Dissertations / Theses on the topic 'Subviral Particles'

Die Spumaretrovirinae, mit ihrer einzigen Gattung der Foamyviren (FV), nehmen aufgrund einer recht ungewöhnlichen Replikationsstrategie und Ähnlichkeiten mit den Hepadnaviren eine Sonderstellung innerhalb der Familie der Retroviren ein. Eine Besonderheit der FV ist, daß sie für die Partikelfreisetzung, im Gegensatz zu den Orthoretroviren, die beiden strukturellen Proteine Gag und Env benötigen. Das Gag- Protein trägt alle für den Kapsidzusammenbau nötigen strukturellen Komponenten, kann jedoch durch eine fehlende Membranbindungsdomäne nicht mit Zellmembranen assoziieren. Der Membrantransport der bereits im Zytoplasma zusammen gebauten FV Kapside wird vermutlich durch das FV Env-Protein vermittelt. Das FV Hüllprotein ist jedoch auch alleine zur Freisetzung von Kapsidlosen, Hüllprotein-haltigen subviralen Partikeln (SVP) fähig. Da eine Envunabhängige Freisetzung virus-ähnlicher Partikel durch ein FV Gag-Protein mit künstlichem Membrananker möglich ist, scheint das FV Gag-Protein auch essentielle strukturelle Elemente für die Partikelfreisetzung zu enthalten. In den letzten Jahren wurden große Fortschritte in der Erforschung der Freisetzung von membranumhüllten Viren und den daran beteiligten viralen Determinanten und zellulären Mechanismen gemacht. Wobei den meist in den viralen Kapsidproteinen vorkommenden Late (L)-Domänen und deren Interaktion mit dem zellulären Proteinsortierungsweg in Multivesikuläre Körperchen (MVB) eine besondere Bedeutung zu kommt. Über die FV virale und subvirale Partikelfreisetzung und die dabei involvierten strukturellen viralen Domänen und zellulären Proteinen war jedoch bisher wenig bekannt. Im Rahmen dieser Arbeit konnte durch Mutationsanalysen von drei potentiellen L-Domän Sequenzmotiven im Prototyp FV (PFV) Gag-Protein ein, innerhalb der Primaten FV konserviertes, PSAP Konsensusmotiv als funktionelle L-Domäne charakterisiert werden. Dessen Mutation führte zu klassischen L-Domän Defekten mit verringerter Partikelfreisetzung, sowie einer elektronenmikroskopisch sichtbaren Arretierung der Virusknospung und seine Funktion war durch homo- und heterologe L-Domän Motive anderer Retroviren teilweise oder vollständig ersetzbar. Ein PPPI Motiv in PFV Gag, mit Ähnlichkeit zur L-Domän PPXY Konsensussequenz, schien jedoch keinen Einfluß auf die FV Freisetzung zu besitzen. Die Charakterisierung eines in allen FV Gag-Proteinen konservierten YXXL Motivs ließ eher auf eine wichtige Rolle beim korrekten Kapsidzusammenbau, als auf eine klassische LDomän Funktion schließen. Eine korrekte Kapsidmorphogenese schien entscheidend für die reverse Transkription des Virusgenoms zu sein. Durch Koexpression verschiedener dominant-negativer Mutanten des zellulären ESCRT-Proteinssortierungsweges konnte gezeigt werden, daß die virale Partikelfreisetzung von PFV augenscheinlich dem generellen Model der Freisetzung vieler membranumhüllter Viren über das VPS-System folgt. Eine spezifische Interaktion des PFV Gag PSAP L-Domän Motivs mit TSG101, einer frühen Komponente der ESCRT-Komplexe, verbindet PFV mit dem VPS-Sortierungsweg der Zelle. Die besondere Fähigkeit des FV Env-Proteins zur Freisetzung von SVPs wurde bereits vor einiger Zeit entdeckt, dennoch war bisher nichts über die viralen und zellulären Determinanten bekannt, die zu einer Knospung des Env-Proteins in Vesikel führten. Durch eine Reihe von Deletions- und Mutationsanalysen des PFV Env-Proteins konnten in dieser Arbeit zwei für die SVP-Freisetzung inhibitorische Abschnitte am N- und C-Terminus der zytoplasmatischen Domänen des Env- Proteins ermittelt werden. Weiterhin wurden essentielle Sequenzen im Leaderpeptid, sowie die Notwendigkeit der Membranspannenden Domäne der Transmembran- Untereinheit für die SVP-Freisetzung festgestellt. Obwohl das PFV Env-Protein kein bekanntes L-Domän Sequenzmotiv enthält, konnte ein Einfluß später Komponenten der ESCRT-Maschinerie auf die SVP-Bildung beobachtet werden. Wobei die genaue Eintrittsstelle in den VPS-Weg im Rahmen dieser Arbeit nicht definiert werden konnte. Die vorgenommen Analysen lassen vermuten, daß die Bildung von SVPs durch die Konzentration der Env-Proteine in der Zellmembranen reguliert wird. Welche genauen Mechanismen dabei zu Grunde liegen und wieweit die zelluläre Ubiquitinylierungsmaschinerie involviert ist, bedarf jedoch weiterer Erforschung. Die Ergebnisse dieser Arbeit verdeutlichen erneut die Sonderstellung der FV innerhalb der Familie der Retroviren. Auf der einen Seite folgt die foamyvirale Viruspartikelfreisetzung den typischen Mechanismen der retroviralen Virusknospung. Andererseits zeigt die Freisetzung von subviralen Partikeln, die bei keinem anderen Retrovirus bisher beobachtet wurde, eine weitere Parallele zur Replikationsstrategie der Hepadnaviren auf.

