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Structure-Function Relationships in Microviridae External Scaffolding ProteinsUchiyama, Asako January 2007 (has links)
Microviruses (canonical members: øX174, G4, and alpha3) are T=1 icosahedral virions with a two scaffolding protein-mediated assembly pathway. The external scaffolding protein D mainly mediates the assembly of coat protein pentamers into procapsids. The results of previous genetic studies suggest that helix 1 of D protein may act as a substrate specificity domain, mediating the initial coat-scaffolding protein recognition in a species-specific manner. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were initially constructed in a plasmid, which over-expresses the protein, between the closely related phages G4 and øX174. The results of biochemical and genetic analyses identify coat-scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.However, protein concentrations higher than those found in typical infections could drive reactions that may not occur under physiological conditions. In order to elucidate a more detailed mechanistic model, the same chimeric external scaffolding gene was placed directly in the øX174 genome, and the chimeric virus was characterized. The results of the genetic and biochemical analyses indicate that helix 1 most likely mediates the nucleation reaction for the formation of the first assembly intermediate containing the external scaffolding protein. Mutants that can more efficiently use the chimeric scaffolding protein were isolated. These second-site mutations appear to act on a kinetic level, shortening the lag phase before virion production.Finally, by using improved protocols, two novel early assembly intermediates, the 9S* and 12S* particles, have been isolated and characterized. The 9S* particle consists of a coat protein pentamer associated with the internal scaffolding protein. The 12S* intermediate is a complex of a 9S* particle with the major spike protein, and the DNA pilot protein. The existence of internal scaffolding and DNA pilot proteins that were absent in previously characterized intermediates suggest that 9S* and 12S* particles are biologically active intermediates. Moreover, preliminary in vitro assembly experiments performed with the 12S* particle and exogenous external scaffolding protein yield empty capsids-like particle, strongly suggesting that these are the physiologically relevant intermediates.
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The DNA replication of bacteriophage [phi]X174 the nucleotide sequence and the chemical nature of the origin /Mansfeld, Alphonsus Dominicus Maria van, January 1979 (has links)
Thesis (doctoral)--Rijksuniversiteit te Utrecht, 1979. / English, summary in Dutch. Includes bibliographies.
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Applications of cryo-electron microscopy in the studies of virus and host interactionsYingyuan Sun (5930315) 17 January 2019 (has links)
<div>Viruses are a group of contagious microbes that have compact structures, containing a nucleic acid core and a protein shell. The replication of viruses requires assistance from hosts which can be almost any cellular organism. Viral infections are often associated with diseases and have been a major threat to the human race. To cope with viral diseases, we need to understand viruses, including their structures, life cycle, pathogenesis and interactions with their hosts. The first structure of a human virus was determined by the Rossmann lab in 1985 using X-ray crystallography.</div><div>Thanks to the recent advances in both hardware and software, cryo-electron microscopy (cryo-EM) has emerged as a powerful tool to study virus structures. Cryo-EM allows structural determination for a wide range of specimens to high resolution comparable to what can be achieved by X-ray crystallography. Currently two techniques of cryo-EM are commonly used in structural virology: single particles analysis (SPA) and electron tomography (ET). </div><div>Single particle analysis has been used to determine the structures of viruses complexed with host factors in three studies that are to be discussed with more details in chapters 2-4. </div><div>The structure of B19 parvovirus complexed with Fabs of a neutralizing human antibody was determined to 3.2 Å resolution. This structure showed that amino acids from three neighboring VP2 proteins form a quaternary structure epitope. In addition, the structure of human rhinovirus-C (RV-C) complexed with its cellular receptor, CDHR3, was determined to 3.9 Å resolution. Despite the low occupancy of the receptors, a “powerful” localized 3D classification procedure helped to select viral particles that had more bound receptors. Furthermore, structures were determined to 10 Å resolution of bacteriophage ΦX174 bound to lipopolysaccharide (LPS) bilayers, before and after genome ejection. These structures showed a series of conformational changes that occurred when a phage penetrated the bacterial membranes. These studies are good examples of applying cryo-EM to investigate virus-host interactions.