Dissertations / Theses on the topic 'Virus particle assembly'

The formation of viral particles requires the coordinated assembly of both nucleic acids and proteins. In the case of Rhabdoviruses, such as vesicular stomatitis virus (VSV), the particles display a characteristic bullet-shape. VSV virions consist of the matrix protein (M), glycoprotein (G), and viral ribonucleoprotein (RNP), which contains the nucleocapsid protein (N) coated RNA bound to the large polymerase protein (L) through the phosphoprotein (P). During assembly, these components are recruited to the plasma membrane where the viral RNP undergoes condensation by M and envelopment with G containing membranes. To address whether formation of the bullet-shape requires a consistent packaging of the viral proteins, the composition of single virions was measured with fluorescence microscopy. We generated autonomously replicating VSV bearing up to 3 fluorescent protein fusions in the disordered N-terminal region of M and N-terminus of P and G. Quantification of single particles reveals that VSV assembles with a range of M, P, and G molecules, suggesting a flexible packaging mechanism. The maintenance of the bullet-shape with significantly less M proposes that condensation does not require the particle to be saturated with M. Our fluorescent VSV clones permit the tracking of viral components in live cells. We observed that assembly of M into particles requires ~2 min and can be broken into 4 stages. First, M forms a small preassembly complex. Second, M rapidly assembles into particles where its incorporation initiates before P, although they are packaged concurrently. This is followed by a delay before final release of particles into the supernatant. Late domains in M were thought to only recruit the endosomal sorting complexes required for transport (ESCRT) pathway to mediate fission. However, using our M fusions we demonstrate that these motifs are required for efficient competition into released particles and a step in assembly prior to pinching off. These constructs have permitted the study of viral assembly at the single particle level and are useful tools for studying viral entry and egress. Specifically, VSV containing M-eGFP and the lassa virus glycoprotein instead of G was used to demonstrate the requirement of a host factor for lassa virus fusion.<br>Medical Sciences

Influenza A Viren besitzen ein segmentiertes, einzelsträngiges RNA-Genom, welches in Form viraler Ribonukleoprotein (vRNP)-Komplexe verpackt ist. Während das virale Genom im Zellkern repliziert wird, finden Assemblierung und Knospung reifer Viruspartikel an der apikalen Plasmamembran statt. Für die Virusbildung müssen die einzelnen viralen Komponenten hierher gebracht werden. Während intrinsische apikale Signale der viralen Transmembranproteine bekannt sind, sind der zielgerichtete Transport und der Einbau des viralen Genoms in neuentstehende Virionen noch wenig verstanden. In dieser Arbeit wurden potentielle Mechanismen des vRNP-Transportes untersucht, wie die Fähigkeit der vRNPs mit Lipidmembranen zu assoziieren und die intrinsische subzellulären Lokalisation des viralen Nukleoproteins (NP), eines Hauptbestandteils der vRNPs. Es konnte gezeigt werden, dass vRNPs nicht mit Lipidmembranen assoziieren, was mittels Flotation aufgereinigter vRNPs mit Liposomen unterschiedlicher Zusammensetzung untersucht wurde. Die Ergebnisse deuten jedoch darauf hin, dass das virale M1 in der Lage ist, Bindung von vRNPs an negativ-geladene Lipidmembranen zu vermitteln. Subzelluläre Lokalisation von NP wurde des Weiteren durch Expression fluoreszierender NP-Fusionsproteine und Fluoreszenzphotoaktivierung untersucht. Es konnte gezeigt werden, dass NP allein nicht mit zytoplasmatischen Strukturen assoziiert, stattdessen aber umfangreiche Interaktionen im Zellkern eingeht und mit hoher Affinität mit bestimmten Kerndomänen assoziiert, und zwar den Nukleoli sowie kleinen Kerndomänen, welche häufig in der Nähe von Cajal-Körperchen und PML-Körperchen zu finden waren. Schließlich wurde ein experimenteller Ansatz etabliert, welcher erlaubt, den Transport vRNP-ähnlicher Komplexe mittels Fluoreszenzdetektion aufzuzeichnen und Einzelpartikelverfolgungsanalysen durchzuführen. Unterschiedliche Phasen des vRNP-Transportes konnten beobachtet werden und ein 3-Phasen-Transportmodell wird skizziert.<br>Influenza A viruses have a segmented single-stranded RNA genome, which is packed in form of viral ribonucleoprotein (vRNP) complexes. While the viral genome is replicated and transcribed in the host cell nucleus, assembly and budding of mature virus particles take place at the apical plasma membrane. Efficient virus formation requires delivery of all viral components to this site. While intrinsic apical targeting signals of the viral transmembrane proteins have been identified, it still remains poorly understood how the viral genome is transported and targeted into progeny virus particles. In this study, potential targeting mechanisms were investigated like the ability of vRNPs to associate with lipid membranes and the intrinsic ability of the viral nucleoprotein (NP) – which is the major protein component of vRNPs – for subcellular targeting. It could be shown that vRNPs are not able to associate with model membranes in vitro, which was demonstrated by flotation of purified vRNPs with liposomes of different lipid compositions. Results indicated, however, that the matrix protein M1 can mediate binding of vRNPs to negatively charged lipid bilayers. Intrinsic subcellular targeting of NP was further investigated by expression of fluorescent NP fusion protein and fluorescence photoactivation, revealing that NP by itself does not target cytoplasmic structures. It was found to interact extensively with the nuclear compartment instead and to target specific nuclear domains with high affinity, in particular nucleoli and small interchromatin domains that frequently localized in close proximity to Cajal bodies and PML bodies. An experimental approach was finally established that allowed monitoring the transport of vRNP-like complexes in living infected cells by fluorescence detection. It was possible to perform single particle tracking and to describe different stages of vRNP transport between the nucleus and the plasma membrane. A model of three-stage transport is suggested.