Les études structurales des protéines membranaires eucaryotes sont importantes mais particulièrement difficiles à effectuer car elles nécessitent non seulement un système efficace et pratique pour produire la protéine dans une conformation native, d’une technique d’étude structurale compatible avec ce dernier. Du fait de leur modularité, les systèmes de production acellulaires in vitro sont adaptés à la production de protéines membranaires. De plus, l’amélioration de leur robustesse et de leur efficacité les rendent maintenant comme une alternative viable à l’expression cellulaire. Parmi ceux-ci, le Système d’Expression Acellulaire à partir de Germes de Blé (SEA-GB) est le plus efficace pour produire des protéines membranaires eucaryotes, et permet de plus un marquage isotopique efficace et spécifique. Ce dernier point est très utile pour la Résonance Magnétique Nucléaire (RMN), et plus spécifiquement la RMN du solide qui permet l’étude structure de protéines membranaires et d’assemblages macromoléculaires. Depuis récemment, la RMN du solide est compatible avec le SEA-GB, formant un outil puissant pour l’étude structurale de protéines membranaires et assemblages macromoléculaires. Dans ces travaux, les deux techniques ont été combinées pour la production et l’étude des protéines d’enveloppe du virus de l’Hépatite B du canard (VHBC), appartenant à la famille des Hepadnaviridae. Ces virus sont capables de sécréter des virions actifs, mais aussi des particules composées uniquement de protéines d’enveloppe, appelées particules sous-virales (PSV). Dans un premier temps, nous montrons que plusieurs milligrammes de petite protéine d’enveloppe (DHBs S) du VHBC sont produits sous forme soluble avec le SEA-GB. DHBs S forme des PSV durant la traduction, ce qui confirme la conformation native de la protéine. Après désassemblage des PSV, la protéine est majoritairement en hélice , synonyme d’un bon repliement. Après isolation par ultracentrifugation sur gradient de sucrose, les PSV ont été sédimentées dans un rotor de 0.7 mm et étudiées par RMN du solide. Des spectres 2D hNH très prometteurs ont été obtenus, avec un bon signal, des pics isolés et une résolution similaire à celle d’autres protéines membranaires sédimentées et étudiées par RMN du solide. De plus, la superposition du spectre de DHBs S avec des spectres simulés de protéines modèles possédant des structures secondaires caractéristiques confirme que DHBs S est principalement en hélice dans le contexte des PSV. Le signal doit cependant être amélioré pour pouvoir réaliser les expériences nécessaires à des études structurales approfondies, c’est pourquoi des tests d’optimisation de la production ont été effectués. D’une part, l’amélioration du rendement de production, via l’utilisation d’un extrait de germes de blé commercial, et de la stabilisation des PSV, par incubation avec du KSCN, ont été testés. D’autre part, différentes méthodes de purification ont été examinées: précipitation à l’ammonium sulfate ou au PEG6000, incubation à haute température, élimination de contaminants via une unité d’ultrafiltration, purification d’affinité ou d’exclusion stérique ainsi qu’un test de désassemblage des particules, suivie d’une purification puis de la reconstitution des PSVs en présence de lipides. Enfin, un marquage isotopique spécifique de certains acides aminés a été évalué. Dans la seconde partie, nous avons étendu les possibilités du SEA-GB via l’expression de la grande protéine d’enveloppe (DHBs L) du VHBC. In vivo, la protéine est phosphorylée spécifiquement et subit aussi une traduction alternative ; nous avons montré que c’était aussi le cas dans le SEA-GB. Nous avons aussi testé la coexpression de DHBs S, DHBs L ainsi que de la capside de DHBV pour inclure DHBs L dans les PSV, voire même reconstituer des virions entiers, ce qui augmenterait les possibilités du système. Enfin, nous avons aussi détaillé certains paramètres critiques pour la formation des PSV dans le système
Structural studies of eukaryotic membrane proteins are of prime importance but notoriously difficult as they not only necessitate an efficient and practical overexpression system that allows for membrane protein expression in a biologically relevant folding, but also a structural technique that you can easily combine with the chosen protein production system. In vitro cell-free systems, due to their modulable nature, are particularly suited for membrane protein expression. Furthermore, they now established themselves as a viable alternative to conventional cell-based expression, notably because of considerable advances in robustness and efficiency. Amongst them, the wheat germ cell-free production system (WG-CFPS) proved to be the most efficient for production of eukaryotic membrane proteins, and allows for efficient and specific isotope labeling. This makes it particularly convenient for Nuclear Magnetic Resonance (NMR), and more specifically solid-state NMR which is particularly appropriate for membrane protein studies and macromolecular assemblies. Thanks to very recent advances that lead to a drastic reduction of the quantity of protein needed, solid-state NMR is now compatible with WG-CFPS, creating a powerful tool for structural studies of macromolecular assemblies and membrane proteins. In this work, these two techniques are combined for the production and study of the envelope proteins from the duck Hepatitis B Virus (DHBV), that belongs to the Hepadnaviridae family. These viruses are able to secrete active virions, but also particles composed only of envelope proteins, which are called subviral particles (SVPs). In the first part, we show here that the DHBV small envelope protein (DHBs S) is produced as soluble in mg amounts using WG-CFPS. Even more, the protein forms SVPs upon translation, and is thus expressed in a biologically relevant form. After SVPs disassembly, the protein displays a mostly -helical folding, which is characteristic of a well-folded protein, and also very similar to the secondary structure of an assembly-incompetent mutant. After further isolation by ultracentrifugation on a sucrose gradient, the SVPs were sedimented in a 0.7 mm rotor and observed by solid-state NMR. Very promising hNH 2D spectra, with a good signal, were obtained. They display numerous isolated peaks and a resolution alike to other sedimented membrane proteins observed by solid-state NMR. Moreover, superimposition of the DHBs S spectrum with simulated spectra from proteins with extreme secondary structure content confirms that the protein is mostly -helical in the context of the SVPs. Nonetheless, the signal still needs to be improved in order to perform the experiments necessary for in-depth structural analysis. To that end, sample optimization assays were conducted. On the one hand, protein yield improvement, by the use of a commercial wheat germ extract, and SVPs stabilization, by incubation with KSCN, were tried. On the other hand, different methods for SVPs purification were tested, including PEG6000 or ammonium sulfate precipitation, incubation at high temperature, contaminant removal with an ultrafiltration device, affinity or size-exclusion purification as well as tests of particles disassembly, purification followed by SVPs reconstitution in lipids. Finally, amino-acid specific isotopic labeling of DHBs S was evaluated. In the second part, we could show extended possibilities of WG-CFPS through expression of DHBV large envelope protein (DHBs L). In vivo, the protein undergo specific phosphorylation as well as alternative translation, and we could show that it is also the case upon wheat germ cell-free expression. We also tested coexpression of DHBs S, DHBs L and of the DHBV capsid in order to assess the possibility of DHBs L inclusion in SVPs, or even complete virion reconstitution, which could even augment WG-CFPS possibilities. Ultimately, we also detail some critical parameters for SVPs formation in the WG-CFPS