</div><div>However, single particle analysis requires samples to be isolated, homogenous and monodispersed. On the contrary, tomography allows in situ studies and is applicable to samples with more flexibility and more heterogeneity. In the case of ΦX174, the structural changes that are involved in the assembly of the H-tube during infection remains a huge mystery. To provide an environment that is more similar to the surface of a bacterial cell, LPS-containing liposomes were mixed with ΦX174 viruses. It was then observed that the ΦX174 particles bound to these liposomes in a very compact manner which was impossible interpret with single particle analysis. Using cryo-ET, 3D volumes of liposome-ΦX174 complexes were reconstructed and structural details were visualized by sub-tomogram classification and averaging.</div><div>The emergence of cryo-EM has not only made high-resolution structural studies possible but also broadened the scope of samples with which virologists could work. Moreover, studies on flexible and heterogeneous complexes between viruses and host factors are now possible using either single particle analysis or electron tomography. These techniques will help us to understand virus-host relationships and finally, to develop effective anti-viral therapies.</div>
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La coévolution antagoniste bactérie - bactériophage : contraintes génétiques appliquées au modèle expérimental Escherichia coli - PhiX174Cavalié Michel, Alix 02 February 2010 (has links) (PDF)
Les bactéries et les bactériophages (phages), sont ubiquitaires. Parasites obligatoires des bactéries, les phages modifient fortement la diversité et la dynamique des populations bactériennes. Comprendre l'interaction bactérie-phage, pour prévoir et contrôler son évolution a de nombreux intérêts en écologie, industrie, et santé publique. La coexistence des bactéries et des phages repose en partie sur la coévolution antagoniste qui s'établit entre eux. Nous avons choisi d'étudier les contraintes génétiques limitant la coévolution entre Escherichia coli et PhiX174. En étudiant la niche écologique de PhiX174, nous avons établi que les souches sensibles à ce phage étaient rares et remarquables par leur diversité phylogénétique. De plus, alors que le lipopolysaccharide (LPS) est le récepteur reconnu par PhiX174, nous avons démontré que PhiX174 peut lyser des bactéries arborant des LPSs très variés, ce qui contraint la population bactérienne à résister autrement qu'en modifiant son récepteur. Puis, en modifiant le génotype de l'hôte bactérien, nous avons profondément altéré la pérennité de la coexistence bactérie-phage. Enfin, la mise en place d'un protocole de coévolution entre E. coli C et PhiX174 nous a permis de discuter de l'évolution de la pression de sélection induite par les phages sur la population bactérienne et de l'existence d'un compromis entre virulence et infectivité. Il semblerait, en définitive, que les fortes relations épistatiques entre les gènes participant à l'interaction bactérie-phage, sont autant de contraintes génétiques qui s'ajoutent aux contraintes écologiques déjà décrites et limitent la coévolution antagoniste qui s'établit entre E. coli et PhiX174.
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Virus removal in ceramic depth filters / Entfernung von Viren mittels keramischer Tiefenfilter : die elektrostatisch begünstigte AdsorptionMichen, Benjamin 05 April 2011 (has links) (PDF)
Diese Arbeit untersucht den Einsatz von keramischen Materialien in der Trinkwasseraufbereitung mittels Filtration und fokussiert dabei die Entfernung von Viren. Herkömmliche, auf Kieselgur basierende Tiefenfilter (Filterkerzen) mit Porengrößen im unteren Mikrometerbereich, werden hinsichtlich ihres Rückhaltevermögens gegenüber Kolloiden (Viren sowie Polystyrolpartikel) untersucht, um deren Einsatzfähigkeit in der Entfernung von Mikroorganismen im Allgemeinen abschätzen zu können. Ferner wird gezeigt, wie durch ein einfaches Verfahren solche Filter modifiziert werden können, um auch kleinste Viren mit ca. 30 nm Durchmessern aus dem Rohwasser zu entfernen. Die Zugabe von MgO während der Granulierungsstufe im Herstellungsprozess der Filterkerzen bewirkt eine erhebliche Verbesserung des Virenrückhalts bis zu über 99.9999%. Die experimentellen Ergebnisse wurden dabei mit theoretischen Modellen verglichen, um Aussagen über die Mechanismen der Virenentfernung treffen zu können.