Viruses are infectious agents that must infect the cells of living organisms in order to reproduce. They have relatively simple genomes which encode few proteins but can compensate for their simplicity by hijacking components of their cellular hosts. Arenaviruses, a family of zoonotic viruses carried by rodents, encode only 4 proteins. One of these proteins, Z, is responsible for several functions during the virus life cycle including driving the formation and release of new virus particles at the plasma membrane of infected cells. Relatively little is known about how this viral protein is regulated or the complement of host proteins it engages in order to produce new virus particles or augment Z's other functions. To address this gap in knowledge, mass spectrometry was used to identify phosphorylation sites in the Old World arenavirus, lymphocytic choriomeningitis virus (LCMV) Z protein. Phosphorylation sites were identified at serine 41 (S41) and tyrosine 88 (Y88). Functional studies using recombinant (r)LCMV containing mutations at these phosphorylation sites revealed that both were important for the production of defective interfering (DI) particles. DI particles are replication-incompetent virus particles that interfere with the production of infectious virus and mitigate its cytopathic effect. While a mutation that mimics phosphorylation at S41 reduced LCMV's ability to produce both infectious and DI particles, this mutation had a much stronger impact on DI particles. Production of DI particles in Y88-mutant rLCMV was drastically reduced while the impact on infectious virus was minimal. Y88 lies within a type of viral late domain (PPXY) also found in matrix proteins of several disparate virus families where it has been shown to drive infectious virus release by recruiting the membrane scission machinery of the cellular endosomal sorting complex required for transport (ESCRT). Inhibition of the ESCRT pathway drastically reduced LCMV DI particle but not infectious virus release indicating that Z's PPXY late domain and the cellular ESCRT complex are required specifically for the production of DI particles. Mass spectrometry was also used to identify host protein partners of Z as well as the host proteins recruited into virus particles for the New World arenavirus, Junin (JUNV). ESCRT complex proteins were enriched in JUNV virus-like particles (VLPs) and bona fide virions. In contrast to LCMV, inhibition of the ESCRT complex resulted in significantly less infectious JUNV release. This indicates that the ultimate role of ESCRT engagement by the Old World arenavirus, LCMV, differs from that of New World, JUNV. This work represents the first demonstration that a viral protein motif and the host machinery it engages selectively drive DI particle production independently of infectious virus. It also suggests that host cell kinases can dynamically regulate the production of DI particles through phosphorylation of Z. Finally, the late domain-mutant rLCMV generated in these studies represents the first LCMV strain known to produce undetectable levels of DI particles which provides the opportunity to assess the impact that a loss of DI particles has on the ability of LCMV to establish or maintain a persistent infection.

Lors d’une infection chronique, le virus de l'hépatite C (HCV) circule sous forme de lipoviroparticule (LVP) : particules hybrides associant des composants viraux (ARN, les protéines structurelles) et des composants cellulaires (apolipoprotéines, cholestérol). Au cours de ma thèse, nous nous sommes intéressés à identifier la plateforme d'assemblage du HCV, et le rôle du rassemblement des protéines virales par NS2 dans sa formation. Nous avons montré que des interactions de natures différentes sur la membrane du RE sont impliquées dans cette association protéique. Nos résultats suggèrent que des interactions complexes (directes ou via des « membranes résistantes aux détergents » (DRM)) entre les protéines complexées par NS2, peuvent immédiatement précéder la formation LVP. Nous avons également démontré que l’hétérodimère E1E2, les apolipoprotéines B et E (ApoB, ApoE) s’associent en un complexe de protéines dans le réticulum endoplasmique (RE) lorsqu'elles sont exprimées ensembles. Ce complexe se forme au début de l'assemblage du HCV, quelle que soit l'expression des autres protéines virales, et est conservée sur les LVP sécrétées. Basé sur ces données, nous avons proposé un mécanisme expliquant l’initiation de la morphogenèse des LVP. Ensuite, nous avons évalué l'importance de l'association E1E2/ApoE pour le cycle de vie du virus. Nous avons initié une étude pour identifier les acides aminés E1E2 impliqués dans l'interaction avec les apolipoprotéines. Avec ces données, nous souhaitons proposer une meilleure compréhension des mécanismes de la morphogenèse du HCV<br>In chronic infection, the hepatitis C virus (HCV) circulates as lipoviral particles (LVP): hybrid particles associating viral (RNA, structural proteins) and cellular components (apolipoproteins, cholesterol). During my PhD, we were interested in identifying the HCV assembly platform, and the role of the association of the viral proteins by NS2 during its formation. We showed that different natures of interactions on the ER membrane are involved in this proteic association. Our results suggest that a complex interplay between proteins of the complex formed by NS2, directly or through “detergent resistant membranes” (DRMs) may be immediately followed by LVPs formation. We also demonstrated that E1E2 heterodimer, apolipoproteins B and E (ApoB, ApoE) associate as a protein complex in the endoplasmic reticulum (ER) when expressed together. This complex is formed early in HCV assembly, regardless the expression of other viral proteins, and is conserved on the secreted LVPs. Based on these data, we proposed a mechanism explaining LVP morphogenesis initiation. Then we assessed the importance of E1E2/ApoE association for viral life cycle. We initiate a study to identify the E1E2 amino acids involved in the interaction with apolipoproteins. With these data, we wished to provide a better understanding of the mechanisms of the HCV morphogenesis

