No description available.

2,3-Dipalmitoyl-S-Glycerylcystein (Pam2Cys), ein Toll-like-Rezeptor 2 (TLR2)-Ligand, aktiviert dendritische Zellen und erhöht die Expression von HLA-DR auf deren Zelloberfläche. Natürliche Killerzellen (NK-Zellen) können über die Ausschüttung von Zytokinen das erworbene Immunsystem stimulieren. Zudem weist die Entdeckung von HLA-DR auf NK-Zellen, besonders auf CD56bright-NK-Zellen, auf eine Verbindung zwischen angeborenem und erworbenem Immunsystem hin. Gekoppelt an ein immundominantes Peptidepitop aus dem Glykoprotein D des Herpes-Simplex-Virus (HSV) Typ 2, wurde Pam2Cys in dieser Arbeit als potentieller lipopeptidbasierter HSV-Impfstoff eingesetzt, um die Effekte dieser Vakzine auf NK-Zellen zu untersuchen. Die Einflüsse der Vakzine wurden anhand der CD69-, HLA-DR- und TLR2-Expression auf und in NK-Zellen nach Inkubation mit Pam2Cys untersucht. Die Analyse der Gesamt-NK-Zellpopulation ließ nur mit Hilfe der quantitativen Real-Time-PCR einen signifikanten Anstieg der TLR2-Expression erkennen, jedoch nicht durchflusszytometrisch. Inkubierte NK-Zellen zeigten deutlich mehr HLA-DR auf der Zelloberfläche, als unstimulierte NK-Zellen. Die Untersuchung der zwei großen NK-Zellsubpopulationen, CD56bright und CD56dim, ergab, dass stimulierte CD56bright-Zellen wesentlich mehr HLA-DR und TLR2 aufwiesen, als stimulierte CD56dim-Zellen. Die Expression des Aktivierungsmarkers CD69 stieg in der Gesamtpopulation der NK-Zellen nicht in signifikantem Maße an, zeigte jedoch eine Zunahme nach Inkubation mit den Pam2Cys-Molekülen. Die Aufnahme der Lipopeptide in NK-Zellen wurde mit der konfokalen Mikroskopie bestätigt. Diese Ergebnisse zeigen, dass die hier verwendete HSV-Vakzine von NK-Zellen aufgenommen wird und sie die Ausbildung von Oberflächenrezeptoren auf diesen Zellen moduliert. Insbesondere die Expressionssteigerung von HLA-DR auf CD56bright-NK-Zellen deutet auf die Fähigkeit dieser Zellen hin, Antigene zu präsentieren und stellt einen weiteren Mechanismus vor, der eine impfstoffbezogene Immunantwort unterstützen kann. Die vorliegende Arbeit bildet eine Basis für weiterführende Studien an NK-Zellen hinsichtlich der Antigenpräsentation im Allgemeinen und der Antigenpräsentation nach Stimulation durch eine Vakzine im Besonderen. / The Toll-like receptor 2 (TLR2) ligand 2,3-Dipalmitoyl-S-Glycerylcystein (Pam2Cys) enhances the surface expression of HLA-DR on dendritic cells and activates them. Natural killer cells (NK cells) may stimulate the adaptive immune system via the production of cytokines. Furthermore the detection of HLA-DR on NK cells, especially on CD56bright NK cells, hints at a connection between innate and adaptive immunity. In this study Pam2Cys was used as a potential lipopeptide-based HSV vaccine attached to an immunodominant epitope in herpes simplex virus (HSV) type 2 glycoprotein D. The effects of this vaccine on NK cells were analysed here. Examination of the influence of this vaccine was performed by measuring the expression of CD69, HLA-DR and TLR2 on and in NK cells after incubating them with Pam2Cys. Using quantitative real-time PCR, a significant increase of TLR2-mRNA in the entire NK cell population was detectable; however flow cytometry was not able to confirm these results. Incubated NK cells showed considerably more HLA-DR than unstimulated NK cells. Analysis of the two big NK cell subpopulations, CD56bright and CD56dim, revealed essentially more HLA-DR and TLR2 on stimulated CD56bright cells, compared to stimulated CD56dim cells. Expression of the activation marker CD69 after incubation was not triggered significantly on NK cells altogether; nevertheless an increase could be measured. Confocal microscopy confirmed an uptake of lipopeptides into NK cells. These data demonstrate the surface receptor modulation on NK cells by a potential HSV vaccine and the ability of NK cells to endocytose Pam2Cys. Particularly, the increase of HLA-DR on CD56bright NK cells points at the ability of these cells to present antigens, introducing a new mechanism able to support a potent immune response to a vaccination. This study provides a basis for further work on NK cells in terms of antigen presentation in general and antigen presentation after stimulation by a vaccine in particular.

