The magnitude and efficacy of CD8+ T cell memory may notably regress, especially if immune induction occurs in the absence of adequate CD4+ help. This report demonstrates that this CD8+ memory malfunction could be remedied if a source of cognate antigen-recognizing helper cells were provided during recall. The inability of adoptive transfer of memory SIINFEKL-specific CD8 cells to reject tumors was overcome if recipients were primed for ovalbumin-specific helper cell responses. Additionally, animals primed for a SIINFEKL-specific memory response and incapable of rejecting the tumor could regain protective immunity if given helper cells. This pattern of CD8+ T cell functional rescue or reprogramming by helper cell transfer was replicated using a Herpes simplex virus antiviral immunity system. Our results could mean that therapeutic vaccine approaches could be designed to compensate situations that have defective CD8+ T cell function.

The herpes simplex virus type 2 (HSV-2) UL21 protein is conserved between all members of the Alphaherpesvirinae subfamily. Although UL21 is essential for virus propagation in HSV-2, its function in viral replication is poorly understood. Cells infected with HSV-2 strains lacking UL21 exhibit an approximate two-hour delay in viral gene expression that cannot be explained by a defect in virus entry or capsid engagement with, or movement along, microtubules. However, we noted a defect in the ability of UL21 knockout (KO21) capsids to associate with the nucleus after infection. We found that the delay in viral gene expression was not directly due to the absence of UL21 insofar as cells stably expressing UL21 could not complement the delay in gene expression. We suggest that the KO21 delay in gene expression is due to alterations in virion composition and that in the absence of UL21, a key virion component required for the timely delivery of capsids to the nucleus fails to be packaged into virions. Interestingly, at late times after infection, levels of viral proteins in KO21 infected cells reach wild-type levels, indicating that a secondary function is responsible for the essential nature of UL21. We found that at late times post-infection KO21 infected cells accumulated capsids in the nucleus but these fail to reach the cytoplasm and mature into infectious virions. Thus, we hypothesize that the essential function of UL21 is to facilitate capsid trafficking from the nucleus to the cytoplasm. Moreover, the viral glycoproteins gD and gC were retained in the endoplasmic reticulum, and were under-glycosylated in KO21 infected cells. It is therefore possible that the absence of UL21 prevents the targeting of glycoproteins to the inner nuclear membrane, preventing the formation or function of the nuclear egress complex. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-09-10 16:20:32.162

Les héparanes sulfates (HS) sont des polysaccharides sulfatés qui participent à de nombreux processus physiopathologiques. L’entrée du virus HSV-1 dans ses cellules cibles nécessite l’intervention de la glycoprotéine gD, qui peut interagir avec quatre récepteurs différents: HVEM (Herpes Virus Entry Mediator), les nectines-1 et -2, et des HS 3-O-sulfatés. De plus, la protéine gD est impliquée dans la protection des cellules cibles contre l’apoptose. Nos travaux ont montré que les réponses anti-apoptotiques induites par la gD nécessitent la coopération entre HVEM et les HS 3-O-sulfatés. Chez l’Homme, sept 3-O-sulfotransférases (3-OSTs) sont impliquées dans la réaction de 3-O-sulfatation, qui se distinguent par des différences d’expression et de spécificité de substrat. Selon l'environnement inflammatoire, les macrophages subissent deux types de polarisation, caractérisés par des phénotypes et des fonctions distinctes. Les travaux du Laboratoire ont montré que la 3-OST3B est fortement induite dans les macrophages pro-inflammatoires, à l’inverse de la 3-OST2. Nous avons alors montré que l’expression de la 3-OST3B est régulée de manière transcriptionnelle et post-transcriptionnelle. En revanche, la diminution de la 3-OST2 fait intervenir des mécanismes différents, dont l’action d’un microARN. La dernière partie de la thèse a porté sur la localisation subcellulaire des 3-OSTs. Nos résultats montrent que la 3-OST3B est localisée dans le Golgi, alors que la 3-OST2 et la 3-OST3A sont retrouvées jusqu’à la membrane plasmique. Dans leur ensemble, nos résultats suggèrent une fonction spécifique pour chaque 3-OST dans la génération de motifs HS 3-O-sulfatés distincts. / Heparan sulfates (HS) are sulfated polysaccharides able to modulate several normal and pathological processes. Binding of Herpes-Simplex virus HSV-1 to its target cells is mediated in part by the glycoprotein gD. This protein can interact with four receptors: HVEM (Herpes Virus Entry Mediator), nectin-1 and -2, and 3-O-sulfated HS. In addition, gD is capable of protecting host cells against apoptosis. In this context, we showed that gD protects macrophages against apoptosis by a mechanism dependent on the cooperation between HVEM and 3-O-sulfated HS. The reaction of 3-O-sulfation can be catalyzed by seven 3-O-sulfotransferases (3-OSTs), which are differentially expressed and exhibit fine substrate specificity. Depending on the immune environment, macrophages can undergo proinflammatory (M1) or alternative (M2) polarization, resulting in distinct phenotypes and functions. A previous study of the laboratory has showed that 3-OST3B is strongly induced in M1 macrophages, while 3-OST2 is poorly detected. We then demonstrated that the induction of 3-OST3B is regulated at the transcriptional and post-transcriptional levels upon M1 polarization. Conversely, the reduction of 3-OST2 expression involved distinct mechanisms, including the action of a microRNA.The last part of the thesis focused on the subcellular localization of 3-OSTs. Our results demonstrated that 3-OST3B is localized in the Golgi apparatus, while 3-OST2 and 3-OST3A reach the plasma membrane. Altogether, our results suggest that each 3-OST isoenzyme may be involved in the generation of distinct 3-O-sulfated HS motifs.