Les virus bactériens (bactériophages), durant leur co-évolution avec les bactéries, ont su trouver de nombreuses voies pour détourner les machineries cellulaires dans le but de se multiplier efficacement. L’infection par le phage dès son entrée dans le cytoplasme est un bouleversement pour la bactérie en termes de ressources monopolisées à ses dépens et probablement de restructuration de l’espace cytoplasmique. Dans ce travail de thèse, l’impact de l’infection de la bactérie Gram-positive Bacillus subtilis par le bactériophage SPP1 a été étudié.La réplication de l’ADN est initiée par des protéines précoces virales. Elle mène au chargement de l’hélicase virale gp40 sur l’origine de réplication de SPP1 dont les brins d’ADN ont été ouverts par la protéine de liaison à l’origine, gp38. Le réplisome bactérien est ensuite recruté de manière massive au sein de l’usine de réplication formant un foyer défini dans le cytoplasme bactérien. L’interaction de gp40 avec les protéines cellulaires DnaX et DnaG assure fort probablement le recrutement du complexe cellulaire au foyer de réplication. La quantité d’ADN viral synthétisée représente presque 500 copies d’ADN viral par bactérie après 30 minutes d’infection, ce qui est équivalent à la taille de 5 génomes de B. subtilis. Des études de FRAP (Fluorescence Recovery After Photobleaching) montrent que l’usine de réplication est très dynamique. Ce comportement est inhibé par la présence de HPUra montrant qu’il dépend de la présence d’un réplisome actif.Les concatémères résultant de la réplication de l’ADN viral sont le substrat pour l’encapsidation du génome de SPP1 dans des procapsides préformées. La maturation de ces procapsides en particules virales infectieuses suit une voie d’assemblage spécifique. Deux protéines rapportrices de différentes étapes de cette voie ont été suivies : la protéine d’échafaudage gp11, présente à l’intérieur de la procapside avant encapsidation de l’ADN, et la protéine auxiliaire gp12, qui se fixe à la surface de la capside pendant l’encapsidation. Les procapsides colocalisent partiellement avec l’usine de réplication du génome viral. Après encapsidation de l’ADN, les capsides vont s’accumuler dans des foyers de stockage qui ont une localisation indépendante du foyer de réplication. Cette organisation est également observée dans des bactéries très allongées où deux régions de stockage sont retrouvées situées de part et d’autre de l’usine de réplication mais éloignées des pôles cellulaires. La microscopie électronique combinée à des immuno-marquages révèlent que cette compartimentation corrèle avec une réorganisation majeure de l’ultrastructure du cytoplasme bactérien.L’assemblage et la dynamique des foyers viraux dans la bactérie ont été suivis pendant toute la durée du cycle viral dans un système de microfluidique. Elle montre que les étapes de réplication de l’ADN viral et la formation de la particule du phage sont des processus compartimentés dans le cytoplasme de la bactérie tant spatialement que temporellement. Bien que la croissance cellulaire soit retardée, les bactéries continuent de s’allonger et de se diviser pendant l’infection par SPP1. Le virus exploite donc de manière efficace les machineries cellulaires et l’architecture de la bactérie pour une multiplication optimale. Ces stratégies sont probablement utilisées par de nombreux phages pour remodeler la cellule bactérienne à leur avantage<br>During the co-evolution of viruses and cells, viruses exploited numerous ways to hijack cell machineries for their optimal multiplication and dissemination. Phage infection is a major challenge to bacteria, exploiting extensively cellular biosynthetic ressources and possibly re-organizing the cytoplasm space. The work in this thesis investigated the cellular impact of infection by SPP1, a well-characterized model tailed bacteriophage that infects the Gram-positive bacterium Bacillus subtilis.Viral DNA replication is initiated by early phage proteins whose activity culminates in loading of the SPP1 helicase gp40 at the melted phage origin of replication. The bacterial replisome is then massively recruited to the phage replication factory that is localized at a defined position of the cytoplasm. The interaction of gp40 with its two cellular partners DnaX and DnaG mediates most likely the hijacking of the B. subtilis replication machinery. More than 500 copies of the viral genome are synthesized within 30 minutes after initiation of infection, which is roughly the equivalent to five B. subtilis genomes. FRAP (Fluorescence Recovery After Photobleaching) experiments showed that the viral DNA factory is highly dynamic, a behavior that depends on active DNA replication.The concatemers resulting from DNA replication are the substrate for encapsidation of the SPP1 genome into preformed procapsids. Maturation of procapsids to infectious viral particles follows a defined pathway. The SPP1 scaffolding protein gp11, that occupies the interior of the procapsid before DNA packaging, and gp12, that binds to capsids during DNA packaging, were followed to dissect the steps of this process. Procapsids partially co-localize with DNA replication factories. After packaging the DNA-filled capsids fully segregate to spatially distinct warehouses where viral particles accumulate. Recruitment of SPP1 proteins to these compartments recapitulates the sequential order of their assembly to build the viral particle. The replication factory is most frequently flanked by two warehouses. Such pattern is also observed in very elongated cells where the viral compartments remain localized nearby each others and far from the bacterial poles. Immuno-electron microscopy of cryo-sections from infected cells highlights a complete remodelling of the bacterial cytoplasm dedicated to virus multiplication.The assembly and dynamics of the SPP1 replication factory and virions warehouses were visualized during the complete phage infection cycle in microfluidics experiments. The viral compartments are well individualized in the cytoplasm both in terms of space and time. Although bacterial growth is retarded, cells continue to elongate and to divide during SPP1 infection. Structuration of viral factories appears as a very efficient way for SPP1 to exploit bacterial resources and cytoplasmic space to optimize its multiplication. This strategy might be widely used by phages for remodelling the bacterial cell

