Transcription and DNA replication of herpes simplex virus type 1 (HSV-1) occur in nuclear domains adjacent to structures named ND10. The HSV-1 single-stranded DNA binding protein ICP8 localizes to these nuclear domains to direct the assembly of the pre- and replication compartments. Inhibition of cyclin dependent kinases with roscovitine inhibits HSV-1 DNA replication, even in the presence of all required HSV-1 proteins, at an unidentified step. Here I show that roscovitine inhibits the localization of pre-expressed ICP8 to new replication sites. Therefore, the inhibition of HSV-1 DNA replication occurs at a step prior to initiation. I next evaluated the mechanisms of inhibition of proper ICP8 localization. ICP8 was extracted at lower salt concentrations from roscovitine-treated than untreated cells, but the affinity of ICP8 for ssDNA in vitro was not affected. I propose that roscovitine inhibits HSV-1 DNA replication by inhibiting DNA accessibility. I also discuss alternative mechanisms.

Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication, the viral genes have adapted the strategy of being recognized and processed by the cellular biosynthetic machineries. Viruses therefore provide an important tool to study the cellular machinery regulating gene expression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, to study RNA processing at the level of pre-mRNA splicing in mammalian cells. During a lytic infection, HSV cause an almost complete shut-off of host cell gene expression. Importantly, HSV infection cause inhibition of pre-mRNA splicing which is possibly advantageous to the virus, as only four HSV genes contain introns. The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptional processes such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor of pre-mRNA splicing. We show that ICP27 inhibits pre-mRNA splicing in vitro in the absence of other HSV proteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27 induced inhibition of splicing can be reversed, either by the addition of purified SR proteins or by the addition of an SR protein specific kinase, SRPK1. We propose that SR proteins are prime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing. In order to learn more about how splicing is organized in the cell nucleus in vivo, we investigated how cellular splicing factors are recruited to sites of transcription and splicing in adenovirus infected cells using confocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Our results demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein is required for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 is redundant and insufficient for the translocation of the protein to active viral polymerase II genes in adenovirus infected cells.

Fracture repair is a regenerative event that involves the precise coordination of a variety of cells for successful healing process. Within the microstructure hierarchy of bone repair, the predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts. Although the role of osteoblasts during fracture healing has been previously shown, their role during the initiation phase of endochondral fracture repair remains unclear. In order to study the role of osteoblasts during fracture repair, we used a transgenic mouse model expressing the herpes simplex virus thymidine kinase gene in early differentiating osteoblasts, which allows conditional ablation of cells in osteoblastic lineage upon treatment with the Gancicolvir drug. Results from this study suggest that not only are osteoblasts required in later stages of fracture repair as the medium for bone synthesis, and osteoclast activation during bone remodelling, but could also be required for the initiation and advancement of the endochondral ossification process.

Fracture repair is a regenerative event that involves the precise coordination of a variety of cells for successful healing process. Within the microstructure hierarchy of bone repair, the predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts. Although the role of osteoblasts during fracture healing has been previously shown, their role during the initiation phase of endochondral fracture repair remains unclear. In order to study the role of osteoblasts during fracture repair, we used a transgenic mouse model expressing the herpes simplex virus thymidine kinase gene in early differentiating osteoblasts, which allows conditional ablation of cells in osteoblastic lineage upon treatment with the Gancicolvir drug. Results from this study suggest that not only are osteoblasts required in later stages of fracture repair as the medium for bone synthesis, and osteoclast activation during bone remodelling, but could also be required for the initiation and advancement of the endochondral ossification process.

