TY - JOUR A1 - Hennig, Thomas A1 - Djakovic, Lara A1 - Dölken, Lars A1 - Whisnant, Adam W. T1 - A Review of the Multipronged Attack of Herpes Simplex Virus 1 on the Host Transcriptional Machinery JF - Viruses N2 - During lytic infection, herpes simplex virus (HSV) 1 induces a rapid shutoff of host RNA synthesis while redirecting transcriptional machinery to viral genes. In addition to being a major human pathogen, there is burgeoning clinical interest in HSV as a vector in gene delivery and oncolytic therapies, necessitating research into transcriptional control. This review summarizes the array of impacts that HSV has on RNA Polymerase (Pol) II, which transcribes all mRNA in infected cells. We discuss alterations in Pol II holoenzymes, post-translational modifications, and how viral proteins regulate specific activities such as promoter-proximal pausing, splicing, histone repositioning, and termination with respect to host genes. Recent technological innovations that have reshaped our understanding of previous observations are summarized in detail, along with specific research directions and technical considerations for future studies. KW - herpes simplex virus KW - RNA polymerase II KW - transcription KW - host shutoff KW - promoter-proximal pausing KW - C-terminal domain KW - polyadenylation KW - splicing Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246165 SN - 1999-4915 VL - 13 IS - 9 ER - TY - THES A1 - Djaković, Lara T1 - The HSV-1 ICP22 protein selectively impairs histone repositioning upon Pol II transcription downstream of genes T1 - Das HSV-1 ICP22 Protein stört selektiv die Repositionierung von Histonen bei der Transkription durch Pol-II unterhalb von Genen N2 - Herpes Simplex Virus type 1 (HSV-1) is an ubiquitous neurotropic human pathogen that infects a large majority of the world’s population. It is the causative agent of the common cold sore but also responsible for life-threatening infections (e.g., encephalitis), particularly in immunocompromised individuals and neonates. Like other herpesviruses, HSV-1 takes over the cellular RNA machinery to facilitate productive infection while efficiently shutting down host gene expression by targeting multiple steps of RNA metabolism. The two viral proteins, vhs and ICP27, play a crucial role in this process. Delivered by the tegument of the incoming virus, the virion host shut-off (vhs) endonuclease rapidly starts cleaving both cellular and viral mRNAs. With the onset of viral gene expression, the HSV-1 immediate-early protein ICP27 promotes the expression of viral early and late genes through various mechanisms, including mRNA processing, export, and translation. Prior research by the Dölken lab demonstrated that lytic HSV-1 infection results in the disruption of transcription termination (DoTT) of most cellular genes by the viral ICP27 protein. This significantly contributes to HSV-1 induced host shut-off. DoTT results in transcription for tens of thousands of nucleotides beyond poly(A) sites and into downstream genes. Interestingly, this was found to be accompanied by a dramatic increase in chromatin accessibility downstream of the affected poly(A) sites. This is consistent with the formation of extensive downstream open chromatin regions (dOCR) and indicative of impaired histone repositioning in the wake of RNA polymerase II (Pol II) downstream of the affected poly(A) sites. In my PhD thesis, I demonstrate that dOCR formation is dependent on the viral ICP22 protein when poly(A) read-through transcription is triggered by the ectopic expression of ICP27 or salt stress. I show that dOCR formation occurs when a high level of transcriptional activity arises downstream of genes due to the HSV-1-induced DoTT. To investigate whether histone composition is affected downstream of genes, I established the ChIPmentation approach to study associated changes and the influence of DoTT and dOCR formation on major histone modification marks. In HSV-1 WT infection, dOCR formation was reflected in alterations of canonical H1 histone downstream of affected genes, which was absent in ICP22 infection. To elucidate the underlying molecular mechanism, two major histone chaperones SPT6 and FACT (SPT16 and SSRP1), which govern histone repositioning and may thus play a