Le virus de l'hépatite C (VHC) est détecté dans les sérums de patients infectés sous forme de particules infectieuses lipidées de très faibles densités. Le VHC est un virus enveloppé dont l'assemblage de particules virales se produit à la membrane du réticulum endoplasmique consécutivement au clivage séquentiel de sa polyprotéine et à sa maturation en protéines structurales et non structurales. Dans ce travail, nous avons cherché à mieux comprendre les mécanismes d'assemblage, d'enveloppement et de sécrétion des particules infectieuses. Dans une première étude, nous avons montré la connexion fonctionnelle entre les complexes de réplication et les sites d'assemblage. Dans une seconde étude, nous avons montré que p7 ralentissait de manière dose-dépendante le trafic ER-Golgi, conduisant à une rétention intracellulaire de la glycoprotéine virale E2. En outre, nous avons montré que le clivage du précurseur protéique E2p7 contrôle l'expression intracellulaire E2 et les niveaux de sécrétion des particules subvirales et des virions infectieux. Enfin, nous avons également mis en évidence que l'extrémité N-terminale de p7 gouverne l'infectivité spécifique des particules en coordonnant la rencontre des composants de la nucléocapside avec les glycoprotéines, mais aussi l'enveloppement de la nucléocapside. Dans une troisième étude, nous avons découvert des fonctions et des facteurs spécifiques du sérum, des cellules productrices et des séquences du VHC qui modulent la lipidation des particules virales au cours de leur assemblage et de leur sécrétion. Au total, ces différents travaux ont contribué à mieux comprendre les étapes de l'assemblage du VHC et les mécanismes modulant i) le transfert des ARN viraux des complexes de réplication vers les sites d’assemblage, ii) la rencontre des nucléocapsides et des glycoprotéines, et enfin, iii) l'acquisition de lipides par des particules virales<br>Hepatitis C virus (HCV) is detected in the sera of infected patients as lipidated infectious particles of very-low density. HCV is an enveloped virus whose assembly of viral particles occurs at the endoplasmic reticulum membrane following sequential cleavage of its polyprotein and its maturation as structural and non-structural viral proteins. In this work, we aimed at better understanding the mechanisms of assembly, envelopment and secretion of infectious particles. In a first study, we highlighted the functional connection between replication complexes and assembly sites. In a second study, we showed that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2 viral glycoprotein. In addition, we showed that cleavage of an E2p7 precursor protein controls E2 intracellular expression and secretion levels of subviral particles and infectious virions. Finally, we also highlighted that p7 N-terminal extremity governs the specific infectivity of the infectious particles by coordinating the encountering of the nucleocapsid components with the glycoproteins and the envelopment of the nucleocapsids. In a third study, we discovered specific functions and factors from serum, producer cells, and HCV sequences that modulate lipidation of viral particles during their assembly and secretion. Altogether, these different works contributed at better understanding the steps of HCV assembly and the mechanisms modulating i) the transfer of viral RNAs from replication complexes to assembly sites, ii) the encountering of the nucleocapsids and glycoproteins followed by virion envelopment, and finally, iii) the acquisition of lipids by viral particles

The Tetraviridae are a family of ss (+) RNA viruses that specifically infect lepidopteran insects. Their icosahedral capsids are non-enveloped and approximately 40 nm in diameter with T=4 quasi-equivalent symmetry. The omegatetraviruses, which are structurally the best characterised in the family, include Helicoverpa armigera stunt virus (HaSV) and Nudaurelia capensis omega virus (NwV). The omegatetravirus procapsid is composed of 240 identical copies of the capsid precursor proteins, which undergo autoproteolytic cleavage at its carboxyl-terminus generating the mature capsid protein (b) and γ-peptide. This process occurs in vitro following a shift from pH 7.6 to pH 6.0. The viral capsid encapsidates two ss genomic RNAs: The larger RNA1 encodes the viral replicase as well as three small ORFs while RNA2 encodes the capsid precursor protein together with an overlapping ORF designated P17. While a wealth of structural data pertaining to the assembly and maturation of omegatetraviruses is available, little is known about how this relates to their lifecycle. The principle aim of the research described in this thesis was to use an experimental system developed in the yeast, Saccharomyces cerevisiae, to investigate the assembly of HaSV and NwV virus-like particles (VLPs) in terms of maturation and encapsidation of viral RNAs, in vivo. The yeast expression system used two promoter systems for expression of capsid precursor protein: in the first, a hybrid promoter (PGADH) was used for high-level expression, while the second, PGAL1, produced substantially lower levels of the virus capsid protein precursors. An increase in the level of HaSV capsid protein precursor (p71) via the PGADH promoter resulted in a dramatic increase in VLP assembly as compared with the PGAL system. A protein equivalent to the mature capsid protein (p64) appeared at later time intervals following induction of transcription. Transmission electron microscopic studies showed that p64 correlated with the presence of mature VLPs as opposed to procapsids in cells containing p71. This confirmed that the presence of p64 denoted maturation of VLPs in vivo. Further investigation indicated that maturation correlated with cell aging and the onset of apoptosis. It was shown that induction of apoptosis resulted in VLP maturation while inhibition of apoptosis prevented maturation. These results suggested that the process of apoptosis might be the trigger for maturation of virus procapsids in their host cells. The increase in the efficiency of VLP assembly observed in the high-level expression system was proposed to be due to an increase in the cellular concentrations of viral RNA. To test this hypothesis, HaSV P71 was co-expressed with either P71 mRNA or full length RNA2. An increase in the solubility of p71 was observed in cells expressing increased levels of both RNAs, but there was no increase in the efficiency of VLP assembly. Northern analysis of encapsidated RNAs revealed that there was no selective encapsidation of either P71 mRNA or viral RNA2. This data indicated that the increase in viral RNA was not the reason for increased efficiency of VLP assembly, but most likely resulted from higher concentrations of p71 itself. It was decided to determine whether a highly efficient nodavirus replication system developed in yeast for heterologous production of proteins, could be used as a method for expressing the capsid protein precursor. The aim of using this system was to determine if VLPs assembled in a replication system specifically encapsidated viral RNA. Transcripts encoding the NwV capsid protein precursor (p70) were generated in yeast cells by replication of a hybrid RNA template by the Nodamura virus (NoV) replicase. Western analysis confirmed the presence of p70 as well as a protein of 62 kDa corresponding to the mature NwV capsid protein. Northern analysis of purified VLPs showed that NoV RNA1 and RNA3 were encapsidated, but no RNA2 was detected. Taken together, the data lead to the conclusion that specific encapsidation of tetraviral RNAs required more than close proximity of the viral RNAs and assembling virus-like particles. Encapsidation specificity in the omegatetraviruses may require additional viral proteins such as p17 during encapsidation or specific viral RNA encapsidation was replication-dependent. Replication-dependent assembly has been shown in the nodaviruses.

