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Singer, Anthony N. "A New Approach to the Development of an RSV Anti-viral Targeted Nanocarrier for Dual Inhibition of Viral Infection and Replication." Scholar Commons, 2018. https://scholarcommons.usf.edu/etd/7712.
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Respiratory Syncytial Virus (RSV) is a potentially life-threatening respiratory pathogen that infects approximately 64 million children and immunocompromised adults globally per year. Currently, there is a need for prophylactic and therapeutic approaches effective against primary and secondary RSV infections. This project focuses on the development of a simple, smart, and scalable anti-RSV nanotherapeutic that combines novel cellular antiviral defense mechanisms targeting the inhibition of viral fusion and replication. An ICAM-1 targeted liposomal nanocarrier will be synthesized and coated with a layer of chitosan containing the anti-fusion HR2-D peptide as an extracellular defense mechanism. Additionally, chitosan complexed to dual expressing short hairpin RNA (shRNA) recombinant plasmids will be encapsulated within the nanocarrier, and provide an intracellular defense mechanism that will interfere with the expression of the NS1 and P proteins. In combination, both defense mechanisms are expected to induce a synergistic anti-RSV effect that will surpass those of conventional therapeutics. Through this research, the NS1 and P containing plasmid (pSH-NS1-P) was cloned, and the nanotherapeutic was successfully synthesized. Based on the acquired results, pSH-NS1-P was shown to express anti-RSV effects, and it was also concluded that both inserts were producing active shRNA. Additionally, the anti-RSV efficiency of HR2-D was confirmed. Overall, this research will lead to development of a dual-mechanistic anti-viral nanotherapeutic.
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Cherry, Elana. "Trans-dominant negative inhibition of human immunodeficiency virus type 1 replication by expression of protease-reverse transcriptase fusion proteins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0019/NQ50069.pdf.
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Timmoneri, Martina. "Inhibition of HCMV replication by small molecules interfering with the dimerization of the DNA polymerase processivity factor UL44." Doctoral thesis, Università degli studi di Padova, 2018. http://hdl.handle.net/11577/3425876.
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Human cytomegalovirus (HCMV) is a leading cause of severe disease in immunocompromised individuals, including AIDS and transplanted patients, and in congenitally infected newborns. Despite the availability of several drugs, pharmacological treatment of HCMV infections is associated with poor bioavailability, toxicity and the emergence of resistant strains. Furthermore, no vaccine is available and no drugs are approved to prevent vertical transmission during pregnancy, therefore, it is essential to identify new potential targets of therapeutic intervention.
HCMV DNA polymerase accessory protein UL44 plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit UL54 by tethering it to the DNA. Binding of UL44 to dsDNA occurs in the absence of ATP and clamp loaders, and depends on UL44 homodimerization. Indeed, our research group previously demonstrated that the protein can dimerize in cells and point mutations disrupting protein self-interaction also prevent DNA binding and abolish viral OriLyt-dependent DNA replication in transcomplementation assay. Therefore, disruption of UL44 homodimerization represents an attractive target for the development of new antivirals. Based on these observations, using the recently published crystal structure of UL44 hodimers our research group previously performed a virtual screening with the Glide software in combination with a library of 1.3 x 106 small molecules (SMs) to identify SMs potentially interfering with UL44 homodimerization. After three rounds of screening (HTVS: high-throughput virtual screening, SP: standard precision, XP: extra precision), followed by an in depth analysis of compounds chemical properties, 18 SMs were selected for further analysis. Selected compounds were obtained from a commercial supplier, to be tested in a variety of assays for their ability to inhibit UL44 homodimerization both in cell and in vitro, as well as on HCMV replication.
For this purpose, we applied two in vitro methods to monitor the effect of our candidates on UL44 dimerization such GST-pulldown Assay and Thermal Shift Assay (TSA). Furthermore, Plaque Reduction Assay (PRA) and Fluorescence Reduction Assay (FRA) were performed to study their inhibitory effects on viral replication.
Initially we validated that GST-pulldown assay was suitable to study UL44 dimerization and its perturbation caused by SMs. Therefore, we performed a first screening to test the effect of the 18 SMs on UL44 dimerization, and we identified 3 of these which reproducibly inhibited the dimerization. Prompted by these results, we selected such SMs to investigate a possible dose-response relationship between the dimerization inhibition and the compounds concentration. Unfortunately, data analysis revealed a high variability, and lack of correlation between the inhibition and the concentration of SMs used in the assays. In parallel, PRAs were performed to validate the effect of selected SMs, but these were unable to inhibit viral replication with high potency.
Subsequently, two recombinant HCMV viruses (TB4-IE2-EYFP and TB4-UL83-EYFP) were obtained from Michael Winkler (Leibniz-Institut für Primatenforschung Goettingen, Germany) in order to study the effect of our molecules on HCMV replication by FRAs. Therefore, after a screening of the 18 SMs, 4 of these were identified as possible inhibitors of viral replication and were selected to further studies. We investigated their and 50% effective dose (ED50) and 50% cytotoxic concentration (CC50) values and their effects on the viral gene expression. Only 2 SMs reproducibly inhibited expression of Early and Late genes.
Then we evaluated an alternative in vitro assay and among the various possibilities, we have chosen TSA that is able to inform whether a SM induce the disruption of constitutive oligomeric interfaces. In a first moment we validated that the assay allow the discrimination between monomeric and dimeric forms of UL44, suggesting its potential for the screening of our SMs. As result of the SMs screening by TSA, one molecule revealed a possible inhibition of dimerization.<br>Cytomegalovirus (CMV) è un importante patogeno di interesse umano. Al momento gli antivirali disponibili ed utilizzati per la terapia contro l’infezione da CMV presentano una serie di problematica quali l’alto costo, bassa biodisponibilità, alta tossicità ed il presentarsi di ceppi virali resistenti. Inoltre non è disponibile un vaccino ed ancora non è stato approvato alcun farmaco per prevenire la trasmissione verticale durante la gravidanza. Per questi motivi, sono necessari nuovi efficaci farmaci antivirali.
La proteina accessoria UL44 della DNA polimerasi di CMV, svolge un ruolo essenziale nella replicazione virale, conferendo processività alla subunità catalitica UL54 ancorando il complesso oloenzimatico al DNA.
Il legame di UL44 al dsDNA avviene in assenza di ATP e dei clamp loaders, e dipende dalla omodimerizzazione di UL44. Il nostro gruppo di ricerca, infatti ha recentemente dimostrato che la proteina può dimerizzare in cellule e che mutazioni puntiformi in grado di inficiare tale dimerizzazione prevengono il legame con il DNA ed aboliscono la replicazione del DNA virale oriLyt-dipendente in saggi di transcomplementazione transiente.
Perciò, la distruzione dell’omodimerizzazione UL44 rappresenta un potenziale ed allettante target per lo sviluppo di nuovi antivirali. Partendo da queste osservazioni ed usando la struttura cristallografica degli omodimeri UL44 che è stata recentemente pubblicata, il nostro gruppo di ricerca ha eseguito un virtual screening con il software Glide in combinazione con una libreria di 1.3 x 10^6 piccole molecole (SMs) per identificare SMs che potenzialmente potessero interferire con l’omodimerizzazione di UL44. Dopo tre rounds di screening (HTVS: high-throughput virtual screening, SP: standard precision, XP: extra precision), seguiti da un’analisi delle proprietà chimiche dei composti, sono state selezione 18 SM per ulteriori analisi. I composti selezionati sono stati acquistati presso un fornitore commerciale, per essere testati in diversi saggi per valutare le loro abilità di inibire l’omodimerizzazione di UL44 in vitro, sia la replicazione virale.
Con questo scopo, abbiamo utilizzato due metodi in vitro per monitorare l’effetto dei nostri candidati sulla dimerizzazione di UL44, quali GST-pulldown assay e Thermal Shift Assay (TSA). Inoltre per studiare l’inibizione sulla replicazione virale sono stati eseguiti saggi di Plaque Reduction Assay (PRA) e Fluorescence Reduction Assay (FRA).
Inizialmente abbiamo confermato che il GST-pulldown assay fosse idoneo per studiare la dimerizzazione di UL44 e la sua perturbazione causata dalle SMs. Pertanto abbiamo eseguito un primo screening per saggiare l’effetto delle 18 SMs sulla dimerizzazione di UL44, ed abbiamo identificato 3 molecole che inibivano la dimerizzazione in modo riproducibile. Incoraggiati da questi risultati, abbiamo selezionato queste SMs per valutare una possibile relazione dose-risposta tra l’inibizione della dimerizzazione e la concentrazione dei composti. Sfortunatamente, l’analisi dei dati hanno rivelato alta variabilità, e perdita di correlazione tra l’inibizione e la concentrazione delle SMs utilizzate nei saggi.
In parallelo, abbiamo eseguito PRAs per validare l’effetto delle SMs precedentemente selezionate, ma queste si sono presentati incapaci di inibire la replicazione virale in modo consistente.
Successivamente abbiamo ottenuto due virus CMV ricombinanti (TB4-IE2-EYFP and TB4-UL83-EYFP) da Michael Winkler (Leibniz-Institut für Primatenforschung Goettingen, Germany) per studiare l’effetto delle nostre molecole sulla replicazione di CMV mediante FRAs. Pertanto, dopo uno screening delle 18 SMs, 4 di queste sono state identificate come possibili inibitori della replicazione virale e sono state selezionate per essere ulteriormente studiate. Abbiamo valutato la loro dose effettiva 50% (ED50) e la loro concentrazione citotossica 50% (CC50) e gli effetti relativi all’espressione genica. Solo due SMs in modo riproducibile inibivano l’espressione dei geni Early e Late.
Poi abbiamo valutato un saggio in vitro alternativo e tra le varie possibilità, abbiamo scelto il TSA che è un saggio in grado di informare se una SM induce la distruzione delle interfacce di un oligomero costitutivo. In un primo momento abbiamo confermato che il saggio permettesse la discriminazione tra le forme monomeriche e dimeriche di UL44, suggerendo il suo potenziale per lo screening delle nostre SMs. Come risultato di questo screening, una molecola ha rivelato possibile inibizione della dimerizzazione.