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Virus removal in ceramic depth filters: the electrostatic enhanced adsorption approachMichen, Benjamin 18 March 2011 (has links)
Diese Arbeit untersucht den Einsatz von keramischen Materialien in der Trinkwasseraufbereitung mittels Filtration und fokussiert dabei die Entfernung von Viren. Herkömmliche, auf Kieselgur basierende Tiefenfilter (Filterkerzen) mit Porengrößen im unteren Mikrometerbereich, werden hinsichtlich ihres Rückhaltevermögens gegenüber Kolloiden (Viren sowie Polystyrolpartikel) untersucht, um deren Einsatzfähigkeit in der Entfernung von Mikroorganismen im Allgemeinen abschätzen zu können. Ferner wird gezeigt, wie durch ein einfaches Verfahren solche Filter modifiziert werden können, um auch kleinste Viren mit ca. 30 nm Durchmessern aus dem Rohwasser zu entfernen. Die Zugabe von MgO während der Granulierungsstufe im Herstellungsprozess der Filterkerzen bewirkt eine erhebliche Verbesserung des Virenrückhalts bis zu über 99.9999%. Die experimentellen Ergebnisse wurden dabei mit theoretischen Modellen verglichen, um Aussagen über die Mechanismen der Virenentfernung treffen zu können.:Contents
Chapter I Introduction 1
Chapter II Removal or inactivation of microorganisms, in particular viruses, for drinking water purposes with focus on small-scale, decentralised systems: A literature review 7
II. I Physical and chemical treatments 8
II. II Filtration processes 10
II. III Conclusions 15
Chapter III Mechanisms of adsorption in depth filtration 17
III.I Surface charge and the electrical double layer 18
III.II van der Waals interactions 22
III.III DLVO theory 23
III.IV Non-DLVO forces 25
III.V Extended DLVO Theory 27
Chapter IV Virus adsorption studies 29
IV.I A literature review 30
IV.I.I Virus concentration by adsorption-elution 33
IV.I.II Improved virus adsorption in filtration 35
IV.II The electrostatic enhanced adsorption approach 37
Chapter V Viruses 39
V.I Literature review 40
V.I.I Structure and morphology 40
V.I.II The viral life cycle 41
V.I.III Human pathogenic viruses in the aquatic environment 42
V.II Experimental 46
V.II.I The choice of viruses for adsorption studies 46
V.II.II Propagation and enumeration of the bacteriophages 48
V.II.III Characterisation of bacteriophages 51
V.III Results and discussion 54
V.III.I Production of high-titre and high-purity phage stocks 54
V.III.II Characteristics of bacteriophages 59
V.III.III Detection of a viral contaminant - the ‘Siphophage’ 64
Chapter VI The diatomaceous earth-based depth filter 69
VI.I Literature review 70
VI.I.I Diatomaceous earth 70
VI.I.II Retention of microorganisms in the DE-based depth filter 71
VI.II Experimental 73
VI.II.I Manufacturing the depth filter 73
VI.II.II Physical characterisation 74
VI.II.III Performing filter retention tests 75
VI.II.IV Latex retention test 76
VI.II.V Studying adsorption kinetics in a batch experiment 79
VI.II.VI Applying (X-)DLVO theory 80
VI.III Results and discussion 83
VI.III.I Characterisation of the depth filter 83
VI.III.II Latex removal in the depth filter 86
VI.III.III Filter performance on virus removal 94
VI.III.IV Batch-sorption experiments 99
VI.IV Summary and conclusions 102
Chapter VII The magnesium oxide modified depth filter 103
VII.I Experimental 104
VII.I.I Choice of the adsorbent material 104
VII.I.II Manufacturing the MgO-modified filter and characterisation methods 105
VII.II Results and discussion 106
VII.II.I The adsorbent: Magnesium oxide powder 106
VII.II.II Physical characterisation of modified depth filters 108
VII.II.III Virus removal in depth filters containing MgO 113
VII.II.IV Ageing behaviour of MgO modified filters 118
VII.II.V Discussion on the removal mechanisms 130
VII.III Summary and conclusions 134
Chapter VIII Summary, conclusions and outlook 135
VIII.I Summary and conclusions 136
VIII.II Outlook 137
Abbreviations, symbols and physical constants 139
Reference list 142
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