Die Spumaretrovirinae, mit ihrer einzigen Gattung der Foamyviren (FV), nehmen aufgrund einer recht ungewöhnlichen Replikationsstrategie und Ähnlichkeiten mit den Hepadnaviren eine Sonderstellung innerhalb der Familie der Retroviren ein. Eine Besonderheit der FV ist, daß sie für die Partikelfreisetzung, im Gegensatz zu den Orthoretroviren, die beiden strukturellen Proteine Gag und Env benötigen. Das Gag- Protein trägt alle für den Kapsidzusammenbau nötigen strukturellen Komponenten, kann jedoch durch eine fehlende Membranbindungsdomäne nicht mit Zellmembranen assoziieren. Der Membrantransport der bereits im Zytoplasma zusammen gebauten FV Kapside wird vermutlich durch das FV Env-Protein vermittelt. Das FV Hüllprotein ist jedoch auch alleine zur Freisetzung von Kapsidlosen, Hüllprotein-haltigen subviralen Partikeln (SVP) fähig. Da eine Envunabhängige Freisetzung virus-ähnlicher Partikel durch ein FV Gag-Protein mit künstlichem Membrananker möglich ist, scheint das FV Gag-Protein auch essentielle strukturelle Elemente für die Partikelfreisetzung zu enthalten. In den letzten Jahren wurden große Fortschritte in der Erforschung der Freisetzung von membranumhüllten Viren und den daran beteiligten viralen Determinanten und zellulären Mechanismen gemacht. Wobei den meist in den viralen Kapsidproteinen vorkommenden Late (L)-Domänen und deren Interaktion mit dem zellulären Proteinsortierungsweg in Multivesikuläre Körperchen (MVB) eine besondere Bedeutung zu kommt. Über die FV virale und subvirale Partikelfreisetzung und die dabei involvierten strukturellen viralen Domänen und zellulären Proteinen war jedoch bisher wenig bekannt. Im Rahmen dieser Arbeit konnte durch Mutationsanalysen von drei potentiellen L-Domän Sequenzmotiven im Prototyp FV (PFV) Gag-Protein ein, innerhalb der Primaten FV konserviertes, PSAP Konsensusmotiv als funktionelle L-Domäne charakterisiert werden. Dessen Mutation führte zu klassischen L-Domän Defekten mit verringerter Partikelfreisetzung, sowie einer elektronenmikroskopisch sichtbaren Arretierung der Virusknospung und seine Funktion war durch homo- und heterologe L-Domän Motive anderer Retroviren teilweise oder vollständig ersetzbar. Ein PPPI Motiv in PFV Gag, mit Ähnlichkeit zur L-Domän PPXY Konsensussequenz, schien jedoch keinen Einfluß auf die FV Freisetzung zu besitzen. Die Charakterisierung eines in allen FV Gag-Proteinen konservierten YXXL Motivs ließ eher auf eine wichtige Rolle beim korrekten Kapsidzusammenbau, als auf eine klassische LDomän Funktion schließen. Eine korrekte Kapsidmorphogenese schien entscheidend für die reverse Transkription des Virusgenoms zu sein. Durch Koexpression verschiedener dominant-negativer Mutanten des zellulären ESCRT-Proteinssortierungsweges konnte gezeigt werden, daß die virale Partikelfreisetzung von PFV augenscheinlich dem generellen Model der Freisetzung vieler membranumhüllter Viren über das VPS-System folgt. Eine spezifische Interaktion des PFV Gag PSAP L-Domän Motivs mit TSG101, einer frühen Komponente der ESCRT-Komplexe, verbindet PFV mit dem VPS-Sortierungsweg der Zelle. Die besondere Fähigkeit des FV Env-Proteins zur Freisetzung von SVPs wurde bereits vor einiger Zeit entdeckt, dennoch war bisher nichts über die viralen und zellulären Determinanten bekannt, die zu einer Knospung des Env-Proteins in Vesikel führten. Durch eine Reihe von Deletions- und Mutationsanalysen des PFV Env-Proteins konnten in dieser Arbeit zwei für die SVP-Freisetzung inhibitorische Abschnitte am N- und C-Terminus der zytoplasmatischen Domänen des Env- Proteins ermittelt werden. Weiterhin wurden essentielle Sequenzen im Leaderpeptid, sowie die Notwendigkeit der Membranspannenden Domäne der Transmembran- Untereinheit für die SVP-Freisetzung festgestellt. Obwohl das PFV Env-Protein kein bekanntes L-Domän Sequenzmotiv enthält, konnte ein Einfluß später Komponenten der ESCRT-Maschinerie auf die SVP-Bildung beobachtet werden. Wobei die genaue Eintrittsstelle in den VPS-Weg im Rahmen dieser Arbeit nicht definiert werden konnte. Die vorgenommen Analysen lassen vermuten, daß die Bildung von SVPs durch die Konzentration der Env-Proteine in der Zellmembranen reguliert wird. Welche genauen Mechanismen dabei zu Grunde liegen und wieweit die zelluläre Ubiquitinylierungsmaschinerie involviert ist, bedarf jedoch weiterer Erforschung. Die Ergebnisse dieser Arbeit verdeutlichen erneut die Sonderstellung der FV innerhalb der Familie der Retroviren. Auf der einen Seite folgt die foamyvirale Viruspartikelfreisetzung den typischen Mechanismen der retroviralen Virusknospung. Andererseits zeigt die Freisetzung von subviralen Partikeln, die bei keinem anderen Retrovirus bisher beobachtet wurde, eine weitere Parallele zur Replikationsstrategie der Hepadnaviren auf.

Die Familie der Retrovirinae wird in zwei Unterfamilien untergliedert, die Orthoretrovirinae und die Spumaretrovirinae. Foamyviren stellen aufgrund einiger besonderer Eigenschaften die einzigen Vertreter dieser Unterfamilie, die sie als Bindeglied zwischen den Retroviren und den Hepadnaviren erscheinen lassen. So erfolgt beispielsweise die reverse Transkription des viralen Genoms nicht erst nach Eintritt in die Zielzelle, sondern, anders als bei Orthoretroviren, bereits in der Produzentenzelle noch während oder kurz nach der Morphogenese. Diese Eigenschaft teilen Foamyviren mit den Hepadnaviren ebenso wie die obligate Koexpression der Kapsidproteine mit den viralen Hüllproteinen für die Freisetzung von Viruspartikeln. Im Gegensatz zu Orthoretroviren sind Foamyviren folglich nicht in der Lage virusähnliche Partikel (VLP) zu sekretieren und die spezifische Funktion des PFV Env Proteins kann nicht durch heterologe Hüllproteine übernommen werden. Die Synthese des PFV Env Vorläuferproteins erfolgt am rER, wobei es eine Typ III Membrantopologie erhält, mit sowohl dem N- als auch dem C-Terminus im Zytoplasma. Während des Transports des Proteins zum Ort der Partikelknospung, wird es posttranslational im Golgi-Apparat, oder dem trans-Golgi Netzwerk, durch Furin oder eine Furin-ähnliche Protease in drei partikelassoziierte Untereinheiten prozessiert. Eine Partikelassoziation retroviraler Signalpeptide ist bislang nur für Foamyviren nachgewiesen worden, genauso wie eine essentielle Rolle dieses Proteins bei der Interaktion zwischen dem

Like all other viruses, a successful egress of functional particles from infected cells is a prerequisite for foamy virus (FV) spread within the host. The budding process of FVs involves steps, which are shared by other retroviruses, such as interaction of the capsid protein with components of cellular vacuolar protein sorting (Vps) machinery via late domains identified in some FV capsid proteins. Additionally, there are features of the FV budding strategy quite unique to the spumaretroviruses. This includes secretion of non-infectious subviral particles and a strict dependence on capsid-glycoprotein interaction for release of infectious virions from the cells. Virus-like particle release is not possible since FV capsid proteins lack a membrane-targeting signal. It is noteworthy that in experimental systems, the important capsid-glycoprotein interaction could be bypassed by fusing heterologous membrane-targeting signals to the capsid protein, thus enabling glycoprotein-independent egress. Aside from that, other systems have been developed to enable envelopment of FV capsids by heterologous Env proteins. In this review article, we will summarize the current knowledge on FV budding, the viral components and their domains involved as well as alternative and artificial ways to promote budding of FV particle structures, a feature important for alteration of target tissue tropism of FV-based gene transfer systems.

Die Familie der Retrovirinae wird in zwei Unterfamilien untergliedert, die Orthoretrovirinae und die Spumaretrovirinae. Foamyviren stellen aufgrund einiger besonderer Eigenschaften die einzigen Vertreter dieser Unterfamilie, die sie als Bindeglied zwischen den Retroviren und den Hepadnaviren erscheinen lassen. So erfolgt beispielsweise die reverse Transkription des viralen Genoms nicht erst nach Eintritt in die Zielzelle, sondern, anders als bei Orthoretroviren, bereits in der Produzentenzelle noch während oder kurz nach der Morphogenese. Diese Eigenschaft teilen Foamyviren mit den Hepadnaviren ebenso wie die obligate Koexpression der Kapsidproteine mit den viralen Hüllproteinen für die Freisetzung von Viruspartikeln. Im Gegensatz zu Orthoretroviren sind Foamyviren folglich nicht in der Lage virusähnliche Partikel (VLP) zu sekretieren und die spezifische Funktion des PFV Env Proteins kann nicht durch heterologe Hüllproteine übernommen werden. Die Synthese des PFV Env Vorläuferproteins erfolgt am rER, wobei es eine Typ III Membrantopologie erhält, mit sowohl dem N- als auch dem C-Terminus im Zytoplasma. Während des Transports des Proteins zum Ort der Partikelknospung, wird es posttranslational im Golgi-Apparat, oder dem trans-Golgi Netzwerk, durch Furin oder eine Furin-ähnliche Protease in drei partikelassoziierte Untereinheiten prozessiert. Eine Partikelassoziation retroviraler Signalpeptide ist bislang nur für Foamyviren nachgewiesen worden, genauso wie eine essentielle Rolle dieses Proteins bei der Interaktion zwischen dem