Herpes Simplex Virus type 1 (HSV-1) is an ubiquitous neurotropic human pathogen that infects a large majority of the world’s population. It is the causative agent of the common cold sore but also responsible for life-threatening infections (e.g., encephalitis), particularly in immunocompromised individuals and neonates. Like other herpesviruses, HSV-1 takes over the cellular RNA machinery to facilitate productive infection while efficiently shutting down host gene expression by targeting multiple steps of RNA metabolism. The two viral proteins, vhs and ICP27, play a crucial role in this process. Delivered by the tegument of the incoming virus, the virion host shut-off (vhs) endonuclease rapidly starts cleaving both cellular and viral mRNAs. With the onset of viral gene expression, the HSV-1 immediate-early protein ICP27 promotes the expression of viral early and late genes through various mechanisms, including mRNA processing, export, and translation. Prior research by the Dölken lab demonstrated that lytic HSV-1 infection results in the disruption of transcription termination (DoTT) of most cellular genes by the viral ICP27 protein. This significantly contributes to HSV-1 induced host shut-off. DoTT results in transcription for tens of thousands of nucleotides beyond poly(A) sites and into downstream genes. Interestingly, this was found to be accompanied by a dramatic increase in chromatin accessibility downstream of the affected poly(A) sites. This is consistent with the formation of extensive downstream open chromatin regions (dOCR) and indicative of impaired histone repositioning in the wake of RNA polymerase II (Pol II) downstream of the affected poly(A) sites. In my PhD thesis, I demonstrate that dOCR formation is dependent on the viral ICP22 protein when poly(A) read-through transcription is triggered by the ectopic expression of ICP27 or salt stress. I show that dOCR formation occurs when a high level of transcriptional activity arises downstream of genes due to the HSV-1-induced DoTT. To investigate whether histone composition is affected downstream of genes, I established the ChIPmentation approach to study associated changes and the influence of DoTT and dOCR formation on major histone modification marks. In HSV-1 WT infection, dOCR formation was reflected in alterations of canonical H1 histone downstream of affected genes, which was absent in ICP22 infection. To elucidate the underlying molecular mechanism, two major histone chaperones SPT6 and FACT (SPT16 and SSRP1), which govern histone repositioning and may thus play a role in H1 homeostasis, were extensively studied. Both histone chaperones have been recently shown to be recruited to the viral genome by interactions with ICP22 protein. To investigate whether the depletion of SSRP1 or SPT6 would complement the loss of ICP22 to induce dOCR, T-HF cells with doxycycline-inducible knock-down of either of the two factors were generated. ATAC-seq analysis revealed that the interaction between the two histone chaperones and ICP22 is not involved in HSV-1-induced dOCR formation, suggesting the involvement of other proteins. In summary, this work sheds new light on a fundamental molecular mechanism of the cellular transcriptional machinery that is manipulated by the concerted actions of the two HSV-1 immediate-early proteins ICP22 and ICP27. / HSV-1 ist ein weit verbreitetes, neurotropisches Virus, mit welchem ein Großteil der Weltbevölkerung infiziert ist. Es verursacht milde Infektionen wie Herpes labialis, aber kann auch lebensbedrohliche Infektionen des Nervensystems (z. B. Enzephalitis) in immunsupprimierten Menschen und Neugeborenen auslösen. Um sich lytisch zu vermehren, programmiert HSV-1 die Transkriptions- und Translationsmaschinerie der Zelle effizient um und hemmt gleichzeitig an mehreren Punkten zelluläre Genexpression. Zwei virale Proteine, vhs und ICP27, spielen dabei eine entscheidende Rolle. Vhs wird im Tegument des Virions mit dem Inokulum in die Zelle geliefert und baut so zelluläre Transkripte ab noch bevor virale Genexpression startet. ICP27 wird also sogenanntes „immediate-early“ Gen als eines der ersten viralen Proteine exprimiert und kann unterstützt auf mehreren Ebenen (RNA Prozessierung, Export und Translation) die Expression der viralen „early“ und „late“ Gene. Unsere Gruppe hat diesbezüglich gezeigt, dass die Terminierung der Transkription (sogenanntes „DoTT“) durch das virale Protein ICP27 in der lytischen Infektion gestört wird. Dies trägt maßgeblich zur Abschaltung der zellulären Genexpression bei. Die Störung der Terminierung führt dazu, dass RNA Polymerase II bis zu >100 Kilobasen nach dem Polyadenylierungssignal weiter transkribiert. Durch die Aktivität der RNA Polymerase II wird in den 3‘ Regionen der betroffenen Gene das Chromatin gelockert (sogenanntes „dOCR“). Dies steht im Einklang mit einer Öffnung des Chromatins durch gehemmte Histon-Neupositionierung, verursacht durch die Transkription der betroffenen Genomregionen. Im Rahmen meiner Doktorarbeit konnte ich mittels Hochdurchsatzsequenzieranalyse von Transposon-zugänglichem Chromatin (ATAC-seq) zeigen, dass offenes Chromatin durch das virale Protein ICP22 verursacht wird. Dieser Effekt konnte unterdessen nur beobachtet werden, wenn die Terminierung der Transkription, entweder durch die gleichzeitige Expression von ICP27 oder stressinduziert z.B. durch hypertonen Lösungen, gestört wurde. Das Ausmaß an offenem Chromatin korrelierte dabei mit der Transkriptionsaktivität der entsprechenden Genomregionen. Durch bioinformatische Analysen von Hochdurchsatzsequenzierungen von Wildtyp Virus und ICP22-defizitären Mutanten infizierten Zellen konnte ich einen Cluster von stark exprimierten Genen mit ausgeprägter DoTT identifizieren, der besonders stark von der Chromatinöffnung betroffen war. Um zu testen, ob die Histone in den betroffenen Regionen durch die Induktion von dOCR verändert wurden, habe ich ein neues Chromatin-Immunpräzipitations Verfahren namens ChIPmentation etabliert und hiermit die mit der Induktion von dOCR assoziierten Histonvarianten untersucht. Dabei fiel auf, dass das H1 Histon gezielt in von dOCR betroffenen Regionen verloren ging. Um den zu Grunde liegenden Mechanismus zu untersuchen, habe ich die Rolle der beiden Histonchaperonen SPT6 und FACT (SPT16 und SSRP1) ausgiebig charakterisiert. Diese regulieren normalerweise die Repositionierung von Histonen im Zuge der Pol II Transkription. Beide Faktoren werden zudem durch ICP22 in die viralen DNA Replikationzentren im Nukleus rekrutiert, was den Positionierungsdefekt von H1 hervorrufen könnte. Um diese Hypothese zu testen, wurden beide Proteine durch induzierbare shRNA Knockdowns depletiert und mittels ATAC-seq untersucht, ob dies in der Infektion mit ICP22-defizitären Mutanten zur Öffnung des Chromatin führt. Hierbei zeigte sich allerdings, dass die Depletion dieser beider Histonchaperone kein offenes Chromatin bei Infektion mit der ICP22 Knockout Mutante erzeugt. Zudem fiel auf, dass SSRP1 selektiv dazu beitrug, Chromatin in transkribierten Regionen geschlossen zu halten. Offensichtlich spielen daher die beiden Histonchaperonen keine Rolle bei der ICP22-induzierten Öffnung des zellulären Chromatins unterhalb von Genen.

