The effect of brn3a and zhangfei on the nerve growth factor receptor, trkA.

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. 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.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. 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.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 Zhangfei’s envolvement in the NGF-trkA signaling pathway. 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. 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

Механизмы длительной персистенции вируса простого герпеса в организме хозяина

Латентність є складовою частиною життєвого циклу вірусу простого герпесу. Резидентні вірусні геноми знаходяться в чутливих нейронах. Під час латентной фази інфекції відзначається експресія лише однієї (LAT) області вірусної ДНК. Невелика її частина довжиною тільки 348 п.н. відповідальна за реактивуцію дрімаючого вірусу. Гени в межах LAT мають антиапоптотические властивості, що забезпечує виживання нейронів під час реактивації. При цитуванні документа, використовуйте посилання http://essuir.sumdu.edu.ua/handle/123456789/33235Латентность является составной частью жизненного цик-ла вируса простого герпеса. Резидентные вирусные гено-мы находятся в чувствительных нейронах. Во время ла-тентной фазы инфекции отмечается экспрессия лишь одной (LAT) области вирусной ДНК. Небольшая ее часть длиной только 348 п.н. ответственна за реактива-цию дремлющего вируса. Гены в пределах LAT имеют антиапоптотические свойства, что обеспечивает выжива-ние нейронов во время реактивации. При цитировании документа, используйте ссылку http://essuir.sumdu.edu.ua/handle/123456789/33235Latency of HSV-1/2 is a complicated virus-host interaction that plays a crucial role in the pathogenic potential of this virus. Numerous studies have indicated that sensory neurons are the primary sites of HSV latency. The ability of these viruses to reactivate from latency is responsible for recurrent disease and virus transmission. Since LAT is only known viral transcript that are abundantly transcribed in latently in-fected neurons, it is reasonable to hypothesize that it regulate latency. LAT protein expression is tightly regulated and may occur only at specific times during latency to prevent immune recognition. Recent studies demonstrating that the genes en-coding LAT have antiapoptotic properties strongly suggest that this function plays a crucial role in promoting neuronal survival and thus latency. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/3323

Towards an Understanding of the Herpes Simplex Virus Type 1 Latency-Reactivation Cycle

Infection by herpes simplex virus type 1 (HSV-1) can cause clinical symptoms in the peripheral and central nervous system. Recurrent ocular shedding can lead to corneal scarring and vision loss making HSV-1 a leading cause of corneal blindness due to an infectious agent. The primary site of HSV-1 latency is sensory neurons within trigeminal ganglia. Periodically, reactivation from latency occurs resulting in virus transmission and recurrent disease. During latency, the latency-associated transcript (LAT) is abundantly expressed. LAT expression is important for the latency-reactivation cycle in animal models, in part, because it inhibits apoptosis, viral gene expression, and productive infection. A novel transcript within LAT coding sequences (AL3) and small nonprotein coding RNAs are also expressed in trigeminal ganglia of latently infected mice. In this review, an update of viral factors that are expressed during latency and their potential roles in regulating the latency-reactivation cycle is discussed

Как вирусу простого герпеса удается пожизненно персистировать в организме хозяина?