The final step of paramyxovirus infection requires the assembly of viral structural components at the plasma membrane of infected cells followed by budding of virions. While the matrix (M) protein of some paramyxoviruses has been suggested to play a central role in the assembly and release of virus particles, the specific viral and host protein requirements are still unclear. Using Newcastle disease virus (NDV) as a prototype paramyxovirus, we explored the role of each of the NDV structural proteins in virion assembly and release. For these studies, we established a virus-like particle (VLP) system for NDV. The key viral proteins required for particle formation and the specific viral protein-protein interactions required for assembly and release of particles were explored in chapter 2. First we found that co-expression of all four proteins resulted in the release of VLPs with densities and efficiencies of release (1.18 to 1.16 g/cm3and 83.8%±1.1, respectively) similar to that of authentic virions. Expression of M protein alone, but not NP, F-K115Q or HN proteins individually, resulted in efficient VLP release. No combination of proteins in the absence of M protein resulted in particle release. Expression of any combination of proteins that included M protein yielded VLPs, although with different densities and efficiencies of release. To address the roles of NP, F and HN proteins in VLP assembly, the interactions of proteins in VLPs formed with different combinations of viral proteins were characterized by co-immunoprecipitation. The co-localization of M protein with cell surface F and HN proteins in cells expressing all combinations of viral proteins was characterized. Taken together, the results show that M protein is necessary and sufficient for NDV budding. Furthermore, they suggest that M protein – HN protein and M protein - NP interactions are responsible for incorporation of HN protein and NP proteins into VLPs and that F protein is incorporated indirectly due to interactions with NP and HN protein. Since the vacuolar protein sorting (VPS) system is involved in the release of several enveloped RNA viruses, chapter 3 describes studies which explored the role of the VPS system on NDV particle release. First, we characterized the effects of three dominant negative mutant proteins of the VPS pathway on particle release. Expression of dominant negative mutants of CHMP3, Vps4 and AIP1 proteins inhibited M protein particle release as well as release of complete VLPs. Mutation of a YANL sequence in the NDV M protein to AANA inhibited particle release while replacement of this sequence with either of the classical late domain motifs, PTAP or YPDL, completely restored particle release. The host protein AIP1, which binds YXXL late domain sequences, is incorporated into M protein particles. These results suggest that an intact VPS pathway is necessary for NDV VLP release and that the YANL sequence is an NDV M protein L domain. The sequence and structure of the Newcastle disease virus (NDV) fusion (F) protein are consistent with its classification as a type 1 glycoprotein. We have previously reported, however, that F protein can be detected in at least two topological forms with respect to membranes in both a cell-free protein synthesizing system containing membranes as well as infected COS-7 cells (J. Virol. 2004 77:1951). One form is the classical type 1 glycoprotein while the other is a polytopic form in which approximately 200 amino acids of the amino terminal end as well as the cytoplasmic domain (CT) are translocated across membranes. Furthermore, we detected CT sequences on surfaces of F protein expressing cells and antibodies specific for these sequences inhibited red blood cell fusion to HN and F protein expressing cells suggesting a role for surface expressed CT sequences in cell-cell fusion. In chapter 4, we extended these findings and found that the alternate form of the F protein can also be detected in infected and transfected avian cells, the natural host cells of NDV. Furthermore, the alternate form of F protein was also found in virions released from both infected COS-7 cells and avian cells by Western analysis. Mass spectrometry confirmed its presence in virions released from avian cells. Two different polyclonal antibodies raised against sequences of the CT domain of the F protein slowed plaque formation in both avian and COS-7 cells. Antibody specific for the CT domain also inhibited single cycle infections as detected by immunofluorescence of viral proteins in infected cells. The potential roles of this alternate form of the NDV F protein in infection are discussed. Virus-like particles (VLPs) generated from different viruses have been shown to have potential as good vaccines. Chapter 5 explored the potential of NDV VLPs as a vaccine for NDV or as a vaccine vector for human pathogens. Significant quantities of NDV VLPs can be produced from tissue culture cells. These VLPs are as pure as virions prepared in eggs. In addition, some rules for incorporation of viral proteins into VLPs were also explored. We found that the cytoplasmic domain of the fusion (F) protein is necessary for its incorporation into VLPs. We found that an HN protein with an HA tag at its carboxyl terminus was incorporated into VLPs. We also found that the HN and F proteins of NDV, strain B1, can be incorporated into VLPs with M and NP of strain AV. The demonstration of specific domains required for protein incorporation into particles is important in using NDV VLPs as a vaccine vector for important human pathogens. In conclusion, this dissertation presents results that show that the M protein plays a central role in NDV assembly and release, a finding that is consistent with findings with other paramyxoviruses. More importantly, this work extends the current knowledge of paramyxovirus assembly and release by providing the first direct evidence of interactions between paramyxovirus proteins. These interactions between viral proteins provide a rational basis for incorporation of viral proteins into particles. This work also provides a clearer understanding of the role of the host vacuolar protein sorting machinery in NDV budding. A clear understanding of virus assembly and budding process contributes to the design of strategies for therapeutic intervention and in the development of safer, more economical and effective vaccines.

