Les héparanes sulfates (HS) sont des polysaccharides sulfatés qui participent à de nombreux processus physiopathologiques. L’entrée du virus HSV-1 dans ses cellules cibles nécessite l’intervention de la glycoprotéine gD, qui peut interagir avec quatre récepteurs différents: HVEM (Herpes Virus Entry Mediator), les nectines-1 et -2, et des HS 3-O-sulfatés. De plus, la protéine gD est impliquée dans la protection des cellules cibles contre l’apoptose. Nos travaux ont montré que les réponses anti-apoptotiques induites par la gD nécessitent la coopération entre HVEM et les HS 3-O-sulfatés. Chez l’Homme, sept 3-O-sulfotransférases (3-OSTs) sont impliquées dans la réaction de 3-O-sulfatation, qui se distinguent par des différences d’expression et de spécificité de substrat. Selon l'environnement inflammatoire, les macrophages subissent deux types de polarisation, caractérisés par des phénotypes et des fonctions distinctes. Les travaux du Laboratoire ont montré que la 3-OST3B est fortement induite dans les macrophages pro-inflammatoires, à l’inverse de la 3-OST2. Nous avons alors montré que l’expression de la 3-OST3B est régulée de manière transcriptionnelle et post-transcriptionnelle. En revanche, la diminution de la 3-OST2 fait intervenir des mécanismes différents, dont l’action d’un microARN. La dernière partie de la thèse a porté sur la localisation subcellulaire des 3-OSTs. Nos résultats montrent que la 3-OST3B est localisée dans le Golgi, alors que la 3-OST2 et la 3-OST3A sont retrouvées jusqu’à la membrane plasmique. Dans leur ensemble, nos résultats suggèrent une fonction spécifique pour chaque 3-OST dans la génération de motifs HS 3-O-sulfatés distincts. / Heparan sulfates (HS) are sulfated polysaccharides able to modulate several normal and pathological processes. Binding of Herpes-Simplex virus HSV-1 to its target cells is mediated in part by the glycoprotein gD. This protein can interact with four receptors: HVEM (Herpes Virus Entry Mediator), nectin-1 and -2, and 3-O-sulfated HS. In addition, gD is capable of protecting host cells against apoptosis. In this context, we showed that gD protects macrophages against apoptosis by a mechanism dependent on the cooperation between HVEM and 3-O-sulfated HS. The reaction of 3-O-sulfation can be catalyzed by seven 3-O-sulfotransferases (3-OSTs), which are differentially expressed and exhibit fine substrate specificity. Depending on the immune environment, macrophages can undergo proinflammatory (M1) or alternative (M2) polarization, resulting in distinct phenotypes and functions. A previous study of the laboratory has showed that 3-OST3B is strongly induced in M1 macrophages, while 3-OST2 is poorly detected. We then demonstrated that the induction of 3-OST3B is regulated at the transcriptional and post-transcriptional levels upon M1 polarization. Conversely, the reduction of 3-OST2 expression involved distinct mechanisms, including the action of a microRNA.The last part of the thesis focused on the subcellular localization of 3-OSTs. Our results demonstrated that 3-OST3B is localized in the Golgi apparatus, while 3-OST2 and 3-OST3A reach the plasma membrane. Altogether, our results suggest that each 3-OST isoenzyme may be involved in the generation of distinct 3-O-sulfated HS motifs.

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. / 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.

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VP16 initiates the HSV replication cycle by activating immediate early (IE) gene expression. It recruits the RNA pol II through an acidic C-terminal domain. The defective VP16 encoded by the V422 mutant of HSV-1 possesses a truncated C-terminal domain. Therefore, V422 replication is suppressed in most cell-lines, except U2OS osteosarcoma cells. The permissive phenotype of U2OS cells stems from a failure to express one or more inhibitory factors that are produced in restrictive cells. The initial project was designed to identify these host inhibitory factors in restrictive cells of V422, using siRNA silencing technology. To facilitate the siRNA screen, a GFP reporter gene has been inserted into the thymindine kinase (TK) gene of the V422 genome and the wild-type KOS genome. This thesis provides information about characterizing the kinetics of GFP expression from recombinant viruses at both protein and mRNA levels, during different infection times in HeLa and Vero cells. / Virology