A major obstacle to the development of dengue virus (DENV) vaccines has been the need to immunise concurrently against each of the four DENV serotypes in order to avoid sensitising recipients to developing severe DENV infections. A problem already encountered with live attenuated tetravalent DENV vaccines has been the difficulty in eliciting adequate immune responses against all four DENV serotypes in human hosts. This could have been due to variations in the antigenicity and/or the replication rates of the four DENV serotypes. Non-replicating DNA vaccines avoid the issue of different replication rates. Currently, only DENV-1 and DENV-2 DNA vaccines have been evaluated. In this study, a number of DNA vaccines for each of the four DENV serotypes were developed and their immunogenicity was evaluated in outbred mice. These vaccines included DNA vaccines encoding the DENV prM-E protein genes derived from the four DENV serotypes (pVAX-DEN1, -DEN2, -DEN3 and -DEN4), and DNA vaccines encoding DENV prM and hybrid-E protein genes derived from multiple DENV serotypes. The hybrid-E protein genes were constructed by substituting either domains I and II, domain III, and/or the stem-anchor region from the E protein of one DENV serotype with the corresponding region from another DENV serotype. A number of superior DNA vaccines against each of the four DENV serotypes were identified based on their ability to elicit high titres (≥40, FFURNT50) of neutralising antibodies against the corresponding DENV in mice. The superior DNA vaccines against DENV-1 were pVAX-DEN1, pVAX-C2M2E211, pVAX-C2M2E122 and pVAX-C2M1E122. The superior DNA vaccine against DENV-2 was pVAX-C2M1E122 and the superior DNA vaccines against DENV-3 were pVAX-DEN3 and pVAX-C2M3E344. The superior DNA vaccines against DENV-4 were pVAX-C2M3E344, pVAX-C2M4E434 and pVAX-C2M4E433. Each of these DNA vaccines could provide effective protection against infection by the corresponding DENV serotypes. This is the first study to describe the development of DNA vaccines against DENV-3 and DENV-4. However, mice immunised with a tetravalent DENV DNA vaccine, composed of a DNA vaccine encoding the prM-E protein genes from each of the four DENV serotypes (pVAX-DEN1-4), elicited high titres of neutralising antibodies against DENV-1 and DENV-3 only. Nevertheless, the results from this study suggested that a tetravalent DENV DNA vaccine, composed of pVAX-DEN1, pVAX-C2M1E122, pVAX-DEN3 and pVAX-C2M4E434, may provide effective concurrent protection against infection by each of the four DENV serotypes. In addition, mice immunised with pVAX-C2M1E122, which encoded a hybrid-E protein gene derived from DENV-1 and DENV-2, elicited high titres of anti-DENV-1 and anti-DENV-2 neutralising antibodies, and mice immunised with pVAX-C2M3E344, which encoded a hybrid-E protein gene derived from DENV-3 and DENV-4, elicited high titres of anti-DENV-3 and anti-DENV-4 neutralising antibodies. This result suggested that the co-immunisation of these two hybrid-E DNA vaccines also may provide effective concurrent protection against infection by each of the four DENV serotypes. Extracellular E proteins, believed to be in the form of recombinant subviral particles (RSPs), were recovered from the tissue culture supernatant of all DNA vaccine-transfected mammalian cells by ultracentrifugation, except for cells transfected with the pVAX-C2M2E122 hybrid-E DNA vaccine. Western blotting with the monoclonal antibody 4G2 (flavivirus cross-reactive) demonstrated that the extracellular E proteins expressed by the DNA vaccines were synthesized and cleaved in a manner similar to that of native DENV E proteins. In addition, mammalian cells transfected with pVAX-DEN1, pVAX-DEN2 or pVAX-DEN3 secreted higher amounts of extracellular E proteins than cells transfected with pVAX-DEN4. The amount of extracellular E protein secreted by pVAX-DEN4-transfected cells increased when the c-region of the prM/E signal peptidase cleavage site was made more polar. In contrast, decreasing the polarity of the c-region of the C/prM signal peptidase cleavage site of pVAX-DEN4 resulted in no detectable extracellular E proteins from pVAX-DEN4-transfected cells. This result suggested that the amount of extracellular E proteins secreted by cells transfected with DNA expressing the DENV prM-E protein genes may be dependent of the efficiency of C/prM and prM/E protein cleavages by host-derived signal peptidases. Mice immunised with the mutated pVAX-DEN4, which was capable of expressing large amounts of extracellular E proteins in vitro, produced significantly higher concentrations of Th1-type anti-DENV-4 antibodies than mice immunised with the unmodified pVAX-DEN4, but failed to produce detectable levels of anti-DENV-4 neutralising antibodies. In contrast, increasing the ratio of CpG-S to CpG-N motifs in the pVAX-DEN2 DNA vaccine by incorporating either an additional CpG-S motif, or an antibiotic resistance gene with a high ratio of CpG-S to CpG-N motifs, resulted in a significant increase in both the concentration of Th1-type anti-DENV-2 antibodies and the titres of anti-DENV-2 neutralising antibodies in immunised mice. This result suggested that increasing the amount of CpG-S motifs in DENV DNA vaccines may present an simple and effective approach to increasing the immunogenicity of the DENV DNA vaccines.

Dengue is the most common mosquito-borne human disease, leading to 2.5 billion people at risk, 50-100 millions infections each year worldwide and among them, 500 000 severe dengue cases (dengue hemorrhagic fever, DHF/ dengue shock syndrome, DSS) plus more than 20 000 deaths. It can be caused by any of four dengue virus serotypes, which are antigenicly distinct and belong to the Flaviviridae family, genus Flavivirus. However, up till now there is no specific drug and vaccine against dengue. Understanding mechanisms developed by dengue virus to exploit host cells during all stages of the replication cycle is a first step towards the rationale design of anti-viral strategies. Very little is known about the late stages, which consist of assembly, budding and secretion of the virus. It is therefore very important to develop tools in order to study the egress of the virus. In this study, I investigated a stable cell line named Hela-prME that expresses serotype 1 dengue virus (DENV-1) prM and E native structural envelope proteins and constitutively produces dengue recombinant subviral particles (RSPs). Biochemical characterization of DENV-1 RSPs has validated that this cell line is a potential tool to study the dengue viral late-stage. Indeed, the maturation process observed with RSPs is similar to the pathway described for real virus (cleavage of prM fragment, homodimerization of E, acquisition of complex sugars). To better understand and depict the dengue virus late-stage secretion, I combined various electron microscopy (EM) techniques e.g. classical transmission electron microscopy (TEM), negative staining, immunogold labeling on cryo-ultrathin sections (Tokuyashu method) and tomography (ET) with such RSPs tool. The EM results obtained illustrate that electron dense particles and tubules labeled by antibodies directed against E and prM proteins were abundantly found in the lumen of endoplasmic reticulum (ER)-related cisternae of HeLa prME cells. Epositive particles were also found in other structures such as Golgi stacks and vesicles nearby as well as in aggregates with electron dense materials inside and surrounded by membrane. These particles are most likely corresponding to DENV-1 RSPs whereas the tubules may be other structures induced by assembly of prM and E proteins. This study has clearly shown that DENV-1 RSPs assemble in the ER and transport through the secretory pathway before being released. This work further validates the use of dengue RSPs and RSPs-producing cells as a model to study viral egress.
published_or_final_version
Pathology
Master
Master of Philosophy

