要。我们发现，在巨细胞病毒感染的前两个小时内，干扰素刺激基因（ISG）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ISG的基础表达相比，爆发的幅度没有变化。最近，DTX 3L/PARP 9泛素连接酶复合物被证明与STAT 1/STAT 2/IRF 9（ISGF 3）复合物结合，并使ISG启动子内的组蛋白泛素化，从而增强ISG的大子集的表达。有趣的是，我们发现大的HSV 1被膜蛋白pUL 36以其N末端去泛素化（DUB）结构域靶向DTX 3L/PARP 9，并抑制DTX 3L/PARP 9归因于泛素介导的两种功能，即诱导ISG和将p53结合蛋白1（53 BP 1）募集到DNA损伤位点。我们假设（i）DTX 3L/PARP 9通过ISGF 3复合物募集到ISG启动子，促进ISG启动子从非允许状态转换到允许状态，从而诱导更频繁的转录爆发，和（ii）HSV-1 pUL 36 DUB抵消这一点以增加生产性感染。本项目的主要目的是阐明pUL 36 DUB对DXT 3L/PARP 9-ISGF 3相互作用在控制ISGs的基因特异性转录输出中的机制作用。所获得的数据将阐明一个令人兴奋的新的细胞机制，通过该机制ISG在转录爆发水平上受到调节，以及HSV-1如何干扰它。最后，我们将采用我们的scSLAM-Seq方法并开发所需的计算框架来研究单细胞水平上的ISG爆发动力学。总之，我们将研究ISG诱导的病毒操纵，以阐明在染色质水平上控制ISG爆发动力学的新细胞机制。

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.