Infection of a host cell by the Herpes Simplex Virus Type 1 leads to the efficient reprogramming of the cells' synthetic machinery to replicate the viral genome ultimately producing progeny virions. Two proteins introduced upon viral fusion are thought to initiate this effect. The potent transactivator of immediate early genes (VP16) and the mRNA destabilizing virion host shutoff protein (vhs), work in concert with one another to invoke the cascade of viral gene expression, and to destroy pre-existing cellular mRNA. Due to the non-specific nature of vhs induced mRNA degradation, its activity is downregulated at later times during infection to spare virally encoded mRNA. Recent evidence has shown that VP16 is responsible for this vhs downregulation, a process thought to occur by mutual interactions between the two proteins and a potential compartmentalization of vhs within the nucleus (Lam 𝘦𝘵 𝘢𝘭., 1996; Smibert 𝘦𝘵 𝘢𝘭., 1994). Furthermore, such an event is also thought to position vhs so it can be efficiently packaged, a supposition supported by the observation that vhs lacking the ability to bind VP16 is not incorporated into new virions (Read 𝘦𝘵 𝘢𝘭. , 1993). To ascertain if VP16 was indeed capable of relocalizing vhs to the nucleus of a cell in the absence of any other viral factors, we created multiple constructs consisting of various portions of vhs fused in frame to the fluorescent marker protein EGFP. In addition, various truncated forms of VP16 were also fused to EGFP for the purpose of delineating the region of VP16 that is responsible for VP16 and possibly vhs nuclear localization. Co-transfection experiments utilizing EGFP-vhs fusions demonstrated that vhs relocalizes to perinuclear regions in the presence of VP16, an effect absolutely dependent upon its ability to interact with VP16. In addition, deletion mapping of VP16 implicated the region spanning amino acids 335 to 355 as being necessary for this localization, with a stretch of 15 amino acids (330 to 344) appearing to constitute a putative bipartite nuclear localization signal. Interestingly, our observation that the vhs/VP16 complex localizes to a region of the cell thought to ultimately encompass the tegument of new virions gives credence to the notion that this interaction and subsequent localization may indeed function to package vhs into new virions. Furthermore, it is also suggested that vhs may in fact be downregulated at intermediate times during infection through VP16 mediated compartmentalization within the nucleus. For these reasons we propose that the disruption of the vhs/VP16 interaction could severely abrogate the infectivity of HSV and as such could present a novel target for antiviral intervention. / Thesis / Master of Science (MS)

Herpes simplex virus 1 (HSV-1) is a human pathogen that modulates many aspects of RNA metabolism and chromatin structure. We have previously reported that HSV-1 infection leads to disruption of transcription termination (DoTT) by cellular RNA Polymerase II (Pol II) of many cellular genes. By employing various mutant viruses, we observed that the HSV-1 immediate early protein ICP27 is a major contributor to DoTT. This termination defect contributes to host cell shut-off as affected read-through transcripts are retained in the nucleus and thus cannot be translated. HSV-1-induced DoTT also leads to a massive increase in transposon-accessible chromatin in downstream gene regions mediated by the viral immediate-early protein ICP22. The interplay of ICP22 with cellular and viral proteins contributes to various processes in host nuclear systems ranging from alterations of Pol II to chaperone relocalization. To better understand the role of ICP22 in open chromatin formation, a deep understanding of its interaction partners and the involved protein domains is required. Here, we report on a detailed list of ICP22 interaction partners and potential sites of interaction by Co-IP mass spectrometry (MS) analysis of both full-length as well as partial ICP22 deletion mutants. We found that many of the identified ICP22 interactors are functionally linked to the phosphorylation of the Pol II C-terminal domain, transcription regulation, and histone modifications. Additionally, we created a set of ICP22 mutants comprising small deletions of highly conserved regions or point mutations in the core region. Analysis of these mutants using ATAC-seq suggests that the ICP22's conserved core region is crucial for the induction of dOCR. Although some mutants were significantly attenuated compared to WT, a correlation between fitness and induction of open chromatin downstream of genes was not observed. Additional work is required to completely understand the molecular mechanism and biological significance of this process, as well as all the cellular factors involved. This research makes important advancements in our understanding of the ICP22 protein, its interactions with cellular factors, the importance of its domains and associated changes, as well as the consequences of those changes. Additionally, the datasets generated in this study will be important to support subsequent research on ICP22 and its role in HSV-1 infection. / Das Herpes-simplex-Virus 1 (HSV-1) ist ein menschlicher Krankheitserreger, der viele Aspekte des RNA-Stoffwechsels und der Chromatinstruktur moduliert. Wir haben in früheren Arbeiten beschrieben, dass eine HSV-1-Infektion zu einer Unterbrechung der Transkriptionstermination (DoTT) durch die zelluläre RNA-Polymerase II (Pol II) bei vielen zellulären Genen führt. Durch den Einsatz verschiedener mutierter Viren konnten wir zeigen, dass das HSV-1-"immediate early protein" ICP27 einen wesentlichen Beitrag zur DoTT leistet. Dieser Terminierungsdefekt trägt zur Abschaltung der Wirtszelle bei, da die betroffenen ReadThrough-Transkripte im Zellkern verbleiben und somit nicht in Proteine übersetzt werden können. HSV-1-induzierte DoTT führt zudem zur Bildung von transposon-zugänglichem Chromatin in downstream gelegenen Genregionen, was durch das Immediate-Early-Protein ICP22 vermittelt wird. Das Zusammenspiel von ICP22 mit zellulären und viralen Proteinen trägt zu verschiedenen Prozessen in Wirtskernsystemen bei, die von Veränderungen von Pol II bis zur Relokalisierung von Chaperonen reichen. Um die Rolle von ICP22 bei der Bildung von offenem Chromatin zu verstehen, ist ein tiefes Verständnis seiner Interaktionspartner und der beteiligten Proteindomänen erforderlich. Daher haben wir mittels Co-IP-Massenspektrometrie (MS)-Analyse eine detaillierte Liste von ICP22-Interaktionspartnern und potenziellen Interaktionsstellen sowohl von Volllängen- als auch von partiellen ICP22-Deletionsmutanten erstellt. Im Folgenden konnten wir zeigen, dass viele der identifizierten ICP22-Interaktoren funktionell mit der Phosphorylierung der C-terminalen Domäne von Pol II, der Transkriptionsregulation und istonmodifikationen verbunden sind. Darüber hinaus haben wir eine Reihe von ICP22 Mutanten mit Deletionen in hochkonservierten Regionen oder Punktmutationen in der Kernregion erstellt. Die Analyse dieser Mutanten mittels ATAC-seq legt nahe, dass die konservierte Kernregion von ICP22 für die Induktion von „downstream open chromatin“ (dOCR) entscheidend ist. Obwohl einige Mutanten im Vergleich zu WT signifikant geschwächt waren, wurde keine Korrelation zwischen der Fitness und der Induktion von offenem Chromatin in den downstream Genen beobachtet. Weitere Arbeiten sind erforderlich, um den molekularen Mechanismus und die biologische Bedeutung dieses Prozesses sowie alle beteiligten zellulären Faktoren umfassend zu verstehen. Diese Forschung bringt wichtige Fortschritte in unserem Verständnis des ICP22-Proteins, seiner Interaktionen mit zellulären Faktoren, der Bedeutung seiner Domänen und ihrer Veränderungen sowie der Folgen dieser Veränderungen für die Infektion der Zelle. Darüber hinaus werden die in dieser Studie erzeugten Datensätze für die weitere Forschung zu ICP22 und seiner Rolle bei der HSV-1 Infektion von Bedeutung sein.

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