博士
國立中央大學
化學工程與材料工程研究所
95
The protein VP2, matured from the polyprotein, which was encoded by the genome of infectious bursal disease virus (IBDV), is the primary host-protective immunogen of IBDV. According to VP2-452H subviral particle (SVP) analysis, the determination crystal structure and enzyme-linked immunosorbent assay (ELSIA), showed thar His-tag was not exposed on the surface of VP2-452H SVPs. Thus illustrate that the His-tag apparently did not attribute to the effective purification of this protein by IMAC. An affinity must have existed between the protein VP2 and the immobilized metal ions to achieve SVP binding with Ni-NTA resin. Accordingly, the IBDV generated from DF-1 cell culture and non-tagged VP2-441 SVP generated from a baculovirus-insect cell expression system were purified by IMAC. The purification of IBDV viron through IMAC obtained a 60.5% recovery, and the IMAC-purified IBDV has a similar morphology to the wild-type IBDV with a diameter of 65 nm through electron microscope observation. For SVP formed by VP2-441 purified by IMAC a recovery 92% and a purity of also 92% of mature VP2 were obtained. SVP formed by VP2-441 exhibited a diameter approximately 25 nm. These results obtained from the above experiments can demonstrate 1) the protein VP2 does have interaction with immobilized nickel ion; and 2) the protein VP2 can assist both IBDV viron and SVPs to have the affinity with Ni-NTA resin. The recombinant protein VP2-441, i.e., a structural protein VP2 of infectious bursal disease (IBD) virus, can self-assemble into T=1 subviral particles (SVPs) in baculovirus expression system. These SVPs are not to have multiple his-tags on the surface which result in an efficient purification by immobilized metal-ion affinity chromatography (IMAC). This study aimed at getting more insight into the interaction between VP2-441 SVPs and immobilized metal (Ni2+) ions at molecular level. First of all, large quantity of highly purified VP2-441 SVPs obtained by a one-step purification process allowed the performance of equilibrium adsorption measurements and subsequent determinations of binding constants by fitting two isotherm models, i.e., Temkin and Langmuir-Freundlich. Two general conclusion are obtained, first, the maximum bound VP2-441 SVPs per volume resin is limited because the pore size of IMA gel (ca. 24 nm in diameter) is similar to that of SVPs (20 – 25 nm) and the diffusion of the latter into the pores is hindered. The other is that SVPs have an extremely high affinity to the immobilized Ni+2 ions because the dissociate constants obtained from different models are in the scale of 10-9 M, which suggested the interaction mimicking that between an antigen and its antibody. The high binding strength is derived from a multiple-site binding between VP2-441 SVPs and Ni2+ ions as demonstrated by a concave-up Scatchard plot. Finally, we found that the adsorption of SVPs can be well described by Temkin model. A detail understanding of SVP-immobilized metal ion interactions can provide useful strategies for conducting preparative-scale separations of SVPs or even a real virus using IMAC.

abstract: Vaccines against the arthropod-borne dengue virus (DENV) are still commercially nonexistent. A subunit immunization strategy may be of value, especially if a safe viral vector acts as a biologically active adjuvant. The DENV envelope protein (E), the main target for neutralizing immune responses, has three conformational domains. The immunoglobulin-like and independently folding domain III (DIII) contains epitopes that elicit highly specific neutralizing antibodies. The hepatitis B small surface antigen (HBsAg, S) was used as a scaffold to display DENV 2 DIII on a virus-like particle (VLP). A measles virus (MV) was engineered to vector HBsAg and the hybrid glycoprotein DIII-HBsAg in two different loci (DIII-S). Despite the relatively deleterious effect on replication caused by the insertion of two transcription cassettes, the recombinant virus MVvac2(DIII-S,S)P induced the secretion of DIII-S hybrid VLP with a similar sucrose density as HBsAg particles (1.10-1.12g/ml) and peaked at 48 h post-infection producing 1.3x106 TCID50/ml infectious MV units in vitro. A second recombinant virus, MVvac2(DIII-S)N, was engineered to vector only the hybrid DIII-S. However, it did not induce the secretion of hybrid HBsAg particles in the supernatant of infected cells. The immunogenicity of the recombinant viruses was tested in a MV-susceptible small animal model, the experimental group which received two 105 TCID50 I.P. doses of MVvac2(DIII-S,S)P in a 28 day interval developed a robust immune response against MV (1:1280), HBsAg (787 mIU/ml) and DENV2 (Log10 neutralization index of 1.2) on average. In summary, it is possible to display DENV E DIII on hybrid HBsAg particles vectored by MV that elicit an immune response. This forms the basis for a potential vaccine platform against DENV.
Dissertation/Thesis
Masters Thesis Biology 2015

碩士
國立中興大學
生物科技學研究所
98
VP2 is the major viral capsid protein of infectious bursal disease virus (IBDV), a causative agent of a highly contagious immunosuppressive disease in young chickens. In vivo expression of VP2 can cause the formation of icosahedral (T=1) subviral particles (SVPs). Monomeric VP2 protein contains three domains, ie., protrusion (P), shell(S) and base (B) domain. Outward P domain (aa.202~341) is to compose the loop and β-sheet. In this study, to simulate the protein structure of iran strain IR01 VP2 base on local strain P3009 VP2 by using homology modeling, then employed Surface Racer 5.0 and Discovery Studio 2.5 to calculate the exposure area and percent solvent accessibility of side chains of P3009 VP2 and IR01 VP2, respectively. Previous results demonstrated that SVPs can bind to Ni ion by His253 on the DE loop of VP2 P domain of SVP, the specific amino acid were substituted with histidine via site-directed mutagenesis to generate few variants. P3009 VP2, IR01 VP2 mutants were separated by sucrose gradient centrifugation. In the results, themutants have same distribution campare with the WT SVP, the follow-up transmission electron microscopy identified P3009 and IR01 VP2 particles structures have similar diameter with wild type SVP . To test the adsorption of each mutants by immobilized metal affinity chromatography, the results showed the D279H, Q324H on the β-sheet structure of P domain of P3009 VP2 provide the adsorptive to Ni ion and S251H, G285H and Q324H of IR01 VP2 were carried out with the nickel ion adsorption, respectively. Conclusion of this study, the D279H, Q324H in the β-sheet structure of P3009 VP2 provide the adsorption with IMAC and adsorption results of IR01 VP2 showed that credibility of IR01 subvirus particle model from homology modeling.

