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)

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.

In order to examine the potential of HSV-1 as a vector to study the expression of DNA repair genes in mammalian cells, a recombinant virus containing the den V gene from bacteriophage T4 has been constructed. This gene encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Transfection studies indicate that excision repair deficient xeroderma pigmentosum (XP) group A cells are able to carry out excision repair initiated by the den V gene product. This gene along with the 3' LTR of Rous Sarcoma Virus and the SV40 polyadenylation signals were inserted into the non-essential glycoprotein I gene of HSV-1. Immunoprecipitation studies confirmed the production of the den V protein in virus infected cells. The uv survival of this HSV-1:den V recombinant virus was examined in various primary cell types. The cells examined in this study were primary fibroblasts from a normal individual, a Trichothiodystrophy patient and five XP patients as well as a mouse L cell line. The ability of the virally encoded den V gene to restore the excision repair deficiency in these cells was measured by monitoring the uv survival of HSV-1:den V as compared to wildtype HSV-1. Increased survival of HSV-1:den V was detected in Trichothiodystrophy cells, and in cells from XP complementation groups A, C and D, but not in XP cells from complementation groups E and F or in mouse L cells. These results demonstrate that HSV can be effectively used to study the expression of a cloned DNA repair gene in a variety of cell types. HSV has a substantial capacity of gene insertion and a wide host range including cells of human and rodent origin. / Thesis / Master of Science (MS)

The den V gene from bacteriophage T4 codes for a small, pyrimidine dimer specific, endonuclease. Recent studies have shown that transfection of the gene into DNA excision repair deficient, Xeroderma Pigmentosum cells, can partially restore the excision repair ability of the cells and results in an increased resistance to UV light. In this study the den V gene has been inserted into Herpes Simplex Virus type 1 (HSV 1) in order to determine if HSV 1 can be used as a suitable vector for studying DNA repair genes. A 1.9 kb cartridge containing the den V gene, the 3' LTR of Rous Sarcoma Virus as the promoter, and the SV40 polyadenylation signals was inserted as the thymidine kinase locus of the virus. Properly initiated transcription form the construct, HDV 1, was verified by primer extension analysis. The Host cell reactivation of this virus and several other strains of HSV 1 were examined in normal and Xeroderma Pigmentosum cells. The results from these experiments suggest that both the viral DNA polymerase and thymidine kinase genes play important roles for the survival of UV irradiated HSV 1. / Thesis / Master of Science (MS)

Many viral transcriptional activators have been shown to activate genes of heterologous systems. To assess the ability of the herpes simplex virus ICP4 trans-activating protein to complement an adenovirus mutant lacking its own trans-activator, the E1a protein, I constructed an adenovirus type 5 vector containing a temperature sensitive ICP4 gene, under control of its own promoter, within the E1 region of the genome. The recombinant virus expresses ICP4 in human cells which are permissive (293) or nonpermissive (KB and R970-5) for E1a⁻ viral replication, and at levels which approximate those obtained in herpes simplex infection. The adenovirus encoded protein is functional in that it complements an ICP4 deletion mutant of herpes simplex virus, however it is incapable of complementing adenovirus E1a⁻ mutants for viral growth or DNA replication. At the level of activation of gene expression, ICP4 stimulates the expression of the adenovirus E2a gene but not that of other early genes. My results indicate that ICP4 does not possess all of the functions of the E1a proteins and, furthermore, that adenovirus early genes differ in their susceptibility to heterologous trans-activators. / Thesis / Master of Science (MS)

Herpes simplex virus (HSV) infections are common in all human populations and for most people they represent relatively mild lifelong infections. To facilitate the persistent infection of hosts, HSV has evolved immune evasion strategies which suppress various aspects of the immune response including the actions of complement and antibodies. Previously in our laboratory, an HSV immediate early protein called ICP47 was shown to inhibit the MHC class I antigen presentation pathway and thereby block recognition of virus infected cells by CD8+ cytotoxic T lymphocytes (CTL). This thesis explores the potential cellular targets of ICP47. Using immunoprecipitation I found ICP47 associates with the transporter associated with antigen presentation (TAP). By blocking the transport of peptide antigens into the endoplasmic reticulum, MHC class I molecules become unstable and are subsequently degraded before displaying HSV antigens on the cell surface. Thus, CTL destruction of cells infected with HSV is blocked. In addition, an interaction between an ICP47 bacterial fusion protein, called GSTICP47-1 and a cellular protein, calcyclin, was examined. The functions of calcyclin are largely unknown. However, based on its association with ICP47, it was possible that calcyclin might play a role in the class I pathway -perhaps as the peptide shuttle. Nevertheless, the results of several experiments were consistent with the notion that calcyclin and ICP47 may not interact in vivo and that calcyclin may not play a role in the MHC class I antigen presentation pathway. / Thesis / Master of Science (MS)

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.