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Saha, Bratati. "Identification and Validation of Small Molecules Inhibiting Human Adenovirus Replication." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39677.
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Human adenovirus (HAdV) mainly causes minor illnesses, but can lead to severe disease and death in both immunocompromised and immunocompetent patients. In such cases, the current standards of treatment often do not improve disease outcome and no approved antiviral therapy against HAdV exists. Since HAdV relies on cellular machinery to assist in the progression of the virus lifecycle, we hypothesized that small molecules targeting certain cellular proteins/pathways, without severely affecting cell health, may serve as effective anti-HAdV compounds. Thus, we aimed to identify novel inhibitors of HAdV, and investigate the molecular mechanism to determine new therapeutic targets for intervention in HAdV infection. We first examined the antiviral properties of pan-histone deacetylase (HDAC) inhibitor SAHA and found that the drug affects multiple stages of the HAdV lifecycle, resulting in significant reductions in virus yield. SAHA was effective in decreasing gene expression from clinically relevant HAdV serotypes. Subsequent investigations on the role of HDACs in HAdV infection led us to determine that class I HDAC activity, mainly HDAC2, is necessary for optimal viral gene expression. Using a wildtype-like HAdV reporter construct that allows us to monitor virus replication by fluorescence microscopy, we then designed an efficient system for screening small molecules to identify novel HAdV inhibitors. We screened over 1300 small molecules, and the screen was sensitive enough to detect compounds with both robust and modest antiviral activity. Several positive hits were validated to reduce HAdV gene expression and yield from infected cells. Further investigation on the efficacy of these compounds and the mechanism behind their inhibition of HAdV can lead to the discovery of new pharmacological targets and the development of more effective antivirals.
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Nguyen, Albert Thu. "The molecular mechanism of action of bevirimat : a prototype HIV-1 maturation inhibitor /." Oklahoma City : [s.n.], 2009.
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Clerzius, Guerline. "Multiple levels of Protein Kinase R inhibition during Human Immunodeficiency Virus replication by double-stranded RNA binding proteins and its relationship to the weak innate cell response to viral infection." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=92289.
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Following infection by most viruses, an antiviral state is induced in the host cells characterized by the expression of interferon (IFN) and several IFN-stimulated genes (ISGs). IFN treatment is effective to inhibit HIV replication in infected cells, but shows no significant improvement of HIV-infected patients. Currently, the discrepancy between the in vitro and the in vivo findings remains largely unresolved. The IFN-induced RNA-dependent protein kinase PKR is activated via trans-phosphorylation and plays a central role in the IFN-induced antiviral pathway. Our results show that PKR is transiently activated following HIV-1 infection of Jurkat and peripheral blood mononuclear cells. The kinase is then inactivated at the viral peak, when HIV replication is highly active. By immunoprecipitation, we found that PKR forms a ribonucleoprotein complex with cellular double-stranded RNA binding proteins (dsRBPs), the TAR RNA Binding Protein (TRBP), the adenosine deaminase acting on RNA (ADAR)1 and the PKR Activator (PACT) during HIV replication. Over-expression of PKR is sufficient to inhibit HIV production in HEK 293T cells. This inhibition is reversed by expression of the ADAR1, another ISG. By using mutants of ADAR1, we show that this activity is linked to the ability of the protein to bind PKR. In astrocytes that do not replicate HIV efficiently due to an enhanced PKR response, ADAR1 partially restores viral expression. Surprisingly, PACT binds to and inhibits PKR activity. All three dsRBPs, TRBP, ADAR1 and PACT prevent PKR activation and the phosphorylation of its downstream target, eIF2alpha. Together, our results highlight the key function of PKR in innate immunity and its multiple-level of regulation during HIV-1 replication.<br>L'infection d'une cellule par un virus induit un état antiviral, caractérisé par l'expression de l'interféron (IFN) et de plusieurs gènes induits par l'IFN. Le traitement par l'IFN est efficace pour inhiber la réplication du virus de l'immunodéficience humaine (VIH) dans des cellules infectées en culture, mais ne montre aucun effet bénéfique chez les patients infectés par ce même virus. Cette disparité qui existe entre les résultats de recherche in vivo et in vitro n'est toujours pas résolue. PKR, une protéine kinase induite par les IFNs, est activée par phosphorylation et joue un rôle central dans le mécanisme antiviral de l'IFN. Nos résultats démontrent que PKR est activée de manière transitoire suite à l'infection de cellules lymphocytaires Jurkat ou de lymphocytes/monocytes primaires du sang périphérique par le VIH. Par contre, la kinase n'est plus activée durant et après le pic d'infection, lorsque la réplication du virus est intense. Par immunoprécipitation, nous avons démontré que PKR forme un complexe ribonucleoprotéique avec plusieurs protéines cellulaires qui lient l'ARN double-brin, soit la protéine liant l'ARN TAR, TRBP, l'adénosine déaminase ADAR, ainsi que la protéine activatrice de PKR, PACT, pendant la réplication virale. La surexpression de PKR est suffisante pour inhiber la production du VIH dans les cellules HEK 293T. Cette inhibition est supprimée par l'expression d'ADAR1, une des protéines induite par les IFNs. Par différentes mutations dans la séquence protéique d'ADAR1, nous avons démontré que cette activité d'ADAR est liée à sa capacité de lier PKR. Dans les astrocytes, qui ne répliquent pas le VIH efficacement en raison d'une activation accrue de PKR, ADAR1 rétablit partiellement l'expression virale. Étonnamment, PACT se lie à PKR et inhibe son activité dans les cellules infectées par le VIH. Testées en parallèle, TRBP, ADAR1 et PACT empêchent l'activation de PKR ainsi que la phos
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Gonzalez, Carlos M. Damania Blossom A. "Viral modulators od KSHV lytic replication." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2009. http://dc.lib.unc.edu/u?/etd,2432.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.<br>Title from electronic title page (viewed Sep. 3, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology Virology." Discipline: Microbiology and Immunology; Department/School: Medicine.
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Morgan, Rachel E. "Is the Cytoskeleton Necessary for Viral Replication?" Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/biology_theses/38.
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The cytoskeleton plays an important role in trafficking proteins and other macromolecular moieties throughout the cell. Viruses have been thought to depend heavily on the cytoskeleton for their replication cycles. However, studies, including one in our lab, found that some viruses are not inhibited by anti-microtubule drugs. This study was undertaken to evaluate the replication of viruses from several families in the presence of cytoskeleton-inhibiting drugs and to examine the intracellular localization of the proteins of one of these viruses, Sindbis virus, to test the hypothesis that alternate pathways are used if the cytoskeleton is inhibited. We found that Sindbis virus (Togaviridae, positive-strand RNA), vesicular stomatitis virus (Rhabdoviridae, negative-strand RNA), and Herpes simplex virus 1 (Herpesviridae, DNA virus) were not inhibited by these drugs, contrary to expectation. Differences in the localization of the Sindbis virus were observed, suggesting the existence of alternate pathways for intracellular transport.
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Monaghan, Alan. "Mechanisms of adenovirus DNA replication." Thesis, University of St Andrews, 1995. http://hdl.handle.net/10023/13935.
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The development of a cell-free system in which adenovirus DNA synthesis can be initiated in vitro by using the viral genome or plasmids containing the origin of replication as template has led to the identification of the sequences important for origin function and the isolation and purification of the proteins required for viral DNA replication. In vitro studies on adenovirus types 2 and 5 have shown that their replication requires the formation of a large nucleoprotein complex. This is composed of three virally encoded proteins: adenovirus DNA polymerase, precursor terminal protein and DNA binding protein, and two cellular proteins nuclear factor I and nuclear factor III. While the presence of DNA helicases in other eukaryotic DNA replication systems have been well characterised, this was not the case for adenovirus DNA replication. Initial attempts to identify DNA helicase activity associated with any of the adenovirus replication proteins were unsuccessful. However, a novel DNA unwinding activity was found associated with the DNA binding protein (Ad.DBP). We examined the interaction of DBP with partial DNA duplexes and demonstrated that it could displace oligonucleotides annealed to single-stranded M13 DNA. In addition, DBP could also unwind small fragments of fully duplex DNA. Unlike a DNA helicase, DBP promoted DNA unwinding was nucleoside-5'-triphosphate and Mg2+ independent and exhibited no directionality. The activity required saturating amounts of DBP and was both efficient and cooperative in nature. The helix-destabilising activity was shown to be situated in the C-terminal domain of the protein. These properties suggest a role for DBP in DNA replication in which DBP destabilises duplex DNA during origin unwinding and replication fork movement. The second part of the thesis dealt with the characterisation of the putative "active site" of the adenovirus DNA polymerase. This experimental approach was prompted by data from earlier studies which indicated that DBP could increase the processitivity of the polymerase as well as its sensitivity to nucleotide analogue inhibitors. The "active site" was labelled with pyridoxal-5'-phosphate (PLP), a substrate binding site directed reagent for DNA polymerases. Treatment of Ad.5 DNA polymerase with PLP followed by reduction of the enzyme-PLP adduct resulted in irreversible inactivation of the polymerase activity while the 3'-5' exonuclease associated with Ad.5 DNA polymerase was minimally affected. Substrate protection studies indicate that PLP inhibition is complex. Neither template-primer nor substrate dNTP alone showed any protective effect from PLP mediated inhibition. However, the presence of both template -primer and complementary dNTP significantly protected against PLP inhibition. Comparative tryptic mapping of labelled enzyme, modified in the presence and absence of substrates by PLP reaction, on a C-18 reverse phase column, indicated the protection of one peptide from pyridoxylation in the presence of substrates. Amino acid sequence analyses found no sequence to be present in this peak.
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Nilsson, Benjamin Erik. "Viral and host factors regulating influenza virus replication." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:b8953952-e6d5-4f6d-a7ba-cd55277611d1.