碩士
國立中興大學
生物科技學研究所
100
Spinner flask plays a key role in insect cells culture operations in laboratory-scale. With the culture volume increased in a spinner flask, gas-liquid mixing effect will decline. Because Oxygen is a key nutrient in insect cell culture bioprocesses, it’s not conducive for the cultivation in spinner in large-scale. In order to increase the dissolved oxygen in the media, sparging it with air, directly enters the culture medium. However, direct sparging can damage insect cells through bursting bubbles and bubble accumulation. To improve the bubble-associated damage, adding a protective agent and antifoam reduce the cell damage. Aeration of Hi-5 cell culture, an additional 0.2% (v/v) antifoam AF emulsion inhibits the bubble accumulation and 0.2% (w/v) Pluronic F-68 protects cells against shear stress. Maximum cell density was 5 x 106 cells/ml in aeration culture, and specific growth rate was 0.032h-1. Infectious bursal disease virus (IBDV) is a double stranded RNA virus and a highly contagious disease of young chickens at 3 to 6 weeks of age. VP2, one of IBDV’s capsid proteins, is achieved by the induction of neutralizing antibodies in the chicken. In order to mass produce and develop safer vaccines, we have expressed the VP2 protein using an insect cell-baculovirus expression vector system (IC-BEVS). The VP2 protein spontaneously forms a dodecahedral T = 1 subviral particle (SVP) and good protection is achieved for young chickens. In previous studies, VP2 SVPs without His-tag can be purified by IMAC. To further study the effect of the exposed surface of a histidine residue on a VP2 SVPs’ interaction with a metal ion, a series of VP2 SVPs variants were generated. We first calculated the exposed area of the residues on the VP2 SVPs loop. Then we selected the residues with an exposed area of more than 100Å, and they were substituted in histidine. VP2 SVPs variants were analyzed for the interaction between surface histidine and the immobilized nickel ion in an IMAC column. These results show that a direct correlation between protein binding affinity and exposed surface area of the histidine. The surface histidine is involved in intramolecular hydrogen bond, reduced the binding affinity, as compared with the variant containing a histidine residue with a similar exposed surface area.

博士
國立中興大學
生物科技學研究所
94
VP2 is the structure proteins of IBDV capsid and the primary host-protective immunogen of IBDV. When expressed in insect cell, VP2-452H spontaneously forms a T=1 subviral particle (SVP). Although the structure of SVP has been determined at 2.6

碩士
國立臺灣大學
微生物學研究所
95
Dengue viruses（DENV）are members of the family Flaviviridae. Among the 80 or so arthropod-borne flaviviruses, the four serotypes of DENV（DENV1, DENV2, DENV3, DENV4）cause the most important arboviral diseases in human, including the classic dengue fever（DF）and severe dengue hemorrhagic fever/dengue shock syndrome（DHF/DSS）. Infection by one serotype of DENV can provide life-long protection against that serotype, but not against other serotypes. Infection of DENV in mononuclear cells has been shown to be enhanced by the presence of non-neutralizing antibody or suboptimal concentration of neutralizing antibody. This is the so called antibody-dependent enhancement, which may explain the epidemiological observation that people with secondary infection have a significant higher risk of developing DHF than people with primary infection. Previous studies of antibody responses against DENV by hemagglutination inhibition, ELISA and neutralization tests can distinguish between primary and secondary infection, but not different DENV antigens recognized. Although some studies have used Western blot analysis to examine antibody responses in DENV patients, the antibody responses against various DENV antigens of homologous or heterologous serotypes in DENV patients remain unclear. The first specific aim of this study is to use Western blot analysis to investigate the antibody responses against four DENV proteins, envelope（E）, precursor membrane（PrM）, capsid（C）, and nonstructural protein NS1 of 4 serotypes of DENV in 69 patients with DF or DHF and with primary or secondary infection during the DENV2 outbreak in southern Taiwan in 2001-2. Several known monoclonal antibodies against these 4 DENV proteins were used to identify the DENV proteins recognized. With the increase in fever days, sera in patients with primary infection can first recognize E protein of DENV2, DENV1 and DENV3, as well as NS1 of DENV2. Anti-DENV4 E responses appeared later. In patients with secondary infection, sera can first recognize E and PrM proteins of three to four serotypes and NS1 of DENV1 and DENV2. After 7 days from the fever onset, the responses reached to the level and extent similar to that of convalescent stage, in which sera can recognize E, PrM and NS1 proteins of all 4 serotypes. Compared with those with primary infection, antibody responses in patients with secondary infection developed quicker and recognized more antigens at higher levels. Statistical analysis reveals that the proportion of patients with secondary infection developed anti-NS1 and anti-PrM antibodies ≧7 days from fever onset were significantly higher than those with primary infection（P<0.005, chi-square test）. There was no difference in the antibody responses against the four DENV proteins tested between patients with DF and DHF. After treating the antigens with reducing agent, β-mercaptoethanol, antibodies responses to E, PrM and NS1 proteins disappeared in the majority of cases, with anti-NS1 responses left in few cases. On the other hand, expression of E and PrM proteins of DENV can form heterodimer, which is important for the production of recombinant subviral particles（RSPs）. RSPs have been shown to be a useful tool to study the function of PrM/E proteins as well as a potential serodiagnostic antigen and vaccine candidate. Previous studies of the heterodimerization and RSPs of PrM/E proteins focused primarily on the E protein, of which the two α-helices in the stem region（E-H1 and E-H2）and the two α-helices in the anchor region（E-T1 and E-T2）at the C-terminal have been shown to play an important role. Few studies examined the involvement of PrM protein in the process. The second specific aim of this study is to study the expression of PrM protein and the involvement of the C-terminal α-helix（M-H）of the stem region of PrM protein in the heterodimerization with E protein and RSPs production by using DENV4 based expression constructs. Amino acid substitutions of the 5 amino acid residues located at a,d position of the M-H in PrM protein with Alanine or Proline revealed no effect on heterodimerization. In contrast, amino acid substitutions of 3 amino acid residues at position 120, 123 and 127 with Proline resulted in severe impairment in RSPs formation. A serious of C-terminal truncation constructs of PrM and E proteins were also generated. In the absence of E protein, PrM protein can not express well, especially its C-terminal truncation mutants. Moreover, in the presence of C-terminal truncated E protein（without E-H2, E-T1 and E-T2）, the expression of PrM protein significantly decreased. These findings suggested that E protein, especially its C-terminal domain E-H2, play a critical role in the expression of PrM protein. During the past three decades, there was a significant increase in the numbers of DHF/DSS cases and the numbers of countries reported to have DHF/DSS, indicating the importance and need for dengue research. The first part of this study suggested that reactivities to PrM and NS1 proteins recognized by Western blot analysis can be used to distinguish primary and secondary infection. Moreover, the majority of anti-E and anti-PrM antibody responses in DENV patients were conformational sensitive. These findings may provide useful information for future design of subunit vaccines. The second part of this study revealed that the E protein, especially its C-terminal domain, was important for expression of PrM protein. In addition, three critical amino acid residues at the M-H region of PrM protein were critical for the formation of RSPs, suggesting that this α-helix has an important function and may represent potential target for future antiviral strategies.