Zellstress in Form von lytischer Herpes-simplex-Virustyp-1-Infektion, Hitze und Salzstress führt dazu, dass die RNA-Polymerase II über das 3'-Ende von manchen Genen hinaus transkribiert. Dies geht bei Herpes-simplex-Virustyp-1-Infektion teilweise mit offenem Chromatin nach dem 3'-Ende einher. In dieser Arbeit wurden verschiedene Methoden getestet, um diese Effekte genomweit zu eruieren. Dabei wurden die Peak-Caller ATAC-seq-Pipeline, F-Seq, Hotspots und MACS2 getestet sowie mit der Hilfsgröße „downstream Open Chromatin Regions“ gearbeitet. Weiterhin wurde das R-Skript „Pipeline for ATAC-seq and 4sU-seq plotting“ entwickelt, mit dem sich die Dynamik der oben beschriebenen Effekte zeigen lässt: Die Offenheit des Chromatins ist bei Herpesinfektion zusätzlich zur Erhöhung nach dem 3'-Ende generell erhöht. Die Transkription der RNA-Polymerase II über das 3'-Ende hingegen nimmt nach 75k Basenpaaren rapide ab. Die Ergebnisse des R-Skripts im Bezug auf Salz und Hitzestress decken sich mit vorbeschriebener Literatur, in der gezeigt wurde, dass eine Erhöhung der Offenheit des Chromatins nach dem 3'-Ende nicht stattfindet. / Cell stress in the form of lytic herpes simplex virus type 1 infection, heat, and salt stress causes RNA polymerase II to transcribe beyond the 3' end of some genes. This is sometimes associated with open chromatin beyond the 3' end in herpes simplex virus type 1 infection. In this work, several methods were tested to elicit these effects genome-wide. The peak caller ATAC-seq-Pipeline, F-Seq, hotspots, and MACS2 were tested, and the auxiliary variable "downstream open chromatin regions" was used. Furthermore, the R script "Pipeline for ATAC-seq and 4sU-seq plotting" was developed to show the dynamics of the effects described above: Chromatin openness is generally increased in herpes infection in addition to being increased after the 3' end. In contrast, RNA polymerase II transcription across the 3' end decreases rapidly after 75k base pairs. The results of the R-script in relation to salt and heat stress are consistent with pre-described literature showing that an increase in chromatin openness after the 3' end does not occur.

In order to facilitate a biophysical analysis of the carboxy terminal acidic transactivation domain (AAD) of Vmw65 from Herpes Simplex Virus Type 1 (HSV-1), an overexpression system in Escherichia coli was constructed and optimized to produce milligram quantities of this polypeptide. Purification of the polypeptide was facilitated by creating a fusion protein to glutathione S-transferase (GST) from Schizosoma japonicum using a commercially available vector. Upon thrombin digestion of the fusion protein, the carrier and AAD products were resolved by anion-exchange chromatography. With typically 15 mg of AAD available from a 12 litre culture, several biophysical studies were initiated. Circular dichroism and fluorescence spectroscopy both described a polypeptide with an extended structure reminicent of a random-coil; that is, it did not possess substantial quantities of known elements of secondary structure such as a-helicies and β-sheets under physiological conditions. A new structure high in α-helical content was induced upon addition of trifluoroethanol to mimic a hydrophobic milieu. Ultracentrifugation data supported the spectroscopic observations by describing an extended, monomeric polypeptide. The ultimate goal of the study, a teritiary structure, was sought by attempting to crystallize AAD with popular salts and organic solvents. Biologically, the described random-coil structure of AAD could be relevant to its role as a promoter and stablizer of the transcriptional pre-initation complex, the determining step in gene expression. A structurally labile domain would support AAD’s ability to interact with several targets including TFIID and TFIIB, though not necessarily by similar mechanisms. The requirement for a drastic conformational change such as a random-coil to α-helical transition currently remains unclear though observations made in this study of AAD in trifluoroethanol have shown that a conformational change is indeed possible. With a means of producing large quantities of AAD, the opportunity now arises to study its interaction with available cloned targets. The ensuing biophysical studies will then provide a greater understanding of AAD’s important role in gene expression. / Thesis / Master of Science (MSc)