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Avian influenza A viruses typically do not replicate very efficiently when exposed to a mammalian host species. One of the reasons for this is the low activity of the viral RNA-dependent RNA polymerase of avian influenza viruses in mammalian cells. This host restrictive effect can be overcome by adaptive mutations in the avian polymerase, many of which are found in the 627-domain of the PB2 polymerase subunit. Deletion of the 627-domain revealed that this domain is not required for enzymatic functions of the polymerase in vitro, but that it essential for viral replication in a cellular environment in a nucleoprotein-independent manner. While the 627-domain is not necessary for viral RNA synthesis, it was demonstrated that it is involved in mediating encapsidation of nascent replication products. Recently the host factor ANP32A was shown to be the main determinant of host range restriction of the viral polymerase. It was demonstrated that during viral infections ANP32A interacts with KPNA2, a host factor strongly linked to host range restriction of the viral polymerase. It was also revealed that avian polymerases specifically are restricted in vRNA synthesis, a defect that was reversed in the presence of avian ANP32A. ANP32A was shown to be an enhancer of vRNA synthesis in vitro. Viral polymerase-polymerase interactions have been reported previously and presumably fulfil several essential functions during viral replication. Here the potential interaction interfaces of two different polymerase dimers were investigated and a role of polymerase dimers in replication and in trans-activation of cRNA-bound polymerases was found. RNA-binding proteins are essential for RNA metabolism and therefore cell physiology. It has been reported that the RNA-binding proteome responds to biological stimuli. Here the response of the RNA-binding proteome to influenza virus infection was investigated using an in vivo UV crosslinking interactome capture technique. It was demonstrated that the RNA-binding proteome is significantly altered and that this effect is independent of protein abundance. Several host RNA-binding proteins were identified that change their RNA-binding behaviour and that could have pro- or antiviral functions during influenza virus infection.
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Deville, Catherine Michele. "Cellular factors and viral elements for parvovirus replication." Diss., Virginia Tech, 1994. http://hdl.handle.net/10919/40174.
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Autonomous parvoviruses, such as bovine parvovirus (BPV), need a factor present at the S-phase of the cell cycle for a productive infection, while dependent parvoviruses, the adenoassociated viruses (AAVs), require a helper virus to complete an infectious cycle. However, AAV can replicate autonomously in synchronized cells, suggesting that an S-phase factor substitutes for the helper virus. To investigate the nature of the cellular S-phase factor, we performed DNA retardation assays with uninfected nuclear extracts of S-phase cells, synchronized by hydroxyurea pretreatment, and radiolabeled parvoviral termini in their hairpinned conformation. We observed that proteins in HeLa cells, a tissue culture host for AAV, specifically interacted with the terminal sequences of this virus, which act as origins of replication (oris). These assays also showed specific binding between S-phase cellular proteins and termini (oris) of heterologous parvoviruses, for which the cells are not a natural host. For example, proteins from bovine fetal lung (BFL) cells, a tissue culture host for BPV, were able to bind to an AAV terminus and HeLa cell proteins interacted with both termini of the BPV genome. All DNA-protein complexes investigated appeared to be specific for S-phase synchronized cells. In order to begin to characterize the protein(s) involved in the complex formation, we performed SDS-PAGE electrophoresis of some retarded complexes. We report that a 54 kd protein was contained in the complex formed with the BPV left terminus and BFL cell extract. [Binding of BFL cell proteins to a BPV left terminus has been reported earlier]. Using a similar technique, we observed that two phosphoproteins of 55 and 90 kd were present in the retarded complex formed between a BPV left terminus and HeLa cell extract. An antibody directed against human p53, an anti-oncoprotein, was shown to compete binding of BFL cell extract and HeLa cell extract to the BPV left terminus. This antibody also competed the binding of HeLa cell extract to the AAV terminus. Our data suggest that proteins with similar characteristics, most probably among which is p53, are involved in the ori-binding complexes, possibly exerting a role as positive regulator of parvoviral replication.
The secondary structure of the viral ends is remarkably conserved among parvoviruses. Of particular interest is the presence of mismatched/unpaired nucleotides, forming a bubble, in the stem of the left hairpin of almost all autonomous parvoviruses. To analyze the possible role of these unpaired/mismatched nucleotides in the BPV life cycle, two mutants clones lacking the bubble region were constructed and their replicative properties were analyzed after electroporation in permissive cells. Infectivity of the mutant clones was determined by three techniques: observation of cytopathic effect, detection of virally-coded proteins by indirect immunofluorescence, and transient DNA replication assays. We report that the mutant clone containing duplicate sequences of the (mismatched) nucleotides numbered 46 to 57 (BLOP) was defective for replication. The other bubbleless clone (BLOM), that contains duplicate sequences of the (mismatched) nucleotides 99 to 105, was able to replicate. The later clone produced monomer-length viral DNA at about 20% of the level of the infectious genomic clone of BPV, when electroporated as a linear excised sequence. This clone was infectious since it could be propagated by subsequent passage. Expression of viral structural proteins was seen by an indirect immunofluorescence assay using anti-capsid antibodies. Our results suggest that the bubble in the left hairpin of BPV is not required for the viral life cycle, but that specific sequences within the mismatched/unpaired region are necessary for viral replication.<br>Ph. D.
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Evans, Braun Tamara Zoe Marie. "The involvement of HCMV and cellular replication proteins in viral and cellular DNA replication." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608577.
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Nishiyama, Yukihiro. "Herpesvirus Genes: Molecular Basis of Viral Replication and Pathogenicity." 名古屋大学医学部, 1996. http://hdl.handle.net/2237/6180.
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Berthold, François. "Grapevine fanleaf virus replication : viral proteins and host factors." Strasbourg, 2015. http://www.theses.fr/2015STRAJ086.
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Baillie, Andrew James. "Cellular and viral determinants for hepatitis C virus replication." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/6865.
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The recent discovery of an HCV isolate which replicates in cell culture has opened up opportunities to study the full viral life cycle in vitro. This genotype 2a isolate (JFH-1) and its derivatives are the only ones known to replicate efficiently in cell culture, and recent work has indicated that viral determinants for efficient replication may lie in the non-structural protein coding region of the genome. In this thesis chimaeric JFH-1 virus containing full length NS3, NS3 helicase and NS3 protease sequences from genotype 1a and 1b were constructed. The replication efficiencies of chimaeric viruses were tested in cell culture, and were shown not to replicate, indicating that vital viral determinants for JFH-1 replication exist in NS3. The JFH-1 model also provides the opportunity to study the effect of the full viral life cycle on the host cell. Microarray analyses were performed to identify gene expression changes in Huh7 and Huh7.5 cells that had been infected with JFH-1 for 6, 12, 18, 24 and 48 hours. A large number of host genes were found to be regulated during JFH-1 infection, including those involved in lipid metabolism, oxidative stress, apoptosis and intracellular transport. The microarray data were validated by quantitative PCR analyses of separate infection experiments. A selection of the most highly regulated genes was assessed for their necessity to HCV replication by RNA interference studies. The knockdown by siRNA of genes ABLIM3, SPTLC3 and CYP1A1 resulted in significant impairment of HCV replication. The knockdown by siRNA of gene TXNIP (thioredoxin interacting protein) resulted in up to 90% reduction in HCV replication. This is a novel finding which may be of importance to the study of HCV as TXNIP plays roles in oxidative stress, lipid metabolism and glucose metabolism, all of which have potential to influence the HCV lifecycle. Magnetic resonance spectroscopy indicated a change in levels of choline metabolites in JFH-1 infected cells, which has implications for the aspects of the HCV lifecycle associated with lipid membranes and other lipid structures.
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Human, Stacey. "Targeted inhibition of arbovirus replication in mosquito cells." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25899.
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Arthropod-borne viruses (arboviruses) belonging to the Togaviridae, Flaviviridae and Bunyaviridae pose a significant threat to human and animal health worldwide. Many of these (re-)emerging viruses have increased in geographic range and severity. Developing vaccine strains of these viruses that cannot infect, or be transmitted by, mosquito vectors would be useful tools to block virus transmission cycles in their vectors. Exploiting the miRNA pathway to generate viruses that are attenuated, or restricted in their replication, has recently received much attention due to the site-specific expression of miRNAs within a host organism. Recently it has been shown for a number of single-stranded and segmented viruses, that virus spread can be restricted in a cell- or tissue-specific manner by engineering miRNA recognition elements (MREs), which are complementary to cellular or tissue-specific miRNAs, into the virus genome. The aim of this proof-of-principle study was to generate recombinant Semliki Forest viruses (rSFVs) that are unable to replicate in aedine mosquito cell lines. RNA was extracted from uninfected and SFV4-infected Aag2 (Ae. aegypti) and U4.4 (Ae. albopictus) cells and analysed using high-throughput Illumina Solexa sequencing in order to identify mosquito-specific miRNAs. Several of the most highly abundant mosquito-specific miRNAs were selected and MRE cassettes were designed. MRE cassettes were cloned into the SFV4 backbone to produce rSFV, which were able to replicate in mammalian BHK-21 cells but unable to replicate in mosquito-derived cells. Each cassette encoded a Gaussia luciferase (Gluc) reporter gene and four copies of each MRE under the control of a duplicated subgenomic promoter. The resulting viruses were viable, infectious and were used to determine the effect of mosquito-specific MREs on virus replication. A significant reduction was observed in both luciferase (~4 – 5 logs) and virus (~3 logs) production in mosquito cells infected with the rSFVs. Further characterisation of the three most inhibited rSFVs (SFV4-MRE276, SFV4-MRE2940 and SFV4-MRE2945) suggested that the insertion of the MRE cassette into the SFV4 backbone had no significant effect on the growth kinetics of these viruses. Each virus replicated to titres comparable to wildtype (wt) SFV. In stability assays, the rSFVs virus maintained high luciferase expression and high virus titre for 5 low multiplicity passages in mammalian cells. Taken together, these results suggest that the incorporation of MRE cassettes into the SFV4 genome did not affect the stability of, or ability for, these recombinant viruses to replicate, efficiently in a mammalian system. rSFV stability in mosquito cells is questionable as luciferase expression was not maintained over 5 low multiplicity passages in these cells. In order to take this work forward, characterisation of the rSFVs in various mosquito species is required in order to determine whether or not these results are replicated in vivo. In order to apply this technology to a virus that is economically important, the three most effective MREs were cloned into an attenuated Rift Valley Fever virus (RVFV) strain, MP12. The Gluc reporter was removed from the MRE cassettes due to size constraints of the RFVF plasmids. Modified MRE cassettes were cloned into the S and L segments, specifically into the 3’ untranslated regions (UTRs) of the NSs and L genes, to generate single or double rRVFV mutants. All viruses were rescued and were viable. Infected cells displayed cytopathic effect characteristic of RVFV-infected cells. Single S segment mutant viruses reached similar titres to, and took the same amount of time to rescue as, wtRVFV. L segment and double segment mutants (recombinant viruses with MREs in both the S and L segments) reached titres two logs lower and took two days longer to rescue than wtRVFV. This suggests that the incorporation of the MRE cassettes into the L segment UTR is affecting virus transcription or translation in some way. Unfortunately, due to time constraints, further characterisation of these viruses could not be carried out. The technology described here may provide an innovative way to create environmentally contained vaccines that are no longer transmitted by their mosquito vectors.