Encephalitis caused by the alphaherpes viruses HSV 1, HSV 2 and VZV can be devastating and rapid, accurate diagnosis is required. Whilst existing molecular techniques are invaluable in diagnosing acute disease, detection of antibody is needed to confirm infection and to make a diagnosis after the acute stage or during post-infectious encephalitis. Current immunoassays are limited by the volume of sample required. The aim of this project was to develop a rapid, accurate, low sample volume assay to improve diagnosis using Luminex technology.The immunodominant proteins of HSV and VZV, glycoprotein D (gD) and glycoprotein E (gE), were expressed in insect cells using a baculovirus expression vector. Expressed proteins were purified, characterised and used to develop in-house enzyme-linked immunosorbent assays (ELISA) to detect HSV and VZV type-specific antibodies. The performance of each newly developed in-house ELISA was compared with commercial ELISA assays using well characterised serum panels. An excellent correlation between the in-house ELISAs and the commercial ELISA assays (100% for HSV gD and 99% for VZV gE) was observed. To differentiate between HSV-1 and HSV-2 a new commercial ELISA assay (Omega) utilising a branched chain peptide (peptide 55 which provides immune selection of HSV-2 specific antibody) was evaluated against two commercially available HSV-2 ELISA assays. The Omega assay showed an overall agreement of 97.6% with Western blot and other ELISA assays. The two expressed proteins, together with peptide 55, were used to develop a triplex fluorescent microbead immunoassay for the simultaneous detection and quantitation of anti-viral antibody in human sera. Initially a monoplex assay for each analyte was developed and optimised individually and then the three assays were mixed together in a triplex assay. Results for HSV-1 gD and VZV gE obtained from the triplex assay showed a 100% agreement with HSV-1 and VZV in-house ELISA results. In the case of peptide 55, the triplex assay results showed better sensitivity than the Omega ELISA assay with an overall agreement with Western blot and other assays of 98.4%. In addition, in order to facilitate the diagnosis of alphaherpesviruses CNS infections the triplex assay was joined together with a biplex fluorescent microbead immunoassay designed for detecting and measuring human IgG and albumin in CSF and serum samples. The sensitivity and reproducibility of the resultant five-analyte multiplex immunoassay and the previous triplex assays were compared and found to have equivalent sensitivity and specificity. The sensitivity and minimal sample requirements of the new assay suggests that it will be a powerful tool for the diagnosis and study of both acute and post-infectious viral encephalitis.