The spread amongst humans of viral diseases such as acquired immunodeficiency syndrome (AIDS), hepatitis and severe acute respiratory syndrome (SARS) is alarming. A plant-based high fidelity production system is being developed with emphasis on producing antigens capable of being orally delivered to humans in plant packets. To test whether transgenic tobacco, carrot and rice plants can correctly process and assemble the hepatitis-B virus (HBV) core particle/antigen (HBcAg), they were transformed with a C-terminal truncated version of the HBcAg subunit coding sequence. Transgenic tobacco, carrot and rice plants processed the HBV subunits accurately indicating that these recombinant expression systems can be extended to produce other proteins at reduced costs. In the wild-type expression construct (H1); the enhanced cauliflower mosaic virus double 35S (CaMV-d35S) promoter was fused to the alfalfa mosaic virus RNA 4 (AMV-RNA4) sequence to achieve greater translation of a C-terminal truncated HBV core particle subunit. A second expression construct (H2) was plant-codon optimized to match the Arabidopsis thaliana plant genome codon preferences. A third codon-optimized expression construct (H3) had a KDEL (lysyl-aspartyl-glutamyl-leucine) encoded sequence. While a fourth expression construct (H4) included an extensin signal sequence in place of the AMV-RNA4 sequence. Western blotting analysis showed the presence of the HBcAg in transgenic tobacco, carrot and rice plants. The HBcAg levels increased from the H1 to the H4 transgenic tobacco lines. Plant codon-optimization of the HBcAg sequence and addition of the KDEL encoded sequence led to higher levels of HBcAg. The most effective modification was observed when the extensin signal sequence replaced the AMV-RNA4 translation enhancer sequence resulting in the highest observed yields of HBcAg in both the leaves and seeds of the best H4 tobacco plant. In edible plants, higher levels of HBcAg were observed in carrot roots as opposed to carrot leaves and in rice seeds as opposed to rice leaves. Further analyses via electron microscopy indicated that the HBV subunits had assembled into virus-like particles of 25--30 nm diameter in all three plant systems. Therefore, these studies may aid in the global quest to develop cheap, safe and effective vaccines.

Dans cette thèse, l’auto-assemblage en molécules colloïdales de virus en forme de filament, les bactériophages M13, est étudié. Comme première approche, l’affinité de la streptavidine pour la biotine ou un Strep-tag est utilisée et quantitativement comparée. Pour ce faire, des virus modifiés génétiquement, M13-AS, présentant des Strep-tag et des virus M13C7C chimiquement bioconjugués par de la biotine ont réagi via leur extrémité proximale avec des nanoparticules fonctionnalisées par de la streptavidine. Il en résulte la formation de molécules colloïdales en étoile, dont la valence ou nombre de virus par structure, peut être simplement contrôlée par l’excès molaire initial. Cependant, la stabilité de ces molécules colloïdales est limitée par la libération progressive et la dégradation de la streptavidine. Nous avons alors développé une seconde approche basée sur l’affinité soufre-métal, qui s’est avérée à la fois pratique expérimentalement et fiable. Grâce aux groupements disulfures présents sur les cystéines de la protéine P3, des nanoparticules métalliques peuvent se lier à l’extrémité des virus. Le caractère générique de cette méthode est vérifié en faisant varier la nature du métal des nanoparticules ainsi que la souche des virus, dont la sauvage. La valence des structures formées est déterminée en fonction de plusieurs paramètres, dont l’excès molaire initial, la taille des nanoparticules et la force ionique. Un modèle rendant compte des résultats expérimentaux a été élaboré, dont les principales variables sont la surface des nanoparticules et le diamètre effectif électrostatique des virus. Cette approche est étendue à la réalisation de diblocs colloïdaux hétéro bifonctionnels, utilisant les virus comme briques constitutives. Comme preuve de concept, des diblocs bicolores à base de virus sont obtenus par auto-assemblage et leur dynamique est étudiée à l’échelle du bloc élémentaire en microscopie optique de fluorescence. Ainsi, nous avons montré dans cette thèse la réalisation par auto-assemblage d’une nouvelle génération de molécules colloïdales, dont l’auto-organisation peut conduire à la formation de superstructures hiérarchiques hybrides de complexité croissante, potentiellement utiles en sciences des matériaux<br>In this thesis, the self-assembly of rod-like viral particles, specifically the M13 bacteriophages, into colloidal molecules is studied. As the first method, the affinity of streptavidin to biotin or Strep-tag is used and quantitatively compared. In this case, both biologically engineered M13-AS displaying Strep-tags and chemically biotinylated M13C7C viruses have reacted with streptavidin activated nanoparticles via their functionalized proximal ends. This results in star-like colloidal molecules, whose valency – or number of viruses par structure – can be solely controlled by tuning the initial molar excess. However, the stability of these colloidal molecules is limited by streptavidin release and degradation. Thus, we develop the second method based on the sulfur—metal interactions, which is more convenient and reliable. Thanks to the exposed disulfide groups located at p3 proteins, metallic nanoparticles are able to bind to proximal ends of the M13 virus. The generic feature of this method is verified by using different metals and two virus strains including wt-M13. Afterwards, the control of the valency is explored by varying the initial molar excess, the nanoparticle size and the ionic strength. A quantitative model is built correspondingly, using the surface area of Au nanobead and the effective electrostatic diameter of the virus as variables, which accounts for the assembly of colloidal molecules with desired valencies. This method is further applied to assemble heterobifunctional diblocks by using filamentous viruses as building units. As a proof-of-concept experiment, bicolored diblocks are produced and tracked by each block simultaneously. Overall, we demonstrate the synthesis of a new generation of hybrid colloidal molecules, whose self-organization could serve as a promising means to create novel hierarchical biologic/inorganic superstructures that may find applications in materials science

Virion assembly and disassembly are crucial aspects of the virus multiplication cycle, however, relatively little is known about these processes in plant viruses. While the former helps to produce multiple copies of stable infectious progeny virions, the latter is required for release of the encapsidated viral genome into a host cell for initiating new rounds of virus multiplication. In this thesis, I aimed to study Cucumber necrosis virus (CNV) particle assembly and disassembly and the role of CNV coat protein (CP) and host HSP70 homologs in these processes. It was found that CNV infection of Nicotiana benthamiana causes a significant upregulation of HSP70 homologs, and that, in turn, HSP70 is co-opted by the virus at several stages of the multiplication cycle to promote various aspects of the infection cycle including viral RNA, CP and particle accumulation. HSP70 homologs were also found to assist CNV CP in chloroplast targeting possibly to attenuate chloroplast-mediated plant defence and thereby allow further spread of the virus. It was also determined that the HSP70 homologue, Hsc70-2 is bound to CNV virions and that this association appears to facilitate the uncoating efficiency of CNV particles likely via triggering a conformational change in particles. This is the first report that a plant virus utilizes HSP70 homologs for disassembly. A highly basic “KGRKPR” sequence in the ε-region of the CNV CP arm was also examined for its role in virion assembly and encapsidation of viral RNA. Through mutational analysis, it was found that the basic residues promote T=3 versus T=1 virion formation and encapsidation of full-length viral RNA in vivo. Moreover, mutants lacking 2-4 of the basic residues encapsidated proportionately greater amounts of host RNA suggesting the role of these basic residues in selection of viral RNA during assembly. It was also shown that heat shock enhances transcription of heat-inducible ONSEN-like retrotransposons known to be induced during CNV infection. Since retrotransposons are known to play an important role in genome variation, the described studies may be helpful in understanding the importance of plant viruses in inducing genome variation and perhaps adaptation of plants to changes in the environment.<br>Land and Food Systems, Faculty of<br>Graduate