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Huitema, Carly. "Screening, characterization and inhibition of viral cysteine proteinases." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/6080.
Full textAbstract:
3C proteinases (3Cpros) are a family of essential cysteine proteinases found in
various viruses of medical and agricultural significance. Three lines of research related
to the characterization of 3Cpros were pursued. In the first, biological selections and
screens were developed to evaluate proteinase activity in a high-throughput fashion. A
selection system based on the cleavage of an engineered transcriptional regulator, XylR,
by hepatitis A virus (HAV) 3Cpro failed. However, this strategy facilitated the
development of a screen based on the cleavage of fused green fluorescent protein
variants, CyPet and YPet. The screen was used to demonstrate that HAV 3Cpro prefers
Ile, Val or Leu at the P₄ position of the cleavage sequence, and Gly, Ser or Ala at the P₁’
position.
In the second project, the 3Cpro from Israeli acute paralysis virus (IAPV), a
dicistrovirus, was investigated. IAPV has been associated with the recent colony collapse
disorder afflicting commercial hives. A portion of the replicase including 3Cpro was
heterologously produced. The resulting autoprocessed fragments were analyzed using
mass spectrometry to identify the 3Cpro cleavage sequence PIVIE/AQT. This cleavage
sequence likely occurs between the 3A/3B proteins of the polypeptide and is the first
within the replicase to be described in this family of viruses.
Thirdly, inhibitors of HAV 3Cpro and SARS 3CLpro were developed. The keto-
glutamine analogue (HIP2-171-2) competitively inhibited SARS 3CLpro with a Kic = 0.17
± 0.03 μM and is among the most potent peptide inhibitors developed against this
proteinase. The azapeptide epoxide (APE) KAE-3-91 irreversibly inhibited SARS 3CLpro
with a kinact/Ki of 1900 ± 400 M−¹s−¹, which is similar in magnitude to that of the first
generation APE’s produced to inhibit caspases. Finally, both SARS 3CLpro and HAV
3Cpro were screened against a library of inhibitory halopyridinyl esters. Each of three
halopyridinyl esters inhibited 3Cpro with apparent Kic’s of 120-240 nM. However, further
study revealed that the inhibitors were slowly hydrolyzed by both proteinases.
Overall, the described screens and inhibitors should facilitate the further
characterization of 3C and related proteinases as well as the development of novel antivirals.
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Lu, Lenette L. "dsRNA Signaling in Innate Immunity and Viral Inhibition." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1220030971.
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Upton, John H. "The role of RNA secondary structure in replication of Nodamura virus RNA2." To access this resource online via ProQuest Dissertations and Theses @ UTEP, 2009. http://0-proquest.umi.com.lib.utep.edu/login?COPT=REJTPTU0YmImSU5UPTAmVkVSPTI=&clientId=2515.
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King, Benjamin. "Arenavirus Transcription, Replication, and Interaction with Host-Cellular Components." ScholarWorks @ UVM, 2018. https://scholarworks.uvm.edu/graddis/830.
Full textAbstract:
Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. Despite decades of research, it is still unclear how these viruses establish a lifelong, asymptomatic infection in their rodent hosts while infection of humans often results in severe disease. Unable to enter a state of bona fide latency, the transcription and replication of the viral genomic RNA is likely highly regulated in time and subcellular space. Moreover, we hypothesize that the viral nucleoprotein (NP), responsible for the encapsidation of the viral RNA and the most highly expressed viral gene product, plays a key role in the regulation of the viral gene expression program. Further, exploring host-virus interactions may elucidate the basic aspects of arenavirus biology and how they cause such severe disease in humans. To explore these questions in greater detail, this dissertation has pursued three main avenues.
First, to better understand lymphocytic choriomeningitis mammarenavirus (LCMV) genome replication and transcription at the single-cell level, we established a high-throughput, single-molecule (sm)FISH image acquisition and analysis pipeline and followed viral RNA species from viral entry through the late stages of persistent infection in vitro. This work provided support for a cyclical model of persistence where individual cells are initially transiently infected, clear active infection, and become re-infected from neighboring reservoir cells within the population.
Second, we used FISH to visualize viral genomic RNA to describe the subcellular sites where LCMV RNAs localize during infection. We observed that, viral RNA concentrates in large subcellular structures located near the cellular microtubule organizing center and colocalizes with the early endosomal marker Rab5c and the viral glycoprotein in a proportion of infected cells. We propose that the virus is using the surface of a cellular membrane bound organelle as a site for the pre-assembly of viral components including genomic RNA and viral glycoprotein prior to their transport to the plasma membrane where new particles will bud.
Last, we used mass spectrometry to identify human proteins that interact with the NPs of LCMV and Junín mammareanavirus (JUNV) strain Candid #1. We provided a detailed map of the host machinery engaged by arenavirus NPs, and in particular, showed that NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. We demonstrated that JUNV antagonizes the antiviral activity of PKR completely, effectively abrogating the antiviral activity of this surveillance pathway.
In sum, the work composing this dissertation has given us fresh insight into how arenaviruses establish and maintain persistence; the nature of the subcellular site where viral genomic RNA is transcribed, replicated, and assembled with other viral components; and a global view of the cellular machinery hijacked by the viral nucleoprotein. This work improves our basic understanding of the arenavirus life cycle and may suggest novel antiviral therapeutic targets that could be exploited in the future.
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Nayak, Ramnath. "Characterization of the role of adenovirus-5 (Ad-5) gene products E2A, E4ORF6 and VA RNA on adeno-associated virus type 5 (AAV5) transcription, translation and replication." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/6005.
Full textAbstract:
Thesis (Ph. D.)--University of Missouri-Columbia, 2007.<br>The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2007" Includes bibliographical references.
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Bull, Rowena Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Mechanisms of replication and genomic diversity in human caliciviruses." Awarded by:University of New South Wales. Biotechnology & Biomolecular Sciences, 2007. http://handle.unsw.edu.au/1959.4/40657.
Full textAbstract:
Norovirus (NoV) and Sapovirus (SaV) are major causes of outbreak gastroenteritis worldwide. NoV and SaV are highly infectious, have multiple transmission routes and have a short incubation period, thereby facilitating rapid intercontinental spread of new variants. Consequently, a treatment would be advantageous for controlling them. However, currently little is known about the replication cycle and evolution of human NoV or SaV as neither are culturable. NoV and SaV are RNA viruses of the Caliciviridae family and have great genetic diversity which is thought to facilitate irnmune evasion. Consequently variants of NoV GI1.4 arose in 1996, 2002, 2004 and in 2006 and resulted in pandemics. Therefore, in this study, the role of the two main mechanisms associated with generating viral diversity; recombination, and point mutation were investigated for NoV and SaV. Physiological and kinetic properties of three NoV RdRps (genotypes, Gll.b, Gll.4, Gll.7) and two SaV RdRps (genogroups GI, GII) were also investigated. RNA recombination is a significant driving force in viral evolution. Increased awareness of recombination within the Calicivirus genus Norovirus (NoV) has led to a rise in the identification of NoV recombinants and they are now reported at high frequency. Despite this no classification system exists for recombinant NoVs As a result, there is duplication in reporting novel recombinants and the precise number of novel NoV recombinant types is unknown. Therefore, in order to elucidate thero!e of recombination in NoV evolution, 121 NoV nucleotide sequences, compiled from the GenBank database and published literature, were analysed for recombination events. NoV recombinants and their recombination breakpoint were identified using three methods: phylogenetic analysis, Simplot analysis and the Maximum Chi-Squared method. In total 19 unique NoV recombinant types were identified in circulation across the globe and they had a common recombination point near the ORF1/2 overlap. Recombination at the ORF1/0RF2 overlap could have important implications in NoV evolution as it enables a virus to swap its antigenic determinates (capsid) and thereby avoid immune clearance in an analogous manner to antigenic shift in influenza virus. This study also examined the role of NoV and SaV replication in generating viral diversity by comparing the physiological, kinetic and biochemical properties of five genotypically distinct RdRps from two different genera of the Caliciviridae. Genetically diverse HuCV RdRps were expressed in Escherichia coli and characterised in an in vitro assay designed for this study. The results indicated that despite high sequence variation between the five enzymes (between 6% and 71% amino acid difference) they shared similar physiological properties. Though there was some variation in their template usage and kinetic properties. SaV was able to perform primer dependent replication on homopolymeric A RNA whereas the NoV RdRps were not. Additionally, NoV RdRps had a higher incorporation rate and were more kinetically efficient than the two SaV RdRps. The incorporation fidelity of the five enzymes was similar (between 2.2x10-5 to 8.9x10-4 ), although interestingly the most prevalent strain, Gll.4, had the lowest fidelity of the caliciviruses. Therefore, suggesting that RdRp fidelity has an important role in NoV evolution. Overall, this study illustrated that NoV and SaV generate genetic diversity in a similar fashion to other RNA viruses, that is, a delicate combination of recombination, point mutation and replication efficiency. Understanding the mechanisms involved in viral replication and genomic diversity of the calicivirus RdRps is essential if a successful control strategy for the human caliciviruses is going to be developed.