Gene expression in eukaryotic cells is regulated by the combinatorial action of numerous gene-regulatory factors, among which microRNAs (miRNAs) play a fundamental role at the post-transcriptional level. miRNAs are single-stranded, small non-coding RNA molecules that emerge in a cascade-like fashion via the generation of primary and precursor miRNAs. Mature miRNAs become functional when incorporated into the RNA induced silencing complex (RISC). miRNAs guide RISCs to target mRNAs in a sequence-specific fashion. To this end, base-pairs are usually formed between the miRNA seed region, spanning nucleotide positions 2 to 8 (from the 5' end) and the 3'UTR of the target mRNA. Once miRNA-mRNA interaction is established, RISC represses translation and occasionally induces direct or indirect target mRNA degradation. Interestingly, miRNAs are expressed not only in every multicellular organism but are also encoded by several viruses, predominately by herpesviruses. By controlling both, cellular as well as viral mRNA transcripts, virus-encoded miRNAs confer many beneficial effects on viral growth and persistence. Murine cytomegalovirus (MCMV) is a ß-herpesvirus and so far, 29 mature MCMV-encoded miRNAs have been identified during lytic infection. Computational analysis of previously conducted photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking immunoprecipitation (PAR-iCLIP) experiments identified a read cluster within the 3' untranslated region (3'UTR) of the immediate early 3 (IE3) transcript in MCMV. Based on miRNA target predictions, two highly abundant MCMV miRNAs, namely miR-m01-2-3p and miR-M23-2-3p were found to potentially bind to two closely positioned target sites within the IE3 PAR-iCLIP peak. To confirm this hypothesis, we performed luciferase assays and showed that activity values of a luciferase fused with the 3'UTR of IE3 were downregulated in the presence of miR-m01- 2 and miR-M23-2. In a second step, we investigated the effect of pre-expression of miR-m01-2 and miR-M23-2 on the induction of virus replication. After optimizing the transfection procedure by comparing different reagents and conditions, plaque formation was monitored. We could demonstrate that the replication cycle of the wild-type but not of our MCMV mutant that harbored point mutations in both miRNA binding sites within the IE3-3'UTR, was significantly delayed in the presence of miR-m01-2 and miR-M23-2. This confirmed that miR-m01-2 and miR-M23-2 functionally target the major transcription factor IE3 which acts as an indispensable regulator of viral gene expression during MCMV lytic infection. Repression of the major immediate early genes by viral miRNAs is a conserved feature of cytomegaloviruses. The functional role of this type of regulation can now be studied in the MCMV mouse model. / In eukaryotischen Zellen wird die Expression von Genen durch das Zusammenspiel vieler verschiedener biologischer Regulatoren, wie microRNAs (miRNAs), kontrolliert. MiRNAs sind einzelsträngige, kurze, nicht-kodierende RNA-Moleküle, die aus sogenannten primären miRNAs und Vorläufer-miRNAs entstehen und die Genexpression auf Ebene der Posttranskription beeinflussen. Um ihre Funktion ausüben zu können, werden reife miRNAs in RNA-induzierte Silencing-Komplexe (RISCs) eingebaut und zu ihren Ziel-mRNAs geführt. Durch Wechselwirkungen zwischen der miRNA "seed-Region , die die Nukleotide 2 bis 8 vom 5'-Ende überspannt und der 3'UTR (3' untranslatierte Region) der Ziel-mRNA, unterdrückt RISC die Translation der Ziel-mRNA und kann deren Abbau durch direkte sowie indirekte Mechanismen induzieren. Die Expression von miRNAs wurde nicht nur in multizellulären Organismen, sondern in bereits zahlreichen Viren, insbesondere in der Virusfamilie der Herpesviridae, nachgew- iesen. Viruskodierte miRNAs kontrollieren dabei zelluläre wie auch virale mRNA-Transkripte und verleihen dem Virus einen Selektionsvorteil bzgl. Wachstum und Persistenz. Das mur- ine Cytomegalievirus (MCMV) ist ein β-Herpesvirus, das nach aktuellem Wissensstand 29 reife miRNAs kodiert, die allesamt während der lytischen Infektion identifziert wurden. Bioinformatische Analysen eines vor dieser Arbeit durchgeführten PAR-iCLIP-Experiments (photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking and immunoprecipitation), zeigten