Unlike RNA viruses, which typically encode their own RNA-dependent RNA polymerases, DNA viruses typically utilize host RNA Polymerase II (RNAPII) to transcribe their genes. Therefore, host factors that interact with RNAPII often maintain important regulatory roles during DNA virus infections. Senataxin (SETX) is a ubiquitously expressed 303 kDa RNA:DNA helicase that associates with RNAPII. It is involved in the resolution of R-loops and plays a role during the DNA damage response. Mutations in SETX are implicated in the neurodegenerative diseases Type 2 Ataxia with Oculomotor Apraxia (AOA2) and juvenile Amyotrophic Lateral Sclerosis (ALS4). Recent work from our group has demonstrated that SETX also acts as an antagonist of the antiviral response during RNA virus infections. Infections, including those caused by Herpes Simplex Virus type I (HSV-1), have been identified as potential environmental triggers of neurodegenerative diseases. Therefore, we elected to study the role of SETX during DNA virus infections since, in addition to regulating host genes, it may also play a role in viral transcription and/or DNA replication. Our data suggests that SETX is involved in the regulation of viral gene expression, and that SETX facilitates DNA replication and contributes to viral biogenesis. SETX attenuates the antiviral response, and in mouse models of infection, is protective against HSV-1 disease pathogenesis. These studies have enhanced our understanding of the role played by SETX during viral infection and may shed light on the mechanism(s) through which SETX dysfunction results in neurodegenerative diseases. / Thesis / Master of Health Sciences (MSc) / DNA viruses utilize host proteins in gene expression, therefore, associated factors play roles during DNA virus infections. Senataxin (SETX) is a RNA:DNA helicase associated with these proteins. SETX mutations are implicated in the neurodegenerative diseases Type 2 Ataxia with Oculomotor Apraxia (AOA2) and juvenile Amyotrophic Lateral Sclerosis (ALS4). Recently, our group demonstrated SETX antagonizes antiviral responses to RNA virus infections. Infections, including those caused by Herpes Simplex Virus type I (HSV-1), are identified as potential triggers of neurodegenerative diseases. We elected to study the role of SETX during DNA virus infections. Our data suggests that SETX is involved in the regulation of viral gene expression, facilitates HSV-1 DNA replication, attenuates the antiviral response, and in mouse models of infection, is protective against HSV-1 disease pathogenesis. These studies enhance understanding of the role of SETX during viral infection and may shed light on the mechanism(s) of SETX role in neurodegenerative disease.

Herpes simplex virus is an important model in the study of temporally regulated gene expression in eukaryotic cells. Three classes of genes - immediate early, early, and late - are sequentially expressed during the course of lytic infection. One immediate early gene product, ICP4, is required for transactivation of most early and late genes; it is also implicated in repression of immediate early gene expression. ICP4's mechanism(s) of action is/are not yet understood; although ICP4 binds to specific sequences of DNA, whether this is necessary for transregulation by ICP4 is not clear. To gain a better understanding of how the ability of ICP4 to bind DNA relates to its transregulatory activities, I introduced ICP4 binding sites into a simple model promoter within the viral genome. Two sets of construct were made in which an ICP4 binding site (or mutant site) was placed either downstream or upstream of a TATA box, reproducing the spacing found in (i) the native 𝘐𝘊𝘗4 promoter and (ii) the native 𝘐𝘊𝘗0 promoter (respectively). The promoter of HSV-1 𝘜𝘓24𝘣 (a nonessential gene in tissue culture) was replaced with these model promoters and levels of transcripts accumulating from these constructs during lytic infection assayed by primer extension. I found that an ICP4 site placed either upstream or downstream of a TATA box shifted kinetics of expression from E/leaky L to true L. Neither the strength of the TATA box nor the helical orientation of the ICP4 binding site with respect to the TATA box affected this result. / Thesis / Master of Science (MS)

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Infection of most cell types with herpes simplex virus (HSV) mutants lacking the activation functions of VP16 and/or ICP0 results in repression of viral gene expression. However, the human osteosarcoma cell line U2OS supports the replication of VP16 and ICP0 mutants to nearly wild type levels. Prior to the studies presented in this thesis, the basis for the permissivity of U2OS cells to VP16 and ICP0 mutants had not been explored. Here, somatic cell fusion assays were used to determine that U2OS cells support the replication of VP16 and ICP0 mutants due to a defect in an innate gene silencing mechanism. The artificial induction of interferon stimulated genes that occurs during the somatic cell fusion assays is not the basis for the observed repression of viral gene expression. As one means of identifying components of the antiviral pathway defective in U2OS cells, restrictive cell types were treated with kinase inhibitors and infected with VP16 and/or ICP0 mutants. Although several compounds were identified which compensate for the defect in gene expression of VP16 mutants, these drugs also stimulate mutant virus gene expression in U2OS. Thus, U2OS are most likely not defective in the cellular signalling pathway(s) targeted by these compound(s). Finally, the importance of VP16 and ICP0 in modulating chromatin structure on the viral genome in both restrictive and permissive cells was examined, uncovering an essential role for both proteins in altering histone occupancy and acetylation levels. Importantly, U2OS cells have a defect in the chromatin-based pathway targeted by ICP0. However, evidence suggests that the ability of VP16 and ICP0 to affect histone occupancy and acetylation levels is not required for viral gene expression. Taken together, the results of this thesis demonstrate that U2OS cells support the replication of VP16 and ICP0 mutants due to a defect in an innate antiviral mechanism which does not involve the targets of several well characterized kinase inhibitors. The significance of the defect in a chromatin-based pathway targeted by ICP0 in U2OS cells remains to be elucidated. / Virology