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MAENO, KOICHIRO. "Replication of Influenza B Virus: Biological Functions of Viral Neuraminidase." Nagoya University School of Medicine, 1994. http://hdl.handle.net/2237/15935.
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Raynes, Joshua R. "Analysis of Kinase Effects on Viral Replication of the Papillomavirus." UNF Digital Commons, 2006. http://digitalcommons.unf.edu/etd/241.
Full textAbstract:
Papillomaviruses are a genera of small tumor viruses in the Papovaviridae family, whose lifecycle and replication ability is directed by epithelial differentiation. During latency, papillomavirus DNA replication occurs synchronously with the host cell's replication by the activation of the El protein. To elucidate the effects upon viral replication, this study utilized chemical inhibition of several kinases predicted to phosphorylate, and subsequently modify the activity of, the papillomavirus' E1 protein. The amount of DNA replicated was observed via autoradiography following DNA extraction and southern blotting of BPV-transformed C127 cells. Sample extracts from cells exposed to specific chemical inhibitors of PKC, CDK, and DNAPK showed a consistent and significant decrease in viral DNA when compared to the DNA abundance of a control set of extracts. Extracts of cells subjected to inhibition of CK2 displayed an observable increase in replicated viral DNA. To ensure that the kinase modification was not effecting the growth or viability of the cells, a neutral red assay was performed and found no significant difference between control and chemically treated samples in cell viability or overall cell number. These findings, in conjunction with the differential viral DNA abundance, implicate that kinases PKC, CDK, CK2, and DNAPK, have a role in viral genome replication.
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Donaldson, Eric F. Baric Ralph S. "Computational and molecular biology approaches to viral replication and pathogenesis." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2008. http://dc.lib.unc.edu/u?/etd,1731.
Full textAbstract:
Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.<br>Title from electronic title page (viewed Sep. 16, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Microbiology and Immunology Virology." Discipline: Microbiology and Immunology; Department/School: Medicine.
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Geller, Ron. "The role of Hsp90 in viral replication : picornaviruses and beyond /." May be available electronically:, 2008. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Chadwick, D. R. "Studies on antisense RNA inhibition of HIV-1 replication." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597386.
Full textAbstract:
Two vector systems were developed expressing antisense RNA (of two different sizes) complementary to three target regions in the 5' leader/LTR of HIV-1: the R (TAR) region, the primer binding site and the splice donor-packaging signal (ψ) region. After cloning these regions into an expression vector, <I>pcDNA3, </I>designed to express these sequences at high levels from the CMV IE promoter, stable constitutive expression in a T cell line was demonstrated by RT-PCR. After directly challenging these cell lines with HIV-1 at various doses cell lines expressing antisense RNA targeting the ψ-region showed significant inhibition of replication compared to cell lines expressing the same sequence in <I>sense</I>. A second, co-transfection assay using COS-1 cells was also developed to assess the antiviral capabilities of these vectors. Both sequences targeting the ψ-region and one sequence complementary to the TAR region inhibited expression of viral protein; furthermore analyses of relative levels of cellular and viral RNA from these assays suggested that each of these antisense molecules was exerting its effect at an early stage in the transcription-translation pathway. CXCR4, an important co-receptor for HIV was also targeted for down regulation by antisense RNA. Inducible expression of a sequence complementary to a region of this gene using the 'tet-on' system was accomplished leading to down-regulation of surface CXCR4 expression in T cells. Down-regulation appeared to result in reduced replication of HIV-1 in these cells. Genetic strategies for inhibiting HIV replication may provide an alternative and complementary approach to chemotherapy for infected individuals.
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Stoner, Terri Dorene. "Indole-3-Carbinol Inhibition of Herpes Simplex Virus Replication." Kent State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=kent1228328838.
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Kabdulov, Timur O. "Mechanisms of retroviral replication." Morgantown, W. Va. : [West Virginia University Libraries], 2001. https://etd.wvu.edu/etd/controller.jsp?moduleName=documentdata&jsp%5FetdId=2256.
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McGivern, David. "A functional analysis of the replication-associated proteins of maize streak virus." Thesis, University of East Anglia, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249710.
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Moncunill, Piñas Gemma. "Potential New Therapeutic Agents: Effects on HIV Replication and Viral Escape." Doctoral thesis, Universitat Autònoma de Barcelona, 2009. http://hdl.handle.net/10803/4538.
Full textAbstract:
El desenvolupament de nous compostos anti-VIH és una continua necessitat degut a l'aparició de soques resistents i a la toxicitat a llarg terme dels fàrmacs actuals. L'entrada del VIH a les cèl·lules requereix la unió de les partícules virals al receptor CD4 i a un coreceptor, principalment CCR5 o CXCR4, a través de la glicoproteïna viral de l'embolcall gp120, induint canvis conformacionals a gp41 que promouen la fusió de la membrana viral i de la cèl·lula hoste, alliberant la càpside al citoplasma de la cèl·lula. Les soques de VIH es poden classificar en diferents tropismes segons quin coreceptor utilitzen: CCR5 (R5), CXCR4 (X4) o ambdós coreceptors (R5X4). Així doncs, el procés d'entrada és un pas essencial que ofereix noves dianes per agents antivirals. Dels compostos desenvolupats, els antagonistes dels coreceptors són uns dels agents més prometedors. Una altra classe de compostos interessants són les estatines, inhibidors de la HMGCoA reductasa, una classe de fàrmacs ben establerts pel tractament de la hipercolesterolèmia. Estudis recents suggereixen que les estatines tenen efectes directes contra el VIH tant in vitro com in vivo, inhibint l'entrada i la gemmació de virions. L'impacte d'ambdues classes de compostos sobre el VIH i les seves conseqüències en els tractaments a llarg terme són encara desconeguts. Vam voler caracteritzar les estatines com a agents antiretrovirals, determinar el paper que juguen els antagonistes dels coreceptors en l'evolució del tropisme del VIH i caracteritzar el nou antagonista de CXCR4 POL3026. Malauradament, no vam poder detectar una activitat antiviral significativa amb cap de les estatines avaluades, degut a la gran citotoxicitat en els cultius cel·lulars. Tampoc vam poder observar cap efecte de la simvastatina en un estudi pilot en 12 pacients VIH+ durant 8-12 setmanes, tot i ser efectiva en disminuir els nivells de colesterol en plasma. Utilitzant un model in vitro per estudiar el canvi de coreceptor de soques R5 a R5X4 o X4, vam observar que la probabilitat de canviar depèn de l'aïllat clínic i també de les condicions de cultiu, com la disponibilitat de CCR5. La pressió selectiva d'un compost anti-VIH podia modificar l'ús coreceptor. Així doncs, agents contra CCR5 i els inhibidors de la transcriptasa inversa (RT) endarrerien l'emergència de variants R5X4 o X4. Tot i això aquestes variants apareixien més ràpid sota pressió dels agents dirigits contra CCR5 que dels inhibidors de la RT, mentre que antagonistes de CXCR4 podien prevenir la seva aparició. Hem caracteritzat el compost POL3026, que ha demostrat ser un potent inhibidor de soques salvatges y resistents a fàrmacs actuals amb fenotip X4 o R5X4. Diferents assajos i el desenvolupament de virus resistents ens ha permès confirmar que POL3026 bloqueja la replicació del VIH mitjançant la interacció específica amb CXCR4 i que actua com un potent antagonista d'aquest coreceptor. En conclusió, els nostres resultats demanen cautela en l'ús de les estatines per tractar la infecció del VIH i en el risc de que el VIH pot escapar dels antagonistes de CCR5 mitjançant la selecció de virus que utilitzin CXCR4. Per una altra banda, els antagonistes de CXCR4, com POL3026, són interessants agents antivirals que podrien prevenir l'aparició de virus X4 o R5X4.<br>Developing new anti-HIV compounds targeting different steps of the HIV cycle is a continuous need due to the emergence of drug-resistant strains and also to the long-term toxicity of current antiretrovirals used for treatment. HIV entry requires the binding of the viral particles to the CD4 receptor and a coreceptor, mainly CCR5 or CXCR4, through the viral envelope glycoprotein gp120, triggering structural changes in gp41 that promote the fusion of the viral and host cell membranes and viral core release into the host cell. HIV strains can be classified into different tropisms depending on which coreceptor they use: CCR5 (R5), CXCR4 (X4) or both coreceptors (R5X4). Therefore, HIV entry is an essential step that offers several potential new targets for antiviral agents. From the compounds developed, coreceptor antagonists are among the most promising agents. Another class of interesting compounds are statins, HMGCoA reductase inhibitors which are a well-established class of drugs prescribed for treatment of hypercholesterolemia. Recent studies suggest that statins have direct anti-HIV effects both in vitro and in vivo targeting HIV entry and budding. The impact of both classes of compounds, statins and coreceptor antagonists, on HIV replication and viral escape are still unknown. and therefore, its consequences in long-term treatment are also unclear. <br/>We wanted to characterize statins as antiretroviral agents, determine the role of coreceptor inhibitors in the evolution of HIV tropism and characterize the novel CXCR4 antagonist POL3026. Unfortunately, we could not detect a significant anti-HIV activity of statins due to a high cytotoxicity in cell culture or any effect of simvastatin in a pilot study with 12 HIV+ patients after 8-12 weeks, although it was effective lowering the plasma cholesterol levels. Using an in vitro model to study coreceptor switch of R5 strains to R5X4 or X4, we found that the probability to change coreceptor use was dependent on the clinical isolate and also on the cell-culture conditions such as the availability of CCR5. We observed that selective pressure of an anti-HIV compound can modify the evolution of coreceptor use. Reverse transcriptase (RT) inhibitors and CCR5 targeting agents delayed the emergence of CXCR4-using variants compared to untreated cultures. However, CXCR4-using variants emerged faster under CCR5 drug pressure than under RT inhibitors, whereas CXCR4 antagonists could prevent its emergence. We also characterized the novel compound POL3026, which proved to be a potent anti-HIV agent against naïve and drug-resistant strains of X4 and R5X4 phenotype. Several assays and development of resistance allowed us to confirm that POL3026 blocked HIV replication through specific interaction with CXCR4 and that it is a potent antagonist of this coreceptor. In conclusion, our results caution on the use of statins to treat HIV infection and on the risk that HIV may escape from CCR5 antagonists by selecting CXCR4 emerging variants. On the other hand, potent and specific CXCR4 antagonists, such as POL3026, are interesting antiviral agents that could prevent the emergence of R5X4 or X4 viruses.