Herpes simplex viruses (HSV) establish latent infections in sensory neurons of their host and are maintained in this state by little understood mechanisms that, at least in part, are regulated by signalling through nerve growth factor (NGF) and its receptor tropomyosin related kinase, trkA. Previous studies have demonstrated that Zhangfei is a transcriptional factor that is expressed in differentiated neurons and is thought to influence HSV replication and latency. Zhangfei, like the HSV trans-activator VP16 and Luman, binds the ubiquitous nuclear protein host cell factor (HCF) inhibiting the ability of VP16 and Luman to initiate HSV replication. <p>Recently, Brn3a, another neuronal factor thought to influence HSV latency and reactivation was found to possess an HCF-binding domain and could potentially require HCF for activity. The neuronal POU IV domain protein, Brn3a, among its many regulatory functions has been described as an enhancer of the NGF receptor trkA, during development in mouse. I therefore investigated the possible link between Brn3a, TrkA, NGF signaling, HCF, Zhangfei and HSV-1 latency and reactivation. I hypothesized that Zhangfei would also suppress the ability of Brn3a to activate the expression of TrkA and that this would have an impact on NGF-TrkA signaling and, consequently on HSV-1 reactivation from latency.<p>My first study determined which Brn3a/trkA promoter interactions were important for trkA transcription. I constructed a plasmid that contains 1043 base pairs of genomic sequences that extend from 30 nucleotides upstream of trkA coding region. In contrast to previous data, a short 190 bp region that lies proximal to the trkA initiation codon was sufficient for Brn3a trans-activation in NGF-differentiated PC12, Vero and human medulloblastoma cells. At least two portions of the 190 bp fragment bind to Brn3a. In addition, Brn3a increased endogenous levels of trkA transcripts in PC12 cells and initiated trkA expression in medulloblastoma cells, which normally do not express trkA. <p>The second step was to determine the effects of HCF and Zhangfei association with Brn3a on trkA trans-activation. I found that Brn3a required HCF for activating the trkA promoter and that Zhangfei has a suppressive effect over Brn3a-trkA activation in non-neuronal cells. In sympathetic neuron-like NGF-treated PC12 cells, Zhangfei did not suppress the ability of Brn3a to activate the TrkA promoter, however, Zhangfei was able capable of inducing the expression of TrkA in the absence of Brn3a. Both Brn3a and Zhangfei induced the expression of endogenous trkA in PC12 cells.<p>Since Vero and PC12 cells are not from human origin I wanted to examine the ability of Zhangfei to induce trkA transcription in medulloblastoma cells, that because of its tumor nature do not express trkA. TrkA transfections in these cells have shown to drive them to cell arrest or apoptosis. Since Zhangfei is not express in medulloblastoma tumors I then used ONS-76 medulloblastoma cells as a model to determine Zhangfeis envolvement in the NGF-trkA signaling pathway.<p> I show herein that in ONS-76 medulloblastoma cells resveratrol, an inducer of apoptosis and differentiation, increased the expression of Zhangfei and trkA as well as Early Growth Response Gene 1 (Egr1), a gene normally activated by NGF-trkA signalling. ONS-76 cells stop growing soon after treatment with resveratrol and a portion of the cell undergo apoptosis. While the induction of Zhangfei in resveratrol-treated cells was modest albeit consistent, the infection of actively growing medulloblastoma cells with an adenovirus vector expressing Zhangfei mimicked the effects of resveratrol. Zhangfei activated the expression of trkA and Egr1 and caused these cells to display markers of apoptosis. The phosphorylation of Erk1, an intermediate kinase in the NGF-trkA signaling critical for differentiation, was observed in Zhangfei infected cells, supporting the hypothesis that Zhangfei is a mediator of trkA-NGF signaling in theses cells leading either to differentiation or apoptosis. Binding of HCF by Zhangfei did not appear to be required for this effect as a mutant of Zhangfei incapable of binding HCF was also able to induce the expression of trkA and Egr1. <p>In in vivo and in vitro models of HSV-1 latency, the virus reactivates when NGF supply to the neuron is interrupted. Based on the above evidence Zhangfei, in HSV-1 latently infected neurons, would have the ability to prolong a state of latency by inducing trkA expression allowing the activation of NGF-trkA signaling pathway. Since NGF is produced by many cell types it is possible that reactivation is triggered not by a decrease in NGF but by a down-regulation of TrkA expression.Therefore, if Zhangfei expression is suppress the trkA signaling could be interrupted or shifted towards apoptosis signaling, this would allow neuronal HCF-binding proteins like Luman, which can activate HSV IE expression, to initiate HSV IE expression and subsequently viral replication.