碩士
國立中興大學
生物科技學研究所
96
In previous studies, we have confirmed that both of infectious bursal disease virus (IBDV) virions and the subviral particles (SVPs) composed of IBDV VP2 protein can be purified by immobilized metal ion affinity chromatography (IMAC). This suggests some amino acid residues on the surface of IBDV virions mediate the interaction with Ni2+ metal ions. Earlier reports indicate the region from residue 206 to 350 in VP2 protein contain the major immunogenic determinants. Therefore, any mutations on this region may result in IBDV antigenic variation. Furthermore, other resides include 253, 279, and 284 may determine the viral pathogenicity or play a key role on viral entry. In this study, we focus on two histidine residues (His249 and His253) and investigate its importance on the interaction with Ni2+ ion and the effect on the immunogenicity. For these purposes, three VP2-441 variants, H249A, H253A, and H249.253A, were constructed and expressed by baculovirus expression system. The particle morphology of these variants examined by transmission electron microscopy (TEM) was similar to the authentic VP2-441 SVPs, exhibiting that the mutation from histidine to alanine doesn’t affect the particle self-assembly. The binding strength of these variants with immobilized Ni2+ ion was measured by quantitating the unbound VP2 protein after a series of wash with various pH and imidazole concentrations. The results demonstrated that the His253 on the particle surface was the major amino acid contributes to the interaction with Ni2+ ion. Afterward, the adsorption of VP2-441 SVP to Ni2+-NTA was increased with the concentration of free VP2. The bound protein per volume of resin (Q) for VP2-441 SVP is 7.9 x 10-10 moles/ml Ni-NTA. The saturated Q of H249A and H253A reached to 7.3 x 10-10 and 3.5 x 10-10 moles/ml Ni-NTA, respectively. The equilibrium adsorption isoterm of H249.253A SVP to Ni-NTA had no accordance to the concentration of SVP and the average Q value was 2.6 x 10-11 moles/ml Ni-NTA, which is the lowest among the four SVPs. For investigating the effect of such mutation on its immunogenicity, the corresponding SVPs were recognized by two neutralizing monoclonal antibodies (MAb SVP-1 and MAb SVP-4) against IBDV in an ELISA test. The data have shown such mutations offer the SVP a better binding affinity to the two neutralizing monoclonal antibodies. The protections afforded by three VP2-441 SVP variants were also compared by immunizing the specific-pathogen-free chickens and following with a homologous virus challenge. The sera collected from the chickens immunized with H253A SVPs have a higher virus neutralizing (VN) titer than authentic VP2-441 SVPs did. These suggested that the His253 may locate on the center of immunogenic determinants and mutation to alanine has fine-tuned the local structure to fit the binding pocket of neutralization antibody much more tightly.

碩士
國立中興大學
微生物暨公共衛生學研究所
101
Abstract Capillary zone electrophoresis (CZE) has been successfully used for characterizing and analyzing proteins. Infectious bursal disease virus (IBDV) is a double-stranded RNA virus with an icosahedral capsid of 55-65 nm in diameter (T=13) and belonging to the Birnaviridae family, causes immunosuppression in young chickens. The major structure protein VP2 of IBDV is response to the induction of efficacious protection. To express the VP2 protein alone in E. coli has been demonstrated to produce icosahedral particles of 25 nm in diameter (T=1), subviral particles (SVP). The integrity of SVP is highly related to the induction of protection against IBD. Therefore, a method used to quality control vaccine containing SVP requires the ability to detect the intact SVP. The objective of this study is to setup a CZE for separation and identification of SVP of IBDV expressed by E. coli. The SVP was expressed by E. coli and confirmed by Western blotting and electron microscopy. The expressed SVP were purified and concentrated by acid precipitation, sucrose gradient, and the size-exclusion column (Amicon). The CZE system with an inner diameter of 75 μm fused silica capillary, pressure injection and 50 mM Boric acid pH 9 separation buffer was used to analyze the SVP. The SVP with complete purification and concentration steps showed single peak in CZE with a migration time of 2.65 min. After adding anti-VP2 antibody, the SVP-antibody complex showed single peak with a migration time of 1.8 min. The CZE, were used to detect various amounts of SVP, anti-VP2 ab, and the R2 of standard curve, were 0.99, 0.98, respectively. The results indicated that CZE is valuable method in separation and identification of the SVP. The method is an important tool of SVP statistical quality control.

碩士
國立中興大學
生物科技學研究所
94
Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. The outer capsid protein VP2 of IBDV has been suggested to play an role in virus binding and cell recongnition. VP2 can form a particle called subvirus particle (SVP) of 25 nm in diameter, when it was expressed in insect cells. VP2 SVP can suppress the infection of IBDV to DF-1 cells, an IBDV susceptible cell line and isolated form chicken embryo fibroblast cell. We design a system to isolate proteins which interact with VP2 SVP in DF-1 cells by immobilized metal affinity chromatography (IMAC) using VP2 SVP as a ligand. The results show that an 80~90 kDa protein molecule (p90) from DF-1 cells with a strong interaction with VP2 SVP was purified. We also demonstrate that p90 was located on the surface of the DF-1 cells and identified p90 as chicken heat shock protein 90 (HSP90) by liquid-chromatography mass spectrometry (LC-MS/MS). The results suggest Hsp90 may be involved in the recognition of host cells and IBDV, and the entry of IBDV to DF-1 cells.

碩士
國立中興大學
生物科技學研究所
93
Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. The outer capsid protein VP2 of IBDV has been suggested to play an important role in virus binding and cell recognition. VP2 can form a particle called subvirus particle (SVP) of 25 nm in diameter, when it was expressed in insect cells. This study attempts to identify the cellular receptors of IBDV susceptible cell lines using SVP. Inhibition infection assay with VP2-formed SVPs supports that SVPs competition with IBDV virons to the attachment of CEF and partially inhibit the replication of IBDV in CEF. Then, DF1 and Vero cells were used for study as host cell lines. Binding of VP2 particles to either DF1 or Vero cells was observed using various biochemical assays. The localization of the VP2 particles on Vero and DF1 cells was also confirmed by immunofluorescence microscope. The binding of the VP2-formed SVPs to Vero and DF1 cell surfaces was specific and occurred in a dose-dependent manner. Furthermore, the neutralizing monoclonal antibody against IBDV inhibits the attachment of SVPs particles to Vero and DF1 cells. The results suggest that the attachment of IBDV to susceptible cell is mediated by VP2.

碩士
國立中興大學
生物科技學研究所
99
The advantage of Immobilized-Metal ion Affinity Chromatography (IMAC) – one- step purification, recycling is easy, etc.- are decisive when developing large-scale purification procedures for industrial applications. Previous results indicated that SVPs can be purified directly by IMAC and the His 253 on DE loop of P domain plays an important role binding Ni2+ ions. Besides His 253, outward superficial residues on P domain of SVPs provide higher chances for Ni2+ ions binding. In this study, to search the residues this can be substituted for the binding of immobilized Ni2+ ions. First, the calculation of exposure areas of residues on the loop was performed by Surface Racer 5.0 software. Six resides - Gln221, Ser222, Ser251, Ser317, Gln320 and Ala321-have been chosen, than been substituted with histidine. Recombinant proteins was expressed in Escherichia coli and Bac-to-Bac baculovirus expression system, and then purified by IMAC. The binding strength of these variants with immobilized Ni2+ ion was measured by quantitating the unbound VP2 protein after a series of wash with various pH and immidazole concentrations. The results have shown some residues on the loop structure of P domains of SVPs substituted and complement the mutation of H253A to bind Ni2+ ion, and don’t affect density and other physical characteristics.

碩士
國立中興大學
生物科技學研究所
95
In previous research, it demonstrated that Immobilized Metal ion Affinity Chormatography column（IMAC）not only can purify the His-tag fusion IBDV VP2-452H subviral particle （SVP）, but also can purify the IBDV VP2-441 subviral particle without his-tag fusion. Therefore, the VP2 protein absorption with IMAC is not depending on the His-tag. It was suggested that SVP purified by IMAC was due to its surface amino acid residues of VP2. Two histidine residues which were located on the most exposed loops (Loop DE) may engage directly in contact with the immobilized Ni2+ ions. In this work, we engineered SVP surface amino acid, His-249 or His-253, which is related with IMAC absorption, and produced three mutants, VP2-441-H249.253A, VP2-441-H253A and VP2-441-H249A. Mutants VP2-441-H249.253A and VP2-441-H249A were expressed in E.coli and VP2-441-H253A was expressed in both E.coli and baculovirus expression system. After purification by ultracentrifugation, the VP2 SVP assembled as 20~23 nm particles which can be visualized under Transmission electromicroscopy (TEM). The results show that histidine mutation will not affect the self assembly of SVP. However, the three SVP mutants are present in the flow-through and pH 7.8 binding buffer eluent after IMAC purification. It indicates that these three mutants had lost the absorption ability with IMAC and provides evidence that the His 249 and His253 of VP2 play an important role in the binding affinity of SVP with Ni2+ ion of IMAC.