role in H1 homeostasis, were extensively studied. Both histone chaperones have been recently shown to be recruited to the viral genome by interactions with ICP22 protein. To investigate whether the depletion of SSRP1 or SPT6 would complement the loss of ICP22 to induce dOCR, T-HF cells with doxycycline-inducible knock-down of either of the two factors were generated. ATAC-seq analysis revealed that the interaction between the two histone chaperones and ICP22 is not involved in HSV-1-induced dOCR formation, suggesting the involvement of other proteins. In summary, this work sheds new light on a fundamental molecular mechanism of the cellular transcriptional machinery that is manipulated by the concerted actions of the two HSV-1 immediate-early proteins ICP22 and ICP27. N2 - HSV-1 ist ein weit verbreitetes, neurotropisches Virus, mit welchem ein Großteil der Weltbevölkerung infiziert ist. Es verursacht milde Infektionen wie Herpes labialis, aber kann auch lebensbedrohliche Infektionen des Nervensystems (z. B. Enzephalitis) in immunsupprimierten Menschen und Neugeborenen auslösen. Um sich lytisch zu vermehren, programmiert HSV-1 die Transkriptions- und Translationsmaschinerie der Zelle effizient um und hemmt gleichzeitig an mehreren Punkten zelluläre Genexpression. Zwei virale Proteine, vhs und ICP27, spielen dabei eine entscheidende Rolle. Vhs wird im Tegument des Virions mit dem Inokulum in die Zelle geliefert und baut so zelluläre Transkripte ab noch bevor virale Genexpression startet. ICP27 wird also sogenanntes „immediate-early“ Gen als eines der ersten viralen Proteine exprimiert und kann unterstützt auf mehreren Ebenen (RNA Prozessierung, Export und Translation) die Expression der viralen „early“ und „late“ Gene. Unsere Gruppe hat diesbezüglich gezeigt, dass die Terminierung der Transkription (sogenanntes „DoTT“) durch das virale Protein ICP27 in der lytischen Infektion gestört wird. Dies trägt maßgeblich zur Abschaltung der zellulären Genexpression bei. Die Störung der Terminierung führt dazu, dass RNA Polymerase II bis zu >100 Kilobasen nach dem Polyadenylierungssignal weiter transkribiert. Durch die Aktivität der RNA Polymerase II wird in den 3‘ Regionen der betroffenen Gene das Chromatin gelockert (sogenanntes „dOCR“). Dies steht im Einklang mit einer Öffnung des Chromatins durch gehemmte Histon-Neupositionierung, verursacht durch die Transkription der betroffenen Genomregionen. Im Rahmen meiner Doktorarbeit konnte ich mittels Hochdurchsatzsequenzieranalyse von Transposon-zugänglichem Chromatin (ATAC-seq) zeigen, dass offenes Chromatin durch das virale Protein ICP22 verursacht wird. Dieser Effekt konnte unterdessen nur beobachtet werden, wenn die Terminierung der Transkription, entweder durch die gleichzeitige Expression von ICP27 oder stressinduziert z.B. durch hypertonen Lösungen, gestört wurde. Das Ausmaß an offenem Chromatin korrelierte dabei mit der Transkriptionsaktivität der entsprechenden Genomregionen. Durch bioinformatische Analysen von Hochdurchsatzsequenzierungen von Wildtyp Virus und ICP22-defizitären Mutanten infizierten Zellen konnte ich einen Cluster von stark exprimierten Genen mit ausgeprägter DoTT identifizieren, der besonders stark von der Chromatinöffnung betroffen war. Um zu testen, ob die Histone in den betroffenen Regionen durch die Induktion von dOCR verändert wurden, habe ich ein neues Chromatin-Immunpräzipitations Verfahren namens ChIPmentation etabliert und hiermit die mit der Induktion von dOCR assoziierten Histonvarianten untersucht. Dabei fiel auf, dass das H1 Histon gezielt in von dOCR betroffenen Regionen verloren ging. Um den zu Grunde liegenden Mechanismus zu untersuchen, habe ich die Rolle der beiden Histonchaperonen SPT6 und FACT (SPT16 und SSRP1) ausgiebig charakterisiert. Diese regulieren normalerweise die Repositionierung von Histonen im Zuge der Pol II Transkription. Beide Faktoren werden zudem durch ICP22 in die viralen DNA Replikationzentren im Nukleus rekrutiert, was den Positionierungsdefekt von H1 hervorrufen könnte. Um diese Hypothese zu testen, wurden beide Proteine durch induzierbare shRNA Knockdowns depletiert und mittels ATAC-seq untersucht, ob dies in der Infektion mit ICP22-defizitären Mutanten zur Öffnung des Chromatin führt. Hierbei zeigte sich