L'estudi de la utilització del sistema immunològic de l'hoste pels virus com a patògens per al seu propi benefici fou el principal objectiu d'aquest treball. Concretament, en aquest treball de tesis es van analitzar dues espècies d'herpesvirus virulents en humans, l'Herpes Simplex Virus-1 (HSV-1) i l'Epstein Barr Virus (EBV). L'entrada d'ambdós virus a les cèl.lules és mediada per receptors del sistema immunològic. Es va estudiar la interacció dels respectius receptors cel.lulars, Herpes Virus Entry mediator A (HveA), i el receptor de complement 2 (CR2), amb les glicoproteïnes virals que s'hi uneixen, i amb llurs lligands naturals, els quals participen en la defensa de l'hoste. Es va caracteritzar la interacció dels receptors HveA i CR2 amb els seus lligands fent servir proteïnes recombinants o purificades de sèrum; en el cas de HveA ens vam centrar en la localització del lloc d'unió de cada lligand al receptor. En el cas de CR2, es va analitzar la cinètica d'unió dels seus lligands naturals. Per a ambdós receptors, es va analitzar si la unió de la proteïna viral al receptor podria interferir i/o modular-ne la unió dels seus lligands naturals. Els resultats es van analitzar dins del marc de la resposta immunològica de l'hoste mediada pel receptor cel·lular, i en relació al possible paper modificador d'aquesta resposta per part de la proteïna viral. Per a facilitar el nostre estudi sobre HveA, es van cercar nous lligands peptídics d'aquest receptor, utilitzant llibreries aleatòries de pèptids expressades en el fag M13. Es van aïllar dos pèptids, i es va estudiar la seva interacció amb el receptor i la seva capacitat d'inhibir l'entrada del virus a les cèl·lules, és a dir, com a possibles agents terapèutics. / Our goal in the present work was to study the manipulation of the host immune system by an infecting virus to its own benefit. Specifically, we studied two herpes viruses; Herpes Simplex Virus-1 (HSV-1) and Epstein Barr Virus (EBV), which infect humans. Entry of both viruses into the cell is mediated by the interaction of a specific viral surface glycoprotein with two receptors that participate in the host immune response, the Herpes Virus Entry mediator A (HveA), and complement receptor 2 (CR2). We studied the interaction of these receptors with the viral glycoproteins as well as their host ligands. The latter play a role in the immune response of the host. We characterized these interactions by using recombinant as well as serum-purified proteins. Our study of HveA sought to localize the specific binding site of each ligand on the receptor, while that of CR2 consisted in the kinetic analysis of its interaction with its ligands. We also analyzed whether binding of the viral glycoprotein to each receptor would interfere or modulate its interaction with its host ligands. Our results were analyzed in the context of the role of these receptors in the host immune response, and specifically whether the viral proteins studied undermined the host's ability to defend itself from infection. To study the relationship between HveA, its natural ligands, and the viral proteins involved in HSV entry, we also screened two phage-displayed combinatorial peptide libraries for peptide ligands of a recombinant form of HveA. We isolated two peptides, and studied their interaction with HveA as well as their ability to block HSV entry into HveA-bearing cells.

Fourier transform infrared (FTIR) microscopy has shown promise as an analytical tool for detecting changes in cells and tissues, such as those due to viral infection, apoptosis induction or malignancy. In many cases, diagnosis via FTIR microscopy can be undertaken on a timescale shorter than that required for other physical or histological techniques. In this work we have used FTIR microscopy to study Vero cells that have been infected with herpes simplex virus (type I) and adenovirus. We have studied cellular samples at various time intervals following exposure to the virus. Several spectral regions were identified that allow discrimination between infected and uninfected Vero cell samples at 24 hours post exposure to both HSV1 and adenovirus. Spectral features were also identified that could be used to discriminate infected cells within 2-6 hours after exposure to both viruses. FTIR microscopy is therefore a useful tool for following the kinetics of viral infection in the 2-24 hours time range, at least at the levels of infection used in this study. In a second type of study, FTIR microscopy was used to study apoptosis induction in acute lymphoblastic leukemia T-cells. Apoptosis was induced in T-cells in three different ways. We show that FTIR microscopy can be used to distinguish T-cells in the early stages of apoptosis from normal cells. We also provide data that may suggest that FTIR microscopy can distinguish cells that have undergone apoptosis via different pathways. For most of the FTIR microscopic studies on cellular samples we have focused on the collection of spectral data in the 1500-800 cm-1 region. Spectra were collected for control cells and variously treated cells. The two sets of cells were then analyzed statistically using: 1) pair-wise comparison, 2) logistic regression, 3) partial least square regression, 4) principle component fed linear discriminant analysis and 5) hierarchical cluster analysis. The statistical analyses rigorously quantify to what extent treated and untreated cells can be distinguished. Since different statistical methods give differing results for the same data, it is important the right statistical method should be applied. The basis for these differences is discussed.