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Arnoldi, Francesca. "Interaction of rotavirus nonstructural protein NSP5 with the viral replication complex." Doctoral thesis, Scuola Normale Superiore, 2008. http://hdl.handle.net/11384/85952.
Full textAbstract:
Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms,
where synthesis of the11 dsRNA genome segments and their packaging in new viral
particles take place. RNA replication is mediated by several viral proteins, of which
VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein,
were shown to be sufficient to provide replicase activity in vitro. In vivo, however,
viral replication complexes also contain the nonstructural proteins NSP2 and NSP5,
which were shown to be essential for replication, to interact with each other and to
form viroplasm-like structures (VLS) when coexpressed in uninfected cells.
In order to gain a better understanding of the intermediates formed during viral
replication, this work focused on the interactions of NSP5 with VP1, VP2 and NSP2.
We constructed a tagged form of VP1 and by coimmunoprecipitation experiments
we demonstrated that VP1 and NSP5 interact in virus-infected cells as well as in the
absence of other viral proteins or viral RNA in cotransfected cells. Using deletion
mutants of NSP5 or different fragments of NSP5 fused to EGFP, we identified the 48
C-terminal amino acids as the region essential for interaction with VP1. On the other
hand, removal of the C-terminal 15 amino acids from tagged VP1 resulted in a less
efficient coimmunoprecipitation with NSP5, suggesting an involvement of the Cterminus
of VP1. Interaction of NSP5 with VP2 was investigated by coexpression of
the two proteins in uninfected cells, which resulted in a strong hyperphosphorylation
of NSP5 and in the formation of VLS, that we named VLS(VP2i) to distinguish them
from those induced by NSP2, here designated as VLS(NSP2i). VLS(VP2i) were shown
to assemble independently of the phosphorylation degree of NSP5 and to recruit the
viroplasm-resident proteins NSP2, VP1, VP2 and VP6 (the protein forming the middle
Abstract
4
layer of the virion). Attempts to coimmunoprecipitate NSP5 and VP2 failed both from
infected and cotransfected cells.
Tagged VP1 was found to localize in VLS (both VP2i and NSP2i) and in viroplasms,
and to be able to replace wild-type VP1 structurally by being incorporated into
progeny viral particles. Coexpression of different combinations of tagged VP1,
NSP5, NSP2 and VP2 showed that the interaction of VP1 with NSP5 is not affected
by the other viral proteins and is stronger than the interaction with NSP2. In addition,
an inhibitory effect of VP1 on the levels of NSP5 hyperphosphorylation induced by
both NSP2 and VP2 was observed.
Altogether, these data confirmed an important role for NSP5 in replication, related
with the interactions with the two structural proteins essentially involved in viral
genome synthesis, and suggested that NSP5 plays a key role in architectural
assembly of viroplasms and in recruitment of the other viroplasmic proteins.
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Hänni, Christine Anne. "The function of the checkpoint-dependent inhibition of replication initiation." Thesis, University of Cambridge, 2016. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709525.
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Al-Hababi, Fadhil H. "Inhibition of HCV replication by host cell innate antiviral defences." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/6828.
Full textAbstract:
RIG-I and MDA5 are the major intracellular sensors for the induction of IFN-β, by dsRNA, in Huh7 cells. It was found that the expression of RIG-I, MDA5 and the signalling mediators TRIF, MAVS and MyD88 were upregulated in Huh7 HCV RNA replicon cells. Similar findings were obtained in Huh7 cells infected with JFH-1 HCV. When replicon and HCV infected cells were further stimulated by dsRNA the induction of these genes were reduced compare to control cells. The expression of NS3 and NS3/4A proteins, using adenovirus vectors, reduced RIG-I, MDA5, MAVS and MyD88 expression levels in response to dsRNA stimulation. Ablation of MDA5, MAVS, MyD88 and TRIF in HCV infected Huh7.5 cells, using siRNAs, increased virus replication demonstrating their importance in inhibiting HCV infection. These results show that HCV inhibits the induction of interferon in response to dsRNA and that NS3 plays an important role in this inhibition. Analysis of gene expression during HCV infection showed significant changes in the expression of genes involved in the innate immune response, interferon, apoptosis and cell cycle regulation pathways. These include SOCS1, 2 and 3, OAS3, MXA1, IRF9, IRF1, IFNAR2 and STAT1. SOCS3 knockdown by siRNAs reduced HCV replication indicating an additional mechanism of IFN inhibition by HCV. Infection with HCV also caused cell cycle arrest leading to DNA fragmentation and apoptosis. A decrease in cyclin B1 and an increase in GADD45-β expression during HCV infection indicates that they may play an important role in HCV induced alterations to the cell cycle.
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Han, Xue. "Inhibition of PRRSV replication by combination of antisense morpholino oligomers." College Park, Md.: University of Maryland, 2008. http://hdl.handle.net/1903/8633.
Full textAbstract:
Thesis (M.S.) -- University of Maryland, College Park, 2008.<br>Thesis research directed by: Dept. of Animal and Avian Sciences. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Temperley, Simon M. "A study of the in vitro initiation of adenovirus DNA replication." Thesis, University of St Andrews, 1992. http://hdl.handle.net/10023/13929.
Full textAbstract:
The development of systems in which adenovirus DNA can be replicated in vitro has led to the elucidation of the sequences essential for origin function and to the identification of the proteins required for viral DNA replication. Much of the information currently available has been derived from investigations carried out using adenovirus types 2 and 5 which in addition to the viral proteins, adenovirus DNA polymerase, precursor terminal protein and DNA binding protein, require cellular proteins nuclear factor I and nuclear factor III for efficient initiation of DNA replication. In contrast adenovirus type 4 replicates Its DNA efficiently without these cellular proteins. Correspondingly its minimal origin of replication is remarkably simple in structure, consisting of only the terminal 18bp of the adenovirus genome ('the core sequence'). The effect of point mutations in the core region on adenovirus type 4 DNA replication in vitro was investigated and it was found that mutations within two discrete domains had a married deleterious effect on initiation of DNA replication. The crude Ad4 infected cell extracts initially used for in vitro DNA replication were fractionated and it was found that only four detectable proteins, three of which were identified as viral DNA polymerase, precursor terminal protein and DNA binding protein gave efficient DNA replication in vitro and furthermore behaved similarly to unfractionated infected cell extracts in the presence of template which contained point mutations. To examine a possible role of the core region of the origin as containing sites for specific interactions with viral replication proteins, purified adenovirus type 5 precursor terminal protein and DNA polymerase were assayed for their ability to recognise the terminal 1-18 sequence. It was found that both proteins independently and as a heterodimer bound specifically to a sequence corresponding to the core origin of replication, suggesting that sequences within this region are important for localisation of DNA replication proteins at the origin via a sequence specific DNA-protein interaction.
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Shapka, Natalia. "IDENTIFICATION OF VIRAL AND HOST FACTORS INVOLVED IN TOMBUSVIRUS REPLICATION AND RECOMBINATION." UKnowledge, 2006. http://uknowledge.uky.edu/gradschool_diss/449.
Full textAbstract:
Rapid evolution of RNA viruses with mRNA-sense genomes is a major concern to health and economic welfare due to the devastating diseases these viruses inflict on humans, animals and plants. Rapid viral RNA evolution is frequently due to RNA recombination, which can be facilitated by recombination signals present in viral RNAs. Among such signals are short sequences with high AU contents that constitute recombination hot spots in Brome mosaic virus (BMV) and retroviruses. We have demonstrated that a defective interfering (DI) RNA, a model template associated with Tomato bushy stunt virus (TBSV), a tombusvirus, undergoes frequent recombination in plants and protoplast cells when it carries the AU-rich hot spot sequence from BMV. Similar to the situation with BMV, most of the recombination junction sites in the DI RNA recombinants were found within the AU-rich region. Our results support the idea that common AU-rich recombination signals might promote interviral recombination between unrelated viruses. To test if host genes can affect the evolution of RNA viruses, we used a Saccharomyces cerevisiae single-gene deletion library, which includes ~80% of yeast genes, in RNA recombination studies based on a small viral replicon RNA derived from TBSV. The genome-wide screen led to the identification of five host genes, whose absence resulted in rapid generation of novel viral RNA recombinants. Thus, these genes normally suppress viral RNA recombination, but in their absence hosts become viral recombination hotbeds. Four of the five recombination suppressor genes are likely involved in RNA degradation, suggesting that RNA degradation could play a role in viral RNA recombination. Overall, our results demonstrate for the first time that a set of host genes have major effect on RNA virus recombination and evolution. Replication of the non-segmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. We have demonstrated that p33 is phosphorylated in vivo and in vitro by a membrane-bound plant kinase. Based on in vitro studies with purified recombinant p33, we show evidence for phosphorylation of threonine and serine residues adjacent to the essential RNA-binding site in p33. Our findings suggest that phosphorylation of threonine/serine residues adjacent to the essential RNA-binding site in the auxiliary p33 protein likely plays a role in viral RNA replication and subgenomic RNA synthesis during tombusvirus infections.
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Pathak, Kunj Bihari. "CHARACTERIZATION OF VIRAL AND HOST PROTEINS AND RNA ELEMENTS IN TOMBUSVIRUS REPLICATION." UKnowledge, 2011. http://uknowledge.uky.edu/plantpath_etds/1.
Full textAbstract:
Two thirds of plant viruses are positive-strand RNA viruses including the family Tombusviridae. One of the best-studied members of this family is Tomato bushy stunt virus (TBSV). Like many other viruses, TBSV has much fewer genes when compared to its hosts’ genome. Nevertheless, TBSV utilizes its genome very judiciously. To compensate for a lack of many proteins of its own, it codes for multi-functional replication protein p33 and also co-opts host factors to facilitate its replication.