Латентність є складовою частиною життєвого циклу вірусу простого герпесу. Чутливі нейрони є місцем перебування резидентних вірусних геномів. Під час латентності помітна експресія лише однієї LAT ділянки вірусної ДНК. Невеличка її частина довжиною лише 348 п.н. відповідальна за реактивацію сплячого вірусу. Гени у межах LAT мають антиапоптотичні властивості, що забезпечує виживання нейронів під час реактивації. При цитуванні документа, використовуйте посилання http://essuir.sumdu.edu.ua/handle/123456789/17617Латентность является составной частью жизненного цикла вируса простого герпеса. Резидентные вирусные геномы находятся в чувствительных нейронах. Во время латентной фазы инфекции отмечается экспрессия лишь одной (LAT) области вирусной ДНК. Небольшая ее часть длиной только 348 п.н. ответственна за реактивацию дремлющего вируса. Гены в пределах LAT имеют антиапоптотические свойства, что обеспечивает выживание нейронов во время реактивации. При цитировании документа, используйте ссылку http://essuir.sumdu.edu.ua/handle/123456789/17617Latency of HSV-1/2 is a complicated virus-host interaction that plays a crucial role in the pathogenic potential of this virus. Numerous studies have indicated that sensory neurons are the primary sites of HSV latency. The ability of these viruses to reactivate from latency is responsible for recurrent disease and virus transmission. Since LAT is only known viral transcript that are abundantly transcribed in latently infected neurons, it is reasonable to hypothesize that it regulate latency. LAT protein expression is tightly regulated and may occur only at specific times during latency to prevent immune recognition. Recent studies demonstrating that the genes encoding LAT have antiapoptotic properties strongly suggest that this function plays a crucial role in promoting neuronal survival and thus latency. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/1761

Use of Adeno-associated virus to investigate transcriptional regulation of the preprotachykinin-A promoter in cultured DRG neurons

The preprotachykinin-A (PPT-A) gene encodes the neuropeptides substance P, neurokinin A, neuropeptide K and neuropeptide y, each derived by alternative mRNA splicing and post-translational processing of precursor polypeptides. The PPT-A gene is regulated by a variety of extracellular stimuli including NGF, GDNF, steroids, and inflammation and it is likely that many of these stimuli act at the level of transcription.This study set out to investigate transcriptional regulation of the rat PPT-A gene promoter in dorsal root ganglia neurons (DRG). The proximal promoter region of the PPT-A gene has been extensively studied however, functional studies have been greatly hindered due to the lack of clonal cell lines, which express endogenous PPT-A or can support PPT-A promoter function in reporter gene constructs. DRG express the PPT-A gene endogenously however neurons are refractory to all methods of transfection. Adeno-associated virus vectors (AAV) were therefore used as a tool for the transduction of PPT-A promoter fragments driving a reporter gene to investigate PPT-A promoter activityAAV vectors were shown to achieve 100% transduction efficiency of both neuronal and nonneuronal cells in DRG cultures and AAV vectors containing the PPT-A promoter demonstrated a more restricted expression pattern. In addition, AAV vectors allowed long-term reporter gene expression in cultured DRG neurons in which expression was predominately neuronal.The definition of functional promoter elements supporting tissue specific expression of the PPT-A gene was firstly analysed by infection of adult and neonate cultured DRG neurons with AAV vectors that contained different lengths of the PPTiii A proximal promoter driving the luciferase reporter gene. The PPT-A promoter was found to contain elements that are differentially regulated between neonate and adult DRG neurons. Secondly, the effect of growth factors NGF, LIF, IL-6, BDNF and GDNF on the PPT-A promoter fragment (nucleotides -865 to +92) were examined in cultured adult DRG neurons. These growth factors are known to alter neuropeptide levels and neuronal phenotype. It was found that NGF, LIF and GDNF independently acted to influence PPT-A promoter activity in adult DRG neurons. This suggests that the proximal PPT-A promoter contains regulatory elements that can mediate the effects of these growth factors.Furthermore, the role of a specific binding motif for the basic helix-loophelix (bHLH) family of transcription factors was addressed. Previous biochemical and cell line models have implicated this motif as an important regulator of PPT-A promoter activity. The activity of the PPT-A promoter fragment -865+92 containing an insertional mutation at nucleotide -60 disrupting the bHLH binding motif was compared to the wild-type promoter fragment in the presence or absence of NGF. It was established that this bHLH binding motif could be bound by a repressor in the absence ofNGF and might be an important regulator of basal levels of PPT-A promoter activity. This suggests a novel mechanism by which bHLH factors might be important for NGF regulation of the PPT-A promoter

Characterization of the Genome of Baboon Cytomegalovirus Strain (Ocom4-37) Isolated from the Olive Baboon, Papio Cynocephalus Anubis