einen PAR-iCLIP Peak in der 3'UTR (3' untranslatierte Region) des immediate early 3-Transkripts (IE3) von MCMV. Unter Verwendung von RNAhbybrid, einem miRNA target prediction tool, fanden sich zwei virale miRNAs, näm- lich miR-m01-2-3p und miR-M23-2-3p mit potentiellen Bindestellen innerhalb der 3'UTR des MCMV IE3 Transkripts. Unsere konsekutiv durchgeführten Luciferase-Assays be- stätigten, dass sowohl miR-m01-2 als auch miR-M23-2 an die 3'UTR von IE3 binden. Beide viralen miRNAs führten zu einer verminderten Luciferaseaktivität unter Verwendung von Reportern, in denen die 3'UTR des IE3-Gens mit dem Luciferase-Transkript fusioniert war. xxiv Summary Das IE3 Protein gilt während des lytischen Zykluses als einer der wichtigsten Transkrip- tionsfaktoren von MCMV. Ebenfalls wurde der Einfluss der beiden viralen miRNAs auf die virale Reproduktion von uns untersucht. Hierfür wurden murine Zelllinien vor Infektion mit miR-m01-2 und miR- M23-2 transziert. Das Transfektionsverfahren optimierten wir zunächst durch Testung verschiedener Reagenzien und experimenteller Bedingungen. Schließlich zeigten wir mittels Plaqueassays, dass eine vor Infektion durchgeführte Transfektion mit miR-m01-2 und miR- M23-2 die Replikation von MCMV signifikant verzögerte. Unter Verwendung einer MCMV- Mutante, die durch Punktmutationen in beiden miRNA-Bindungsstellen innerhalb der IE3- 3'UTR charakterisiert war, ließ sich dieser Effekt aufheben. Unsere Experimente weisen somit stark darauf hin, dass miR-m01-2 und miR-M23-2 die Expression des IE3 Proteins regulieren und damit indirekt Einfluss auf die Genexpression während der lytischen Phase des Replikationszykluses von MCMV nehmen. Die miRNA-mediierte Repression der immediate early Genexpression stellt ein evolutionär konserviertes Merkmal von Zytomegalieviren dar. Für eine weitere Einordnung der Rolle dieser Genexpressionskontrolle bedarf es zukünftige Untersuchungen im MCMV-Tiermodell

OBJECTIVE: The overarching goal of this study was to examine the immunostimulatory potential of oHSV-1 rQNestin34.5v2 in syngeneic murine GBM models, perform in vitro screens for upregulation of immune checkpoint molecules in infected glioma cells, and evaluate the antitumor activity of the most promising combination immunovirotherapies. METHODS: The oncolytic activity of HSV-1 rQNestin34.5 was evaluated in CT-2A and GL261 syngeneic murine glioma models. Immunoassays were conducted to assess secretion of damage associated molecular patterns including ATP, HMGB1, Calreticulin, HSP70 and other proinflammatory mediators by infected glioma cells. In vitro screens for expression of inhibitory ligands by glioma cells following HSV-1 rQNestin34.5v2 infection at various doses were analyzed by flow cytometry. Intratumoral HSV-1 rQNestin34.5v2 administration and/or intraperitoneal anti-TIGIT (clone 1B4)/anti-NK1.1 treatments were performed in C57BL/6 mice bearing orthotopic CT-2A glioma to determine effect on overall survival. RESULTS: HSV-1 rQNestin34.5v2 exhibited greater capacity to infect CT-2A and minimal capacity to infect GL261 cells suggesting differences in permissiveness in HSV- 1 replication between the two GBM models. Infection stimulated immunogenic cell death as evidenced by surface expression of calreticulin and HSP70 and elevated extracellular release of ATP and HMGB1 in the GL261 model. CD155 and CD112 (both ligands of TIGIT) as well as PD-L1 were significantly highly expressed in glioma cells. TIGIT was found to be overexpressed in tumor infiltrating NK, CD4 and CD8 T cells suggesting systemic therapy with TIGIT blockade antibodies could have therapeutic utility in combination with HSV-1 rQNestin34.5v2 in GBM. Benefit in overall survival was not observed by anti-TIGIT monotherapy, and combination treatment with HSV-1 rQNestin34.5v2 exhibited modest therapeutic effect with a cure rate 25% in mice bearing intracranial CT-2A tumors. Depletion of NK cells prior to HSV-1 rQNestin34.5v2 administration attenuated brain edema and synergized with rQNestin34.5v2 virotherapy. CONCLUSION: Our findings show that the combination of HSV-1 rQNestin34.5v2 virotherapy with anti-TIGIT checkpoint blockade immunotherapy and/or NK cell inhibition represents a promising strategy to overcome primary resistance to immune checkpoint inhibitors in GBM. / 2025-02-03T00:00:00Z

No description available.