Nanotechnology and its potential for biomedical applications has become an area of increasing interest over the last few decades. Specifically, ultrasmall nanoparticles, ranging in size from 5 to 50 nm, are highly sought after for their physical and chemical properties and their ability to be easily transmitted though the bloodstream. By adjusting the material properties, size, surface potential, morphology, surface modifications, and more, of nanoparticles, it is possible to tailor them to a specific use in biomedical areas such as drug and gene delivery, biodetection of pathogens or proteins, and tissue engineering. The aim of this study was to fabricate ultrasmall poly-(lactic-co-glycolic acid) nanoparticles (PLGA NPs) using a quick and easy nanoprecipitation method1, with some modifications, for general use in various biomedical areas. Nanoprecipitation of two solutions – PLGA dissolved in acetonitrile and aqueous poly(vinyl alcohol) (PVA) – at varying concentrations produced ultrasmall nanoparticles that range in size, on average, from 10 to 30 nm. By the data collected from this study, a selection method can be used to choose a desired PLGA nanoparticle size given a potential biomedical application. The desired nanoparticle can be fabricated using specific concentrations of the two nanoprecipitation solutions. Size of the ultrasmall PLGA NPs was characterized by dynamic light scattering (DLS) and confirmed by transmission electron microscopy (TEM). Spherical morphology of the PLGA NPs was also proved by TEM. By generalizing the ultrasmall PLGA NP fabrication process, the idea is that these NPs will be able to be used in various biomedical applications depending on the goal of the furthered study. As an example of potential application, ~15 to 20 nm PLGA NPs were consistently fabricated for use as virus-like particle (VLP) scaffolds. Following formation, PLGA NPs were introduced to modified human papillomavirus (HPV) protein during protein refolding and assembly into virus-like particles (VLPs) via buffer exchange. The size of the VLPs was monitored with and without PLGA nanoparticles present in solution during the refolding process and TEM images were collected to confirm encapsulation.<br>Master of Science<br>Nanotechnology, the manipulation of materials on an atomic or molecular scale, and its potential for biomedical applications has become an area of increasing interest over the last few decades. Nanoparticles, spherical or non-spherical entities of sizes approximately one-billionth of a meter, have been used to solve a wide variety of biomedical problems. For reference, a human hair is about 80,000 to 100,000 nm in size and the nanoscale typically ranges in size from 1 to 1000 nm. This size range is not visible to the naked eye, so methods of analysis via scientific equipment becomes paramount. Specifically, this study aims to fabricate ultrasmall nanoparticles, ranging in size from 5 to 50 nm, which are highly sought after for their physical and chemical properties and their ability to easily travel though the bloodstream. By adjusting the material properties, size, shape, surface charge, surface modifications, and more, of nanoparticles, it is possible to tailor them to a specific use in biomedical areas such as drug delivery, detection of viruses, and tissue engineering. The specific aim of this study was to fabricate ultrasmall poly-(lactic-co-glycolic acid) nanoparticles (PLGA NPs), a type of polymer, using a quick and easy nanoprecipitation method1, with some modifications. Nanoprecipitation occurs by combining two liquid solutions – PLGA and aqueous poly(vinyl alcohol) (PVA) – which interact chemically to form a solid component – a polymer nanoparticle. These two solutions, at varying concentrations, produced ultrasmall nanoparticles that range in size, on average, from 10 to 30 nm. Data collected from this study can be used to select a desired nanoparticle size given a potential application. The desired nanoparticle can be fabricated using specific concentrations of the two nanoprecipitation solutions. By generalizing the ultrasmall PLGA NP fabrication process, the idea is that these NPs can be used for a variety of biomedical applications depending on the goal of the furthered study. Two PLGA NP example applications are tested for in this work – in DNA loading and in encapsulation of virus-like particles (VLPs), which are synthetically produced proteins that can be neatly folded to resemble a virus. These VLPs can be used to as an alternative to live vaccines and they can be designed to stimulate the immune system. Positive initial results from this study confirm the potential of these nanoparticles to have a wide impact on the biomedical field depending on specific tailoring to a given application.

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<br>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

博士<br>國立成功大學<br>基礎醫學研究所<br>102<br>Hepatitis C virus (HCV) infection not only induces hepatic diseases but also leads to disorder of lipid and glucose metabolism. HCV depends on lipid droplets (LDs) for viral particle assembly and very-low density lipoproteins (VLDLs) for virion egression. However, the components and locations for this process remain unidentified. Apolipoprotein J (apoJ) serves as a Golgi-resident molecular chaperone which can be upregulated by glucose, and is secreted to extracellular space along with the route of VLDLs. This study investigated the effects of apoJ on HCV life cycle. HCV infection could elevate intracellular apoJ expression in primary human hepatocytes. Silencing of apoJ expression by siRNA strategy reduced intracellular and extracellular HCV infectivity and extracellular HCV RNA in HCV-infected Huh7.5 hepatoma cells, whereas intracellular HCV RNA was accumulated in HCV-infected cells. ApoJ was shown to interact with HCV core and NS5A proteins by immunoprecipitation and could further stabilize the protein complex of HCV core and NS5A. HCV infection facilitated the dispersion of intracelluar apoJ along with Golgi to encircle LDs, and the dispersed apoJ colocalized with the core, NS5A, HCV RNA, LDs, endoplasmic reticulum (ER), Golgi, and ER-Golgi membrane contact site. Furthermore, the interplay among glucose, apoJ and HCV particle production was investigated. Serum apoJ was positively correlated with fasting blood glucose concentration and HCV RNA titer in chronic hepatitis C patients. Increase of glucose concentration in culture medium of HCV-infected Huh7.5 cells could upregulate apoJ expression. In conclusion, the glucose-stimulated apoJ protein facilitates infectious HCV particle assembly via stabilization of core-NS5A interaction which encircles LDs and locates at the ER-Golgi membrane contact site.