This project involved cloning, sequencing, and analyzing the genome of baboon cytomegalovirus (BaCMV) strain OCOM4-37. After isolation, cloning and sequencing the coding sequence of the BaCMV genome, comparisons were made with other CMV genomes. These analyses showed that the OCOM4-37 strain is most closely related to CMVs isolated from primates most closely related to baboons.Department of Biochemistry and Molecular Biolog

Vergleichende Analysen zur Replikation und zum intraaxonalen Transport des Pseudorabiesvirus und des Herpes Simplex Virus Typ 1 in primären Rattenneuronen

Nach dem Eintritt in den Wirtsorganismus und initialer Replikation infizieren Alphaherpesviren Neuronen zur weiteren Ausbreitung im Nervensystem und zur Etablierung einer Latenz. Dazu werden die Viruspartikel innerhalb der Axone retrograd von der Peripherie zum neuronalen Zellkörper transportiert. Die umgekehrte Richtung beschreibt den Weg des anterograden Transports vom Zellkörper zur Synapse für weitere Infektionen von Neuronen höherer Ordnung oder zurück zur Peripherie. Der retrograde intraaxonale Transport ist gut untersucht. Dagegen wird über den anterograden Transport kontrovers diskutiert. Zwei verschiedene Transportmodelle werden vermutet. Das „Married Model“ postuliert, dass umhüllte Virionen innerhalb von Vesikeln entlang des Axons transportiert werden. Die Freisetzung der Partikel erfolgt an der jeweiligen Synapse durch Endocytose. Das „Subassembly Model“ geht dagegen davon aus, dass einzelne Virusstrukurkomponenten (Nukleokapsid, Hülle) entlang des Axons transportiert werden. Der Zusammenbau und die Freisetzung erfolgt am Axonterminus bzw. an der Synapse (in vivo) oder am Wachstumskegel (in vitro) oder an speziellen Auftreibungen des Axons, den sogenannten Varicosities. Nach Infektion eines neuronalen Explantatsystems mit dem Pseudorabiesvirus (PrV) konnten ultrastrukturell umhüllte Virionen in Vesikeln detektiert werden und so der Nachweis der Gültigkeit des „Married Model“ als vorherrschendes Transportmodell geführt werden. Dagegen ist die Situation beim prototypischen Alphaherpesvirus, dem Herpes Simplex Virus Typ 1 (HSV-1), weiterhin ungeklärt. Aufgrund der zahlreichen unterschiedlichen Analysemethoden und -systeme war ein direkter Vergleich der beiden Viren bislang nicht möglich. Daher sollte in dieser Arbeit ein standardisiertes neuronales Kultursystem genutzt werden, um vier verschiedene HSV-1 Stämme im Vergleich zu PrV zu untersuchen. Für die Infektionen wurden sowohl Neuronen aus dem oberen Cervikalganglion als auch aus Spinalganglien genutzt. So konnte gezeigt werden, dass in Neuronen, welche mit den HSV-1 Stämmen HFEM, 17+ und SC16 infiziert waren ca. 75% als umhüllte Virionen in Vesikeln und ca. 25% als nackte Kapside vorlagen. Ingesamt war die Anzahl der Viruspartikel in HSV-1 infizierten Neuronen signifikant geringer als in PrV infizierten Kulturen. Überraschenderweise zeigten mit HSV-1 KOS infizierte Neuronen ein reverses Bild. Hier lagen nur 25% der Viruspartikel als umhüllte Virionen in Vesikeln vor, während 75% als nackte Kapside detektiert wurden. Dieser unerwartete Phänotyp sollte auf molekularbiologischer Ebene genauer untersucht werden. Dabei wurde auf die Genregion von US9 fokussiert. Das von US9 codierte Membranprotein spielt eine wichtige Rolle während des Zusammenbaus der Virionen und bei anschließenden axonalen anterograden Transportvorgängen. In dieser Arbeit konnte gezeigt werden, dass das HSV-1 KOS Genom durch verschiedene Basenaustausche an der vorhergesagten TATA-Box von US9 eine Mutation aufweist. Zusätzlich trägt das offene Leseraster durch eine weitere Mutation ein vorzeitiges Stopcodon auf und wird dadurch auf 58 Kodons reduziert, im Gegensatz zu anderen HSV-1 Stämmen, wo es 91 Kodons umfasst. Die Mutation an der TATA-Box verändert auch das ursprüngliche Stopcodon vom US8a Gen, was zur einer Verlängerung von ursprünglich 161 zu 191 Kodons führt. In Northern Blot Analysen konnte eine reduzierte Transkription von US9 in HSV-1 KOS infizierten Zellen detektiert werden. In HSV-1 KOS infizierten Zellen konnten mittels eines spezifischen Antiserums gegen US9 im Western Blot kein Genprodukt nachgewiesen werden. Auch Immunfluoreszenzanalysen zeigten, dass das abgeleitete verkürzte Protein offenbar nicht stabil exprimiert wird. Dagegen konnten Western Blot Analysen die Vergrößerung des pUS8a bestätigen. Der beobachtete auffällige intraaxonale Phänotyp könnte somit durch die Mutation des US9 Protein erklärt werden. Zusammenfassend wurde in dieser Arbeit gezeigt, dass auch bei HSV-1 vorwiegend das „Married Model“ für den anterograden intraaxonalen Transportweg bevorzugt wird und somit beide Alphaherpesviren, HSV-1 und PrV, denselben Transportweg nutzen