Host cell reactivation (HCR) of UV-irradiated (UV'd) herpes simplex virus type 2 (HSV-2), capacity of UV'd cells to support HSV-2 plaque formation and UV enhanced reactivation (UVER) of UV'd HSV-2 were examined in human fibroblasts. The cells were derived from four Cockayne's Syndrome (CS) patients, 5 xeroderma pigmentosum (XP) patients and 5 normal patients. Survival curves for HCR of HSV-2 plaque formation showed 2- components. HCR was not significantly different in the CS strains and an XP variant strain compared to normal, whereas all excision deficient strains showed a significant reduction in HCR. The o37 values for the delayed capacity curves were in the range 8.6-12.4 J/m2 for the normal strains, 3.1-5.1 J/m2 for the CS strains, 6.7 J/m2 for an XP variant strain and between 0.40-1.98 J/m2 for the XP excision deficient strains examined. UVER was also examined for HSV-2 UV-irradiated to survival levels of 10-2 and 10-3 in unirradiated cells. Maximum delayed UVER was observed in normal strains at a UV dose of 15 J/m2 to the virus. Maximum UVER in CS cells was detected at a UV dose of 5 J/m2 to the cells, in XP excision deficient cells maximum UVER occurred at doses ranging from 0.5-2.5 J/m2 to the cells, and in XP variant maximum UVER occurred at 10 J/m2 to the cells. In all cell strains the level of UVER increased with increasing UV dose to the virus. Results are discussed in terms of the repair defects of CS and XP cells and their relationship to possible viral repair functions. In addition, the possible existence of an inducible DNA repair response is discussed in terms of the results of this study. / Thesis / Master of Science (MSc)

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are major human pathogens. HSV establishes latency in the nervous system and reactivates to cause recurrent disease, resulting in transmission of progeny virions to naïve individuals. Though HSV-1 and HSV-2 share similar structure and genes, they have distinctive recurrence profiles. Generally, HSV-1 reactivation is associated with disease 'above the waist' and HSV-2 reactivation is associated with disease 'below the waist'. This phenomenon was described decades ago but still remains unexplained. The mechanism of HSV latent infection in the peripheral nervous system (PNS) has been extensively investigated, especially with in sensory neurons. Another component of the peripheral nervous system (PNS), autonomic neurons, were also known to be infected with HSV productively and latently, but largely ignored because of the assumption that there is no difference in the pathogenesis of HSV in the neurons and that both HSV-1 and HSV-2 behave in the same way in different types of neurons. However, autonomic neurons differ in physiological function compared to sensory neurons. Activation factors of autonomic neurons, such as emotional stress, trauma and hormonal fluctuation, are also known HSV reactivation triggering factors. Therefore, I hypothesized that autonomic neurons innervating the site of HSV infection are responsible the different reactivation frequencies of HSV-1 and HSV-2 after peripheral invasion. In this report, the role of autonomic neurons in HSV pathogenesis were examined using the female guinea pig reactivation model. Major findings of this report are that 1) parasympathetic ganglia innervating the ocular region support latent infection of HSV-1 selectively, thus contributing the more frequent HSV-1 reactivation, 2) mixed autonomic ganglia in the genital area support HSV-2 latent infection selectively, and 3) sympathetic neurons in the genital region supported productive and latent infection of HSV-1 and HSV-2 differently. All of the results in this report indicate that autonomic neurons play a distinctive role in HSV pathogenesis compared to the sensory neurons and are responsible for the different reactivation frequencies of HSV-1 and HSV-2. This report raises the importance of autonomic neurons in HSV pathogenesis and challenges the paradigm of HSV pathogenesis. / Ph. D.