By using recombinant replication proteins p33 and p92 containing single amino acid changes in protein-protein interaction domains (S1 and S2), I demonstrated that the replication proteins are required in sequential steps during virus replication. The in vitro cell-free extract(CFE) based TBSV replication assays revealed that mutations in S1 and S2 domains affected RNA template selection, recruitment and assembly of replicase complex. TBSV replicates on the cytosolic surface of peroxisomal membranes.
To identify the host factor involved in this process of transporting viral replication proteins to peroxisome, I tested the peroxisomal transporter proteins for their ability to bind to p33 in vitro, which led to the discovery of Pex19p. Pull-down and co-purification experiments revealed transient nature of p33-Pex19p binding as expected from a transporter. When pex19p was retargeted to mitochondria, a large fraction of p33 was also re-distributed to the mitochondria validating the importance of Pex19p in p33 localization.
TBSV also utilizes its genomic RNA for non-template activities during its replication. Accordingly, TBSV RNA serves as a platform for the assembly of replicase complex. To further characterize the regulatory cis-elements involved in this process, I utilized CFE and different TBSV RNA mutants together with recombinant p33 and p92 in vitro replication assays. These experiments revealed the role of RNA recruitment element [RIISL(+)] and 3’ non-coding regions as minimal cis-elements required to assemble functional replicase complex. The experiments also indicated that the RIISL(+) and 3’ non coding regions could be physically separated on two different RNA molecules to assemble TBSV replicase, suggesting insights into viral evolution.
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Deng, Ruitang. "Molecular cloning, nucleotide sequencing and genome replication of bovine viral diarrhea virus /." The Ohio State University, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487779914825349.
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Dible, Michele Sturgill. "The effect of rolling on viral replication and detection in cell culture /." The Ohio State University, 1994. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487856906257721.
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Chen, Bojun. "Nitric Oxide Production: A Mechanism for Inhibition of Chlamydia Trachomatis Replication." Digital Commons @ East Tennessee State University, 1993. https://dc.etsu.edu/etd/2653.
Full textAbstract:
Chlamydia trachomatis (CT) replicates in macrophages, but is inhibited by IFN-$\gamma$ or LPS. IFN-$\gamma$ and/or LPS induced nitrite production in mouse peritoneal macrophages, macrophage cell lines (RAW264.7 and J774A.1) and McCoy cells. Kinetic studies indicated that peak production occurred 48 hours post-treatment. CT infection itself was insufficient to induce nitrite production, but resulted in enhancement of nitrite production in IFN-$\gamma$-treated cells. Treatment with IFN-$\gamma$ or LPS resulted in significant inhibition of CT replication in these cells. Strong correlation between nitrite production and inhibition of CT replication was observed in RAW264.7 and J774A.1 cells (correlation coefficients: $-$0.93 and $-$0.94, p $<$ 0.001). N$\sp{\rm g}$- monomethyl-L-arginine (L-NMMA) specifically inhibited nitrite production and partially reversed inhibition of CT replication in macrophage cell lines. NOS mRNA was measured in RAW264.7 cells by Northern blot and Dot blot hybridization. Strong correlation between NOS mRNA expression and inhibition of CT replication (correlation coefficient: $-$0.97, p $<$ 0.05) was observed. Anti-TNF-$\alpha$ antibody completely neutralized the biological activity of TNF-$\alpha$ secreted by LPS-treated RAW264.7 cells, yet the antibody neither reduced nitrite production nor restored CT replication. Combination of the antibody and L-NMMA significantly enhanced restoration of CT replication. In peritoneal macrophages, inhibition of CT replication induced by IFN-$\gamma$ was partially restored by L-NMMA or anti-TNF-$\alpha$ antibody. In McCoy cells, inhibition of CT replication induced by IFN-$\gamma$ and LPS was not significantly restored by L-NMMA. Great restoration of CT replication by 1 mM L-NMMA was observed in LPS-treated J774A.1 cells (31%), but not in IFN-$\gamma$-treated cells (5%). Our data indicate that (1) NO production is one of the mechanisms for inhibition of CT replication in IFN-$\gamma$-activated peritoneal macrophages and RAW264.7 cells; (2) NO plays a significant role in CT inhibition in LPS-treated macrophage cell lines, but not peritoneal macrophages; (3) TNF-$\alpha$ may be associated with inhibition, but the mechanism(s) may not involve NO production; (4) NO production may not be the mechanism for CT inhibition in McCoy cells treated with IFN-$\gamma$ and LPS.
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Lu, Jia. "Norovirus translation and replication." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/278610.
Full textAbstract:
Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. Despite the significant disease and economic burden, currently there are no licensed vaccines or antivirals. The understanding of norovirus biology has been hampered by the inability to cultivate HuNoV in cell culture. To establish a tissue culture system, infectious HuNoVs were purified from clinical stool samples. HuNoV replication was tested in different cell types. The B-cell and intestinal organoids culture systems were validated. In addition, using organoids culture a DNA-based reverse genetic system was shown to recover infectious HuNoV. Due to the challenges associated with cultivating HuNoV, murine norovirus (MNV) was used as a surrogate system to understand the role of eIF4E phosphorylation in norovirus pathogenesis, and VP1-RdRp interaction in regulating viral genome replication. MNV infection results in the phosphorylation of the translation initiation factor eIF4E, re-programming host-cell translation during infection. Inhibiting eIF4E phosphorylation reduces MNV replication in cell culture suggesting a role in viral replication. A mouse model with eIF4E S209A, a phosphor-ablative mutation, was established to understand the role of eIF4E phosphorylation in MNV pathogenesis. In vitro and in vivo characterisations demonstrated that eIF4E phosphorylation may have multiple roles in norovirus-host interactions, but overall has little impact on MNV pathogenesis. The shell domain (SD) of norovirus major capsid protein VP1 interacts with viral RNA-dependent RNA polymerase (RdRp) in a genogroup-specific manner to enhance de novo initiation of RdRp, and to promote negative-strand RNA synthesis. To understand how VP1 regulates norovirus genome replication, chimeric MNVs with genogroup-specific residues mutagenised were characterised in vitro and in vivo. A single amino acid mutation was shown to destabilise viral capsid. SDs with reduced VP1-RdRp interaction showed less capacity to stimulate RdRp, resulting in delayed virus replication. In vivo, the replication of an MNV-3 with homologous mutations was abolished, highlighting the crucial role of this interaction.
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Dao, Luan D. "Human papillomavirus segregation and replication /." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008p/dao.pdf.
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Tai, Hung, and 戴雄. "The role of the non-structural protein, NS1, in influenza virus replication." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44660303.
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Pirlo, Steven Dominic. "Identification of viral-based replicating vectors suitable for the development of a sugarcane bioreactor." Thesis, Queensland University of Technology, 2007. https://eprints.qut.edu.au/16548/1/Steven_Dominic_Pirlo_Thesis.pdf.
Full textAbstract:
The circular, single-stranded (ss) DNA genomes of plant viruses in the families Geminiviridae and Nanoviridae are replicated within the nucleus of a host cell by a mechanism called rolling circle replication (RCR). Although this process relies almost exclusively on the replication machinery of the host cell, initiation occurs via the interaction of the viral replication initiation protein (Rep) with regulatory DNA sequences within the viral genome. The use of a virus-based episomal amplification technology as a plant bioreactor platform exploits the process of Rep-mediated RCR for the high-level amplification of virus-based episomes in plants and subsequent expression of heterologous proteins; such an approach offers advantages over existing gene expression technologies. This PhD thesis describes research towards the development of a virus-based episomal amplification system for use in sugarcane. Such a crop is ideally suited for a plant bioreactor system due to the efficient high-level production of plant biomass and the existence of established production, harvesting and processing infrastructure.
In order to rapidly assess the potential of a virus-based episomal amplification system in sugarcane, a transient assay system was established. Sugarcane callus was identified as the most suitable cell preparation; providing rapid cell regeneration, uniform experimental samples and upon isolation, total DNA suitable for Southern analysis. This assay system once established, proved effective in rapidly identifying virus-based episomes capable of undergoing RCR within sugarcane host cells.
This transient assay system was then used to test the functionality of a virus-based episomal amplification system based on the ssDNA virus, Banana bunchy top virus (BBTV) in sugarcane. BBTV-based episomal amplification vectors were constructed with a reporter gene expression cassette flanked by two copies of the BBTV regulatory DNA sequences. The episomal amplification vectors were bombarded into sugarcane and banana host cells in various combinations and evidence of RCR was assessed through Southern blot analysis. RCR products were identified in banana host cells bombarded with the BBTV-based episomal amplification vectors in combination with vectors encoding BBTV Master-Rep (M Rep). RCR products were not identified within sugarcane cells bombarded with the same construct combinations.
Integrated InPAct (In Plant Activation) episomal vectors based on BBTV were then employed to confirm the transient results, in addition, the functionality of an InPAct vector based on an alternate virus, Tobacco yellow dwarf virus (TYDV) was also assessed. InPAct vectors based on BBTV were constructed with an untranslatable expression cassette for integration within the sugarcane genome. Transient experiments were performed to assess the ability of BBTV M-Rep and TYDV Rep to initiate RCR of their respective InPAct vectors. Visual observation of GFP expression indicated that BBTV M-Rep was capable of initiating RCR of the BBTVbased InPAct vectors within banana host cells but no evidence was observed in sugarcane host cells. TYDV Rep was capable of initiating RCR of the TYDV-based InPAct vector within sugarcane host cells with a 100-fold increase in the number of fluorescent foci compared to cells bombarded with the TYDV InPAct vector alone. The BBTV-based InPAct vector was stably integrated within the sugarcane genome and the ability for BBTV M-Rep to initiate episome formation and RCR was assessed by Southern blot analysis. Evidence of BBTV M-Rep mediated RCR was not detected within the transgenic sugarcane bombarded with BBTV M-Rep. Transgenic sugarcane containing the TYDV-based InPAct vectors was assessed for the ability to be activated by TYDV Rep and undergo RCR. Southern blot analysis demonstrated that TYDV Rep was capable of recognising the integrated TYDVbased InPAct vector and RCR was detected within the transgenic sugarcane.