Herpes Simplex virus (HSV) virions contain the protein vhs (virion host shutoff), which is known to trigger rapid shutoff of host protein synthesis and accelerated decay of viral and cellular mRNAs. HSV-1 strains generally cause weaker shutoff than HSV-2 strains. HSV viruses lacking the VP16 viral transactivator gene show uncontrolled shutoff late in infection (Lam et al., 1996); vhs is known to bind to VP16 (Smibert et al., 1994). In vitro experiments demonstrated that HSV-1 vhs protein (vhsTl) and an intertypic HSV-2 G vhs protein (vhsT2) did not differ in ability to effect mRNA degradation in a rabbit reticulocyte lysate (RRL) assay system, suggesting that virion factors might influence vhs shutoff phenotype. To investigate the possibility that VP 16 influences early shutoff during HSV infection, a virus was constructed containing the HSV-2 strain G VP16 in place of HSV-1 VP16. This virus (8MA2R) grew in a noncomplementing cell line and was unaltered in shutoff phenotype compared to wt strain. Cotransfection assays demonstrated vhsT2 had greater shutoff ability than vhsT1, and this was confirmed in a viral system by constructing intertypic viruses containing portions of the HSV-2 G vhs ORF in a vhs-null HSV-1 tk locus. All intertypic constructs conferred increased shutoff ability relative to vhsT 1, indicating that the strong shutoff ability of HSV -2 vhs is distributed along much of the vhs ORF. Also, to confirm the observation that UL13 null viruses show a lack of shutoff which is not due to lack of vhs in the virion (Overton et al., 1994) a number of wildtype viruses and their UL13-null derivatives were tested for ability to shutoff host translation. All showed near-wt or wt levels of shutoff. / Thesis / Master of Science (MS)