allerdings, dass die Depletion dieser beider Histonchaperone kein offenes Chromatin bei Infektion mit der ICP22 Knockout Mutante erzeugt. Zudem fiel auf, dass SSRP1 selektiv dazu beitrug, Chromatin in transkribierten Regionen geschlossen zu halten. Offensichtlich spielen daher die beiden Histonchaperonen keine Rolle bei der ICP22-induzierten Öffnung des zellulären Chromatins unterhalb von Genen. KW - HSV-1 KW - Open chromatin KW - ICP22 KW - Read-through transcription Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246709 ER - TY - JOUR A1 - Hennig, Thomas A1 - Michalski, Marco A1 - Rutkowski, Andrzej J. A1 - Djakovic, Lara A1 - Whisnant, Adam W. A1 - Friedl, Marie-Sophie A1 - Jha, Bhaskar Anand A1 - Baptista, Marisa A. P. A1 - L'Hernault, Anne A1 - Erhard, Florian A1 - Dölken, Lars A1 - Friedel, Caroline C. T1 - HSV-1-induced disruption of transcription termination resembles a cellular stress response but selectively increases chromatin accessibility downstream of genes JF - PLoS Pathogens N2 - Lytic herpes simplex virus 1 (HSV-1) infection triggers disruption of transcription termination (DoTT) of most cellular genes, resulting in extensive intergenic transcription. Similarly, cellular stress responses lead to gene-specific transcription downstream of genes (DoG). In this study, we performed a detailed comparison of DoTT/DoG transcription between HSV-1 infection, salt and heat stress in primary human fibroblasts using 4sU-seq and ATAC-seq. Although DoTT at late times of HSV-1 infection was substantially more prominent than DoG transcription in salt and heat stress, poly(A) read-through due to DoTT/DoG transcription and affected genes were significantly correlated between all three conditions, in particular at earlier times of infection. We speculate that HSV-1 either directly usurps a cellular stress response or disrupts the transcription termination machinery in other ways but with similar consequences. In contrast to previous reports, we found that inhibition of Ca\(^{2+}\) signaling by BAPTA-AM did not specifically inhibit DoG transcription but globally impaired transcription. Most importantly, HSV-1-induced DoTT, but not stress-induced DoG transcription, was accompanied by a strong increase in open chromatin downstream of the affected poly(A) sites. In its extent and kinetics, downstream open chromatin essentially matched the poly(A) read-through transcription. We show that this does not cause but rather requires DoTT as well as high levels of transcription into the genomic regions downstream of genes. This raises intriguing new questions regarding the role of histone repositioning in the wake of RNA Polymerase II passage downstream of impaired poly(A) site recognition. KW - DNA transcription KW - dogs KW - thermal stresses KW - chromatin KW - histones KW - gene expression KW - cellular stress responses KW - transcriptional termination Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176350 VL - 14 IS - 3 ER - TY - JOUR A1 - Whisnant, Adam W. A1 - Jürges, Christopher S. A1 - Hennig, Thomas A1 - Wyler, Emanuel A1 - Prusty, Bhupesh A1 - Rutkowski, Andrzej J. A1 - L'hernault, Anne A1 - Djakovic, Lara A1 - Göbel, Margarete A1 - Döring, Kristina A1 - Menegatti, Jennifer A1 - Antrobus, Robin A1 - Matheson, Nicholas J. A1 - Künzig, Florian W. H. A1 - Mastrobuoni, Guido A1 - Bielow, Chris A1 - Kempa, Stefan A1 - Liang, Chunguang A1 - Dandekar, Thomas A1 - Zimmer, Ralf A1 - Landthaler, Markus A1 - Grässer, Friedrich A1 - Lehner, Paul J. A1 - Friedel, Caroline C. A1 - Erhard, Florian A1 - Dölken, Lars T1 - Integrative functional genomics decodes herpes simplex virus 1 JF - Nature Communications N2 - The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution. Here, using computational integration of multi-omics data, the authors provide a detailed transcriptome and translatome of herpes simplex virus 1 (HSV-1), including previously unidentified ORFs and N-terminal extensions. The study also provides a HSV-1 genome browser and should be a valuable resource for further research. KW - infected-cell protein KW - messenger RNA KW - binding protein KW - type 1 KW - identification KW - ICP27 KW - translation KW - expression KW - sequence KW - domain Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229884 VL - 11 ER -