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

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.

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.

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus, often acquired during childhood, that currently infects more than 50% of the human population. The symptoms of infection are herpetic lesions that frequently appear throughout a host's life in response to stress in the orofacial or genital region. As a pathogen, HSV-1 replicates rapidly in epithelial cells, but it is also capable of infecting neurons where it can pursue a lytic or latent infection. Latency is a state of viral quiescence where the virus can persist indefinitely yet remain poised to reactivate. Latency is unique to herpesviruses and key to HSV's success, but the molecular mechanisms that govern this state are unclear. A virus-encoded E3-ubiquitin ligase, Infected Cell protein 0 (ICP0), is often correlated with latency establishment but is detected in opposition to the state of latency. During lytic infection, ICP0 has many biological roles but primarily catalyzes the addition of ubiquitin to target substrate, marking proteins for degradation or altering their function. This ubiquitination ability allows ICP0 to alter the intracellular environment making neurons conducive to lytic or latent HSV-1 infection. ICP0's neuron-specific targets, however, are unknown, representing a significant gap in knowledge. Through the studies presented in this dissertation, we identified some of the neuron-specific ubiquitination targets of ICP0 in neurons. We utilized primary adult sensory neurons of the dorsal root ganglia and HSV-1 viral strains KOS, wild-type virus encoding a fully functional ICP0, and HSV-1 n212, encoding a truncated ICP0 protein, to illuminate the mechanisms involved in establishing and maintaining HSV latency. By using adult primary neurons and functional HSV-1 strains with and without ICP0, we were able to show that ICP0 regulates host and viral proteins during the initial onset of neuronal infection. We also show that based on neuronal conditions set forth before HSV-1 initial infection, host proteins will influence HSV-1 viral proteins to repress viral gene expression, thereby promoting the establishment of latency. / Doctor of Philosophy / Herpes simplex virus (HSV-1) is a virus, often acquired during childhood, that more than 50% of people have. Those who are infected with HSV-1 often have cold sores that appear in response to stress on the face or on the genitals. As a virus, HSV-1 replicates around the eyes, nose, and mouth but can also infect neurons where it can continue to replicate or establish latency. Latency is when the virus is inside the neurons but is unnoticeable and can reappear in response to stress. The state of latency is unique to herpesviruses and key to the success of HSV-1, but scientists are unsure of how it works. A protein made by the virus, Infected Cell Protein 0 (ICP0), is often correlated with the state of latency but is often present when the virus is not latent. ICP0 does a lot to support HSV-1, but it primarily destroys proteins that prevent the virus from replicating. By destroying proteins that prevent HSV-1 replication, ICP0 can help the virus make more viruses. The proteins that are destroyed by ICP0 are currently unknown, which represents a significant gap in knowledge. Through the research conducted in this dissertation, we identified some of the proteins that ICP0 destroys in neurons. We utilized neurons from the dorsal root ganglia and HSV-1 viral strain KOS, which encoded a functional ICP0, and n212, which encodes a nonfunctional ICP0, to study the mechanisms used by the virus to infect neurons. By using HSV-1 viruses with and without ICP0, we were able to show what proteins ICP0 destroys during infection in neurons. We were also able to show that HSV-1's ability to establish latency is dependent on how the neurons handle the initial onset of infection. Overall, a combination of host and viral proteins coordinates the virus's ability to establish latency and persist within a host.