The observation that episomal vectors based on TYDV were functional within sugarcane host cells and BBTV-based vectors were not, was unexpected. It had been hypothesised that an episomal vector based on a monocot-infecting virus would replicate in an alternate monocot host, while an episomal vector based on a dicot infecting virus would not. Virus replication is thought to be host-specific however most host range studies have been conducted with full length infectious clones and not deconstructed virus-based episomes. The implication that viral Reps may be functional in plant cells of non-host species was then investigated. The ability for viral Reps to recognise their cognate IR and initiate RCR of virus-based episomes in different host cells was assessed through cross-replication experiments. Four ssDNA plant viruses; BBTV, TYDV, Chloris striate mosaic virus (CSMV) and Tomato leaf curl virus - Australia (ToLCV-Au) were assessed via Southern blot analysis for their ability to initiate both autonomous replication of infectious clones and episomal amplification within three different plant hosts; tobacco, sugarcane and banana.
Results from cross replication studies indicated a complex interaction between viral and host replication components. BBTV infectious clones and episomal vectors were restricted to replication within banana host cells providing a clear indication that episomal amplification vectors based on BBTV are restricted to Musa spp.
BBTV M-Rep was unable to recognise the viral regulatory DNA sequences of the other three ssDNA viruses. TYDV infectious clones and episomal vectors were capable of replicating within all three host cells tested, indicating that TYDV is capable of undergoing RCR within a broad range of plant hosts. TYDV Rep was also capable of recognising the viral regulatory DNA sequences of both CSMV and BBTV given favourable conditions within specific plant hosts. Replication of the CSMV infectious clone was not detected in any of the three host cells, although fidelity of this clone requires further confirmation. CSMV episomal vectors were functional within banana host cells only, indicating that although closely related to TYDV, episomal amplification vectors based on CSMV have a restricted host range.
CSMV Rep could not initiate RCR of episomal amplification vectors containing the viral regulatory DNA regions of the other three viruses in any of the plant host cells. ToLCV-Au infectious clones were capable of replicating within banana and tobacco host cells. Episomal amplification vectors based on ToLCV-Au extended the host range to sugarcane. ToLCV-Au Rep was unable to recognise the viral regulatory
DNA sequences of the other three viruses in any of the plant host cells. The ability for a viral Rep to recognise its own cognate regulatory DNA sequences within alternate plant host cells is variable. Episomal amplification vectors based on TYDV and ToLCV-Au appear to be the most suitable for the further development of a virusbased bioreactor system in sugarcane.
This study details the initial steps taken towards the development of a virus-based episomal amplification system in sugarcane. In doing so, fundamental knowledge into the mechanisms involved in Rep recognition of viral regulatory DNA sequences has been gathered. These research findings will provide a solid foundation for the further development of a sugarcane-based bioreactor.
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Pirlo, Steven Dominic. "Identification of viral-based replicating vectors suitable for the development of a sugarcane bioreactor." Queensland University of Technology, 2007. http://eprints.qut.edu.au/16548/.
Full textAbstract:
The circular, single-stranded (ss) DNA genomes of plant viruses in the families Geminiviridae and Nanoviridae are replicated within the nucleus of a host cell by a mechanism called rolling circle replication (RCR). Although this process relies almost exclusively on the replication machinery of the host cell, initiation occurs via the interaction of the viral replication initiation protein (Rep) with regulatory DNA sequences within the viral genome. The use of a virus-based episomal amplification technology as a plant bioreactor platform exploits the process of Rep-mediated RCR for the high-level amplification of virus-based episomes in plants and subsequent expression of heterologous proteins; such an approach offers advantages over existing gene expression technologies. This PhD thesis describes research towards the development of a virus-based episomal amplification system for use in sugarcane. Such a crop is ideally suited for a plant bioreactor system due to the efficient high-level production of plant biomass and the existence of established production, harvesting and processing infrastructure. In order to rapidly assess the potential of a virus-based episomal amplification system in sugarcane, a transient assay system was established. Sugarcane callus was identified as the most suitable cell preparation; providing rapid cell regeneration, uniform experimental samples and upon isolation, total DNA suitable for Southern analysis. This assay system once established, proved effective in rapidly identifying virus-based episomes capable of undergoing RCR within sugarcane host cells. This transient assay system was then used to test the functionality of a virus-based episomal amplification system based on the ssDNA virus, Banana bunchy top virus (BBTV) in sugarcane. BBTV-based episomal amplification vectors were constructed with a reporter gene expression cassette flanked by two copies of the BBTV regulatory DNA sequences. The episomal amplification vectors were bombarded into sugarcane and banana host cells in various combinations and evidence of RCR was assessed through Southern blot analysis. RCR products were identified in banana host cells bombarded with the BBTV-based episomal amplification vectors in combination with vectors encoding BBTV Master-Rep (M Rep). RCR products were not identified within sugarcane cells bombarded with the same construct combinations. Integrated InPAct (In Plant Activation) episomal vectors based on BBTV were then employed to confirm the transient results, in addition, the functionality of an InPAct vector based on an alternate virus, Tobacco yellow dwarf virus (TYDV) was also assessed. InPAct vectors based on BBTV were constructed with an untranslatable expression cassette for integration within the sugarcane genome. Transient experiments were performed to assess the ability of BBTV M-Rep and TYDV Rep to initiate RCR of their respective InPAct vectors. Visual observation of GFP expression indicated that BBTV M-Rep was capable of initiating RCR of the BBTVbased InPAct vectors within banana host cells but no evidence was observed in sugarcane host cells. TYDV Rep was capable of initiating RCR of the TYDV-based InPAct vector within sugarcane host cells with a 100-fold increase in the number of fluorescent foci compared to cells bombarded with the TYDV InPAct vector alone. The BBTV-based InPAct vector was stably integrated within the sugarcane genome and the ability for BBTV M-Rep to initiate episome formation and RCR was assessed by Southern blot analysis. Evidence of BBTV M-Rep mediated RCR was not detected within the transgenic sugarcane bombarded with BBTV M-Rep. Transgenic sugarcane containing the TYDV-based InPAct vectors was assessed for the ability to be activated by TYDV Rep and undergo RCR. Southern blot analysis demonstrated that TYDV Rep was capable of recognising the integrated TYDVbased InPAct vector and RCR was detected within the transgenic sugarcane. The observation that episomal vectors based on TYDV were functional within sugarcane host cells and BBTV-based vectors were not, was unexpected. It had been hypothesised that an episomal vector based on a monocot-infecting virus would replicate in an alternate monocot host, while an episomal vector based on a dicot infecting virus would not. Virus replication is thought to be host-specific however most host range studies have been conducted with full length infectious clones and not deconstructed virus-based episomes. The implication that viral Reps may be functional in plant cells of non-host species was then investigated. The ability for viral Reps to recognise their cognate IR and initiate RCR of virus-based episomes in different host cells was assessed through cross-replication experiments. Four ssDNA plant viruses; BBTV, TYDV, Chloris striate mosaic virus (CSMV) and Tomato leaf curl virus - Australia (ToLCV-Au) were assessed via Southern blot analysis for their ability to initiate both autonomous replication of infectious clones and episomal amplification within three different plant hosts; tobacco, sugarcane and banana. Results from cross replication studies indicated a complex interaction between viral and host replication components. BBTV infectious clones and episomal vectors were restricted to replication within banana host cells providing a clear indication that episomal amplification vectors based on BBTV are restricted to Musa spp. BBTV M-Rep was unable to recognise the viral regulatory DNA sequences of the other three ssDNA viruses. TYDV infectious clones and episomal vectors were capable of replicating within all three host cells tested, indicating that TYDV is capable of undergoing RCR within a broad range of plant hosts. TYDV Rep was also capable of recognising the viral regulatory DNA sequences of both CSMV and BBTV given favourable conditions within specific plant hosts. Replication of the CSMV infectious clone was not detected in any of the three host cells, although fidelity of this clone requires further confirmation. CSMV episomal vectors were functional within banana host cells only, indicating that although closely related to TYDV, episomal amplification vectors based on CSMV have a restricted host range. CSMV Rep could not initiate RCR of episomal amplification vectors containing the viral regulatory DNA regions of the other three viruses in any of the plant host cells. ToLCV-Au infectious clones were capable of replicating within banana and tobacco host cells. Episomal amplification vectors based on ToLCV-Au extended the host range to sugarcane. ToLCV-Au Rep was unable to recognise the viral regulatory DNA sequences of the other three viruses in any of the plant host cells. The ability for a viral Rep to recognise its own cognate regulatory DNA sequences within alternate plant host cells is variable. Episomal amplification vectors based on TYDV and ToLCV-Au appear to be the most suitable for the further development of a virusbased bioreactor system in sugarcane. This study details the initial steps taken towards the development of a virus-based episomal amplification system in sugarcane. In doing so, fundamental knowledge into the mechanisms involved in Rep recognition of viral regulatory DNA sequences has been gathered. These research findings will provide a solid foundation for the further development of a sugarcane-based bioreactor.
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ANGUS, STEVEN PATRICK. "THE MECHANISM OF RB-MEDIATED CELL CYCLE INHIBITION." University of Cincinnati / OhioLINK, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1061224578.
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Behjatnia, Seyyed Ali Akbar. "Characterisation of DNA replication of tomato leaf curl geminivirus /." Title page, contents and abstract only, 1997. http://web4.library.adelaide.edu.au/theses/09ACP/09acpb419.pdf.
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Lasecka, Lidia. "Studies on the replication of Nairobi sheep disease virus in cultured cells." Thesis, Royal Veterinary College (University of London), 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.618322.
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Hanson, Stephen F. "Inhibition of geminiviral DNA replication by trans-dominant rep gene mutants." 1997. http://catalog.hathitrust.org/api/volumes/oclc/37876375.html.
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