The regulation of neuronal cell fate by the interaction of the Brn-3a transcription factors and the p73 family of proteins.

The Brn-3a and Brn-3b POU transcription factors are expressed in the developing nervous system where Brn-3a is associated with sensory neuronal differentiation and survival. It has been shown that Brn-3a directly interacts with the p53 protein and this interaction resulted in differential regulation of gene targets which affect cell fate i.e. death or differentiation. The p53 related protein, p73, is involved in neuronal development and is expressed as multiple alternatively spliced C-terminal isoforms (TAp73a-£), and N-terminally deleted, dominant negative proteins (ANp73a-P) that show reciprocal function to the TA forms and p53. In this study we show that the Brn-3a/Brn-3b proteins also physically interacted with the p73 isoforms via the POU domain of Brn-3 and the region containing the OD domain of p73 and this interaction is modulated by the different C-terminals of the p73 isoforms. The effect of the Brn-3a/p73 interaction was tested on p53 target genes where co-expression of Brn-3a was shown to potentiate the transcriptional effect of TAp73 on the p2icn>1/Wafl promoter whilst antagonizing TAp73/p53 mediated activation of the pro-apoptotic genes box and noxa. Additionally Brn-3a uses a variety of different mechanisms to modulate the expression of different p53 target genes as well as playing a role in determining the selectivity of individual TAp73 members in controlling the expression of the bax promoter. In agreement with a functional effect, co-expression of Brn-3a and TAp73 increased cell cycle arrest and survival in the ND7 neuronal cell line, whereas co-expression of Brn-3a and ANp73 had no effect on cell cycle arrest but increased cell survival. Similar to p53, p73 (TA and AN) co-localised to a subset of Brn-3a positive neural crest cells (NCC) fated for a sensory lineage. Some of these Brn-3a/p73 co-expressing cells suggested a differentiated cell type and co-localization of TAp73 but not ANp73 with the differentiation marker NF-160, suggested that TAp73, like p53 is associated with differentiation of Brn-3a positive NCC effects of Brn-3a-p73/p53 co-expression was analysed in NCC cultured from Brn-3a-/- embryos showed significantly increased apoptosis upon induction of p53/p73 compared with WT cultures, suggesting that Brn-3a is necessary to overcome the p73/p53 apoptotic pathway. Thus, interaction with Brn-3a in sensory neurones may be critical for modulating p73/p53 mediated gene expression and hence cell fate