Periodic Reporting for period 4 - HERPES (Herpesvirus Effectors of RNA synthesis, Processing, Export and Stability) Reporting period: 2021-11-01 to 2022-04-30 Summary of the context and overall objectives of the project Herpes simplex virus 1 (HSV-1) is an important human pathogen for which no vaccine is currently available to prevent primary infection or reduce the frequencies and severity of virus reactivations. HSV-1 intensively interacts with the cellular transcriptional machinery at multiple levels during lytic infection. Employing next-generation sequencing to study RNA synthesis, processing and translation in short intervals throughout lytic HSV-1 infection, my laboratory made the surprising observation that HSV-1 triggers widespread disruption of transcription termination of cellular but not viral genes. Transcription commonly extends for tens-of-thousands of nucleotides beyond poly(A)-sites and into downstream genes. In contrast to textbook knowledge, HSV-1 infection does not inhibit splicing but induces a broad range of aberrant splicing events associated with disruption of transcription termination. Furthermore, I found poly(A) read-through to be accompanied by a selective defect in histone repositioning downstream of genes resulting in the formation of extensive open chromatin regions. Exploring these fascinating phenomena will provide fundamental insights into RNA biology of human cells.Within the frame of HERPES, I will elucidated the molecular mechanism by which HSV-1 disrupts transcription termination of cellular genes while maintaining transcription termination of viral genes. I will employ cutting-edge methodology ranging from large-scale omics approaches to single cell analysis. I will develop reporter assays to visualise and track poly(A) read-through in single cells. I will identify novel cellular proteins governing transcription termination using a genome-wide Cas9-knockout screen. I hypothesize that the alterations in RNA processing are depicted by specific changes in RNA Polymerase II CTD phosphorylation and in the associated proteins. I will characterise these dynamic changes using mNET-seq and quantitative proteomics. Finally, data-driven quantitative bioinformatic modelling will detail how the coupling of RNA synthesis, processing, export, stability and translation is orchestrated by HSV-1. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We identified the viral ICP27 protein to be responsible for disruption of transcription termination in HSV-1 infection (Wang et al., Nature commun. 2020). ICP27 directly interacts with the cellular CPSF complex resulting in the formation of a dead-end 3’ processing complex that is unable to cleave the nascent RNA at its 3’-end. Remarkably, ICP27 also acts as a sequence-dependent activator of mRNA 3' processing for viral and a subset of host transcripts by directly binding to the nascent mRNAs. We found that poly(A) read-through in HSV-1 infection, but not in cellular stress responses, is accompanied by a selective defect in histone repositioning downstream of genes resulting in the formation of extensive open chromatin regions (Hennig et al., PLoS Pathogens 2019). We identified the viral ICP22 protein to be responsible for this effect (Djakovic et al, Nature commun. in revision). We provided a state-of-the-art reannotation of the HSV-1 genome resulting in the identification of >200 previously unknown viral ORFs and 201 viral transcripts. This include novel viral immediate early proteins and gene products missing in mutant viruses approved for oncolytic therapies.To study specific changes in RNA polymerase II CTD phosphorylation, we succesfully employed mNET-seq. Integrative analysis with published PRO-seq data revealed unexpected manipulations of promoter-proximal pausing by HSV-1 (manuscript in prep.).We extensively tested the usefulness of the RNA aptamer Broccoli for live-cell RNA imaging and found it to be not sufficiently stable and bright even upon concatemerization. We found that it is not sufficiently stable and bright to serve as a reliable reporter for single cell analyses. We pioneered metabolic RNA labeling combined with chemical nucleotide-conversion for single cell RNA sequencing (scSLAM-seq; Erhard et al., Nature 2019). This adds a temporal dimension to single cell RNA sequencing and now facilitates dose-response analyses at single cell level. This was only made possible due to the development of the computational approach GRAND-SLAM, which provides reliable new/total RNA ratios for thousands of genes in individual cells (Jürges et al, Bioinformatics 2018). We filed a patent on GRAND-SLAM.Exploiting new technologies developed in the frame of this project, we identified miRNA-mediated inhibition of miRNA processing as a so far unknown cellular mechanism by which human herpesvirus 6 disrupts mitochondrial architecture, interferes with intrinsic cellular defense mechanisms and governs the latent-lytic switch (Hennig et al., Nature 2022). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Since the publication of our scSLAM-seq approach, we have achieved compatibility with state-of-the-art droplet-based scRNA-seq approaches, i.e. 10x Chromium sequencing. Furthermore, we developed the computational framework of GRAND-SLAM to handle data for thousands of genes in tens-of-thousands of individual cells. We successfully applied our improved scSLAM-seq approach to study transcriptional regulation and cellular reprogramming during SARS-CoV-2 infection of human lung epithelial cells (manuscript in preparation).