博士
國立清華大學
生命科學系
93
Abstract The formation of flavivirus prM-E complex is an important step for the biogenesis of immature virions, followed by a subsequent cleavage of prM to M protein through cellular protease to result in the production and release of mature virions. The intracellular formations of the prM-E complexes of Japanese encephalitis virus (JEV) were investigated by baculovirus co-expression of prM and E in trans and prM and E in cis in Sf9 insect cells analyzed by anti-E antibody immunoprecipitation and sucrose gradient sedimentation analysis. A series of the carboxyl-terminally truncated prM mutant baculoviruses were constructed to demonstrate that the truncations of the transmembrane (TM) region resulted in a reduction of the formation of the stable prM-E complex. Alanine scanning site-directed mutagenesis on the prM99-103 region indicated that the His99 residue was the critical prM-binding element for the prM-E heterodimeric stable complex formation. The single amino acid mutation at the His-99 residue of prM abolishing the prM-E interaction was not due to the reduced expression or different subcellular location of the mutant prM protein involved in prM-E interactions as characterized by pulse chase labeling and confocal scanning microscopic analysis. Recombinant subviral particles were detected in the Sf9 cell culture supernatants by baculovirus co-expression of prM and E proteins but not by the prM-H99A mutant. Sequence alignment analysis was further conducted in different groups of flaviviruses to show the prM-H99 residues are generally conserved. The TM regions of prM protein on prM-E heterodimeric complex formation and recombinant subviral particles formation of JEV were further characterized and the TM1 region of prM, especially the specific amino acid sequence-GXXXG motif, was demonstrated using deletion, alanine insertion and glycine substitution mutagenesis. The GXXXG motif located in the TM1 region of prM of JEV were identified for the influence of heterodimerization and virus assembly. Furthermore, the TM regions of E protein on prM-E heterodimeric complex formation and recombinant subviral particles formation of JEV were characterized and the TM1 regions and the conserved charged residues (RDR) of the connecting segment of E were demonstrated using alanine insertion and replacement mutagenesis. These data indicate that the TM regions and His99 of prM and the TM regions and the conserved charged residues (RDR) of the connecting segment of E proteins play a crucial role of the biogenesis of JEV envelope. This information, concerning a molecular framework for the prM and E proteins, is considered to elucidate the structure/function relationship of the prM-E complex synthesis and provide the proper trajectory for flavivirus assembly and maturation.

碩士
中臺科技大學
食品科技研究所
97
Abstract VP2 is a major structural protein of Infectious bursal disease virus (IBDV), which contains only two Cysteine residues, Cys-99 and Cys-197, located at SD domain and PA’ domain, respectively. In this study, VP2 was engineered by site-directed mutagenesis to investigate the role of Cysteines on the formation of VP2 subviral particle (SVP). Mutants VP2-C99A, VP2-C197A, VP2-C197T, VP2-C197S and VP2-C99.197A were expressed in E. coli and VP2-C99A and VP2-C197A were also expressed in insect cell/ baculovirus system. VP2-C99A expressed in both systems remains its ability of SVP formation and can be purified by immobilized metal-ion affinity chromatography (IMAC), with a particle size of 20-25 nm. The other mutants expressed in E. coli lost their affinity to Ni-NTA resin, indicating that they do not exist in SVP form. Most of the insect cell-derived VP2-C197A was present in the flow-through and pH 7.8 binding buffer eluant, suggesting that they have lost the absorption ability with Ni-NTA. The small amount of VP2-C197A purified by IMAC exhibits swelling and incomplete particle form observed under electron microscope (EM). Our results indicate that Cys-197 is an essential and important site for the self-assembly of VP2 into subviral particle.

博士
國立陽明大學
生化暨分子生物研究所
96
Crystal Structure of Infectious Bursal Disease Virus VP2 Subviral Particle at 2.6 Å Resolution: Implications in Virion Assembly and Immunogenicity Abstract Infectious bursal disease virus (IBDV) is responsible for the hightly contagious and immunosuppressive disease in young chicken. The structural protein VP2 of IBDV spontaneously forms a dodecahedral T=1 subviral particle (SVP), and is a primary immunogen of the virus, understand of its structure is efficient for vaccine development. In this study, the structure of IBDV SVP was determined in a cubic crystal and refined to 2.6Å resolution. It contains 20 independent VP2 subunits in a crystallographic asymmetric unit. Each subunit is folded mainly into a shell domain and a protrusion domain, both with the Swiss-roll topology, plus a small helical base domain. Three VP2 subunits constitute a tight trimer, which is the building block of IBDV (sub)viral particles. The structure revealed a calcium ion bound to three pairs of symmetry-related Asp31 and Asp174 to stabilize the VP2 trimer. To investigate the effect of Ca2+ on the IBDV SVP structure, we used EGTA to remove the divalent ion and analyzed the particle morphology by gel electrophoresis and electron microscopy, and the results indicated that the metal-ion may be important not only in maintaining highly stable quaternary structure but also in regulating the swelling and dissociation of the icosahedral particles. A Ca2+-dependent assembly pathway was thus proposed, which involves further interactions between the trimers. The 20 independent subunits showed conformational variations, with the surface loops of the protrusion domain being the most diverse. These loops are targets of the neutralizing antibodies. Several common interactions between the surface loops were clearly observed, suggesting a possible major conformation of the immunogenic epitopes. Knowledge of the three-dimensional structure of SVP may be useful in rationally incorporating important foreign epitopes into the loop region to create engineered recombinant SVP as new potent immunogens or vaccines. Structural Basis of Metal-conjugated Complexes as 3C and 3C-like Protease Inhibitors Abstract Viral proteases have been pursued for anti-virus therapy, and their crystal structures were used to assist the design of inhibitors. Some metals (Cu2+, Hg2+, Zn2+) and metal-conjugated compounds showed cysteine protease inhibition activity. Here, to elucidate the metal-inhibitor binding mode and to synthesize better inhibitors, 3C-like protease from Coronaviridae and 3C protease from Piconaviridae complexed with metal-conjugated inhibitors were analyzed crystallographically. Five active metal-conjugated inhibitors (PMA, TDT, EPDTC, JMF1586 and JMF1600) bound with the 3C-like protease (3CLpro) of severe acute respiratory syndrome (SARS)-associated coronavirus (CoV) were determined. The complex structures reveal two major inhibition modes: Hg2+-PMA is coordinated to C44, M49 and Y54 with a square planar geometry in the S3 pocket, whereas each Zn2+ of the four zinc-inhibitors is tetrahedrally coordinated to the His-Cys catalytic dyad. 3CLpro of human coronavirus 229E (HCoV-229E) and 3C proteases of Coxsackie B viruses type 3 (CVB3) also have the His-Cys catalytic residues as 3CLpro of SARS-CoV. The first crystal structures of CVB3 3Cpro, and the crystal structure of 3Cpro from CVB3 and 3CLpro from HCoV-229E in complex with the inhibitor EPDTC were also determined. The zinc ion of EPDTC is again tetrahedrally coordinated to the His-Cys catalytic residues of CVB3 3Cpro and HCoV-229E 3CLpro. For anti-virus drug design, this Zn2+-centered coordination pattern would serve as a starting platform for inhibitor optimization.