In human infection, herpes simplex virus (HSV) navigates two distinct life cycles; lytic and latent. The latent cycle takes place in sensory neurons, and is characterized as a dormant period punctuated by stress-induced episodes of viral reactivation. Understanding the mechanisms by which HSV latency and reactivation occur has been hindered by the lack of a model that faithfully recapitulates the environment of a human sensory neuron. Systems ranging from rat neurons to human fibroblasts have been developed to host HSV latency, however few available models have been able to investigate the role of human neuron-specific factors. To address this need, human medulloblastoma tumour cell lines, which derive from neuronal precursor cells, were differentiated and examined for their ability to host the HSV latency-reactivation cycle—in a manner similar to the differentiated PC-12 cell model. ONS-76 and UW228 medulloblastoma cell lines were screened for differentiation capacity. The differentiated cells were demonstrated to possess neuronal character as several neuron-specific proteins were found to be expressed. Differentiated ONS-76 cells were not compatible with hosting HSV latency, however, infection with a viral mutant impaired for lytic cycle initiation exhibited a deviant pattern of gene expression that resembles what has been observed in reactivation. Differentiated UW228 cells were found to host a low frequency, stable infection with the HSV mutant, characterized by the absence of infectious virus and viral lytic gene expression in the presence of persisting viral DNA. This DNA could further be induced to re-enter the lytic cycle through heat shock treatment and removal of differentiating agents from cell cultures. These results depict differentiated medulloblastoma cells as a novel tool in the study of HSV latency and reactivation, as these cells derive from the central nervous system and provide a new cellular perspective through which HSV biology can be viewed.

Traffic Control Signs or destination boards on roadways offer significant information for drivers. Regulation signs tell something like your speed, turns, etc; Warning signs warn drivers of conditions ahead to help them avoid accidents; Destination signs show distances and directions to various locations; Service signs display location of hospitals, gas and rest areas etc. Because the signs are so important and there is always a certain distance from them to drivers, to let the drivers get information clearly and easily even in bad weather or other situations. The idea is to develop software which can collect useful information from a special camera which is mounted in the front of a moving car to extract the important information and finally show it to the drivers. For example, when a frame contains on a destination drive sign board it will be text something like "Linkoping 50",so the software should extract every character of "Linkoping 50", compare them with the already known character data in the database. if there is extracted character match "k" in the database then output the destination name and show to the driver. In this project C++ will be used to write the code for this software.

No description available.

VP16 initiates the HSV replication cycle by activating immediate early (IE) gene expression. It recruits the RNA pol II through an acidic C-terminal domain. The defective VP16 encoded by the V422 mutant of HSV-1 possesses a truncated C-terminal domain. Therefore, V422 replication is suppressed in most cell-lines, except U2OS osteosarcoma cells. The permissive phenotype of U2OS cells stems from a failure to express one or more inhibitory factors that are produced in restrictive cells. The initial project was designed to identify these host inhibitory factors in restrictive cells of V422, using siRNA silencing technology. To facilitate the siRNA screen, a GFP reporter gene has been inserted into the thymindine kinase (TK) gene of the V422 genome and the wild-type KOS genome. This thesis provides information about characterizing the kinetics of GFP expression from recombinant viruses at both protein and mRNA levels, during different infection times in HeLa and Vero cells. / Virology