Herpes simplex viruses (HSV) establish latent infections in sensory neurons of their host and are maintained in this state by little understood mechanisms that, at least in part, are regulated by signalling through nerve growth factor (NGF) and its receptor tropomyosin related kinase, trkA. Previous studies have demonstrated that Zhangfei is a transcriptional factor that is expressed in differentiated neurons and is thought to influence HSV replication and latency. Zhangfei, like the HSV trans-activator VP16 and Luman, binds the ubiquitous nuclear protein host cell factor (HCF) inhibiting the ability of VP16 and Luman to initiate HSV replication. <p>Recently, Brn3a, another neuronal factor thought to influence HSV latency and reactivation was found to possess an HCF-binding domain and could potentially require HCF for activity. The neuronal POU IV domain protein, Brn3a, among its many regulatory functions has been described as an enhancer of the NGF receptor trkA, during development in mouse. I therefore investigated the possible link between Brn3a, TrkA, NGF signaling, HCF, Zhangfei and HSV-1 latency and reactivation. I hypothesized that Zhangfei would also suppress the ability of Brn3a to activate the expression of TrkA and that this would have an impact on NGF-TrkA signaling and, consequently on HSV-1 reactivation from latency.<p>My first study determined which Brn3a/trkA promoter interactions were important for trkA transcription. I constructed a plasmid that contains 1043 base pairs of genomic sequences that extend from 30 nucleotides upstream of trkA coding region. In contrast to previous data, a short 190 bp region that lies proximal to the trkA initiation codon was sufficient for Brn3a trans-activation in NGF-differentiated PC12, Vero and human medulloblastoma cells. At least two portions of the 190 bp fragment bind to Brn3a. In addition, Brn3a increased endogenous levels of trkA transcripts in PC12 cells and initiated trkA expression in medulloblastoma cells, which normally do not express trkA. <p>The second step was to determine the effects of HCF and Zhangfei association with Brn3a on trkA trans-activation. I found that Brn3a required HCF for activating the trkA promoter and that Zhangfei has a suppressive effect over Brn3a-trkA activation in non-neuronal cells. In sympathetic neuron-like NGF-treated PC12 cells, Zhangfei did not suppress the ability of Brn3a to activate the TrkA promoter, however, Zhangfei was able capable of inducing the expression of TrkA in the absence of Brn3a. Both Brn3a and Zhangfei induced the expression of endogenous trkA in PC12 cells.<p>Since Vero and PC12 cells are not from human origin I wanted to examine the ability of Zhangfei to induce trkA transcription in medulloblastoma cells, that because of its tumor nature do not express trkA. TrkA transfections in these cells have shown to drive them to cell arrest or apoptosis. Since Zhangfei is not express in medulloblastoma tumors I then used ONS-76 medulloblastoma cells as a model to determine Zhangfeis envolvement in the NGF-trkA signaling pathway.<p> I show herein that in ONS-76 medulloblastoma cells resveratrol, an inducer of apoptosis and differentiation, increased the expression of Zhangfei and trkA as well as Early Growth Response Gene 1 (Egr1), a gene normally activated by NGF-trkA signalling. ONS-76 cells stop growing soon after treatment with resveratrol and a portion of the cell undergo apoptosis. While the induction of Zhangfei in resveratrol-treated cells was modest albeit consistent, the infection of actively growing medulloblastoma cells with an adenovirus vector expressing Zhangfei mimicked the effects of resveratrol. Zhangfei activated the expression of trkA and Egr1 and caused these cells to display markers of apoptosis. The phosphorylation of Erk1, an intermediate kinase in the NGF-trkA signaling critical for differentiation, was observed in Zhangfei infected cells, supporting the hypothesis that Zhangfei is a mediator of trkA-NGF signaling in theses cells leading either to differentiation or apoptosis. Binding of HCF by Zhangfei did not appear to be required for this effect as a mutant of Zhangfei incapable of binding HCF was also able to induce the expression of trkA and Egr1. <p>In in vivo and in vitro models of HSV-1 latency, the virus reactivates when NGF supply to the neuron is interrupted. Based on the above evidence Zhangfei, in HSV-1 latently infected neurons, would have the ability to prolong a state of latency by inducing trkA expression allowing the activation of NGF-trkA signaling pathway. Since NGF is produced by many cell types it is possible that reactivation is triggered not by a decrease in NGF but by a down-regulation of TrkA expression.Therefore, if Zhangfei expression is suppress the trkA signaling could be interrupted or shifted towards apoptosis signaling, this would allow neuronal HCF-binding proteins like Luman, which can activate HSV IE expression, to initiate HSV IE expression and subsequently viral replication.

No description available.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 89-103). Also available on the Internet.