The contrasting infection strategy of herpes simplex virus (HSV) consists of an initial primary lytic infection in epithelial cells, followed by establishment of lifelong latency in sensory and autonomic neurons of the peripheral nervous system that innervate the site of infection. Any cellular stress trigger, ranging from external stimuli such as UV radiation or nerve injury to psychological and physiological stress, can reactivate HSV from latency in the neurons, resulting in recurrent disease episodes. Stress hormones and deprivation of neurotrophic factor (NTF) both have a strong correlation with HSV reactivation from neurons. However, neuronal signaling pathways cardinal to HSV latency and reactivation are still not clear. This dissertation provides new understanding of HSV latency and reactivation in response to two orthogonal stress stimuli, viz. stress hormones epinephrine (EPI) and corticosterone (CORT), as well as NTF deprivation that simulates a nerve injury in primary neuronal cultures. In this dissertation, we demonstrate that physiological stress hormones EPI and CORT differentially regulate HSV-1 and HSV-2 reactivation in adult neurons. Both EPI and CORT treatment reactivated only HSV-1 in sympathetic superior cervical ganglia (SCG) neurons, while HSV-2 was reactivated only by CORT in both sensory trigeminal ganglia (TG) neurons and sympathetic superior cervical (SCG) neurons. EPI utilized the combination of α and β adrenergic receptor complex, while CORT signaled through glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) to reactivate HSV in the neurons. NTFs are tissue-target derived growth factors required for neuronal protection and survival. Neurotrophins are also required for maintaining HSV latency, as NTF deprivation reactivates both HSV-1 and HSV-2 in adult sensory TG and sympathetic SCG neurons. In addition, assessing the temporal kinetics of HSV gene expression showed differential expression profiles of viral immediate-early (IE) genes ICP0, ICP4, ICP27 and trans-activator VP16 following treatment with stress hormones and NTF deprivation in HSV-1 and HSV-2 infected neurons. We also show that different molecular mechanisms are involved in HSV latency and reactivation, which are dependent on the stimuli and the type of neurons. Tyrosine kinase receptor-mediated PI3K-Akt-mTORC signaling cascades have been studied for their role in maintaining HSV latency. Activation of β-catenin signalosome expression has also been implicated during HSV latency and following reactivation. GSK3β is a key effector molecule that inter-connects Akt and β-catenin mediated pathways, forming an Akt-GSK3β-β-catenin signaling axis. Analyzing the Akt-GSK3β-β-catenin signaling in response to stress hormone and NTF deprivation revealed significant differences in protein expression levels between HSV-1 and HSV-2 infected sensory and sympathetic neurons. In HSV-1 infected neurons, the Akt-GSK3β-β-catenin maintains the signal transmission in order to keep the neurons alive, but HSV-2 infections obliterated the entire axis in the adult neurons, particularly in sympathetic neurons. In summary, we demonstrate that HSV-1 and HSV-2 do not have a 'one for all' infection mechanism. Establishment of latency and reactivation by HSV is virus specific, stimulus specific and neuron specific. / Doctor of Philosophy / Herpes simplex viruses (HSVs) are common global viral pathogens that are responsible for causing lifelong painful infections and debilitating disease. The two serotypes of HSV include HSV-1, which is associated with oral or ocular disease but can also cause genital disease, and HSV-2, which is predominantly associated with genital herpes. Once infected, both HSV-1 and HSV-2 are present as lifelong reservoirs in our peripheral neurons. Stress stimuli mediated by our stress hormones or external triggers, such as nerve trauma or an axonal injury, can periodically reactivate the latent virus to cause recurrent disease. Clinical manifestation of HSV recurrences range from asymptomatic viral shedding to painful blisters, cold sores, or herpetic keratitis. In some cases, the virus can spread to the central nervous system, causing encephalitis or recurrent meningitis. No vaccines have been approved yet, and the current treatment utilizes nucleoside analogs, such as acyclovir and its prodrug valacyclovir, to ameliorate the symptoms of HSV infection by halting viral replication and if taken as a daily prophylaxis, reduces the chances of clinical recurrence. Given the route and transmission efficiency of HSV, it is practically impossible to prevent herpes infection. To develop strategic therapeutic interventions to lock the virus in its latent phase in the neurons and prevent it from reactivation, a better understanding of neuronal signaling pathways cardinal to HSV latency and reactivation is necessary. However, neuronal signaling pathways cardinal to HSV latency and reactivation are still not clear. In this dissertation, we make contributions to better understand HSV latency and reactivation in response to stress stimuli. We show that different stress stimuli exert preferential reactivation between HSV-1 and HSV-2, and are further dependent upon the neurons where they establish latency. Our study specifically focuses on three neuronal stressors that have been associated with HSV recurrences: two stress hormones, epinephrine (EPI) and corticosterone (CORT), as well as deprivation of neurotrophic factors (NTF) that simulates nerve injury. We also focused on a neuronal signaling cascade involved in the response to all of these stimuli, Akt-GSK3β-β-catenin, and viral gene transcripts that respond to these stimuli during reactivation. Comprehensive understanding of the neuronal processes and viral gene transcripts involved during HSV-1 and HSV-2 reactivation in neurons will help the herpes virology field towards development of targeted therapies and vaccines to prevent reactivation and recurrent disease.