碩士<br>國立陽明大學<br>公共衛生研究所<br>100<br>Background: Coronavirus encodes four structural proteins, i.e. membrane (M), nucleocapsid (N), spike (S) and envelope (E). A number of studies have indicated that the M protein plays a key role in virion assembly and it can secret into medium as membrane-enveloped vesicles. M coexpression with N or E can lead to formation of virus-like particles (VLPs). Objective: This study aims to map the domain functionally involved in severe acute respiratory syndrome coronavirus (SARS-CoV) M and human coronavirus 229E (HCoV-229E) M self-assembly and M-N interaction. Methods: SARS-CoV/229E and 229E/SARS-CoV M chimeras were constructed by swapping the correspending domains between SARS-CoV and HCoV-229E M. Each of the M mutants was transiently expressed alone or co-expressed with N. The release efficency of VLPs was assessed by Western blot. The M-N interaction domain was determined by co-ip assay. The localization of M mutants was observed by fluorescence confocal microscopy. Results: Results suggest that transmembrane domain deletion impaired M self-assembly and release, but domain swapping did not. 229E/SARS-CoV chimera containing the 229E M first transmembrane domain co-loclized with wild-type 229E M in both plasma membrane and perinuclear areas. However results of western blot suggest that transmembrane domain swapping does not significantly affect M-N interaction. Conclusions: These results suggest that transmembrane domain swapping between SARS-CoV and HCoV-229E M has no detrimental effects on M self-assembly and release, and it does not significantly affect M-N interaction either.

碩士<br>國立陽明大學<br>公共衛生研究所<br>101<br>HIV-1 protease (PR) is encoded by pol, which is initially translated as a Pr160gag-pol polyprotein by a ribosomal frameshift event. Pr160gag-pol is incorporated into virions via interactions with assembling Pr55gag. The PR-mediated proteolytic cleavage of Pr55gag and Pr160gag-pol, known as virus maturation, is essential for the acquisition of viral infectivity. Within the Gag-Pol, the p6gag is truncated and is replaced by a transframe domain referred to as p6* or p6pol. Removal of p6pol improves Gag-Pol autoprocessing, suggesting that p6pol is involved in regulation of PR activation. However, overlapping of p6gag/p6pol reading frame hampers generic approach to studying p6pol biological function. To assess the p6pol contribution to PR-mediated virus maturation without affecting p6gag reading frame, we introduced an extra copy of p6pol-PR or PR coding sequence at the PR C-terminus. Each of the constructs was transiently expressed in 293T cells, and virus assembly and processing were analyzed by Western blot. The results indicate that HIV-1 mutants containing tandem repeat PR domains were severely defective in virus particle production due to enhanced Gag cleavage. Placement of p6pol between the tandem repeat PR domains resulted in diminished Gag cleavage efficiency. Inactivation of the proximal PR affects Gag cleavage efficiency at a greater extent than inactivation of the distal PR. Our study indicates that the Gag cleavage enhancement effect incurred by over-expressed HIV-1 PR is reduced following the placement of p6pol between the tandem repeat PR domains. This supports the proposal that p6pol plays a negative role in the process of PR activation.

碩士<br>中山醫學院<br>醫學研究所<br>89<br>Abstract The human polyomavirus, JC virus, contains three capsid proteins, VP1, VP2, VP3 and a viral minichromosome. Interactions of these three capsid proteins for virus assembly are not well understood. In the current study, the major capsid protein VP1 and minor capsid protein VP2 of JC virus, have been cloned and expressed in yeast cells. Yeast expression system was employed to co-express VP1 and VP2 proteins for facilitating understanding the function of minor structural protein VP2. When VP1 and VP2 gene were co-transformed into yeast cells, both proteins were expressed and detected by their own monospecific antibodies. VP1 and VP2 proteins co-expressed in yeast were able to cause hemagglutination. Self-assembled capsid-like particles were purified by 10-50% sucrose gradient centrifugation and morphology of the particles were observed by electron microscopy. The VP1 capsid could be disrupted into pentameric capsomeres in the presence of both EGTA and DTT but the VP1VP2 capsid was more resistant to the disruption at the same conditions. These findings indicate that VP2 may play a role in stablizing capsid structure. Furthermore, the efficiency of DNA packaging and delivery into monkey kidney COS-7 cell of VP1VP2 capsid-like particles was increased. The yeast co-expression system will be further employed to investigate protein-protein and DNA-protein interactions during virus maturation.

碩士<br>國立中興大學<br>農業生物科技學研究所<br>90<br>Abstract Gene encoding a structural protein (VP2) of infectious bursal disease virus (IBDV) was cloned and expressed using the host, Escherichia coli (E. coli), to investigate its capability of the formation of particulate structure. Although the previous attempt to produce unfused VP2 in E. coli was reported to be unsuccessful, in this report, the soluble E. coli-derived rVP2 (erVP2) proteins were found to form particles of approximately 20 nm in diameter. Those particles were partially purified employing CsCl density gradient ultracentrifugation, and confirmed by direct observation under the electron microscope. To facilitate the purification of the particles, the VP2 protein was incorporated a metal ion binding site (His)6 at its C-terminus. The chimeric erVP2H proteins also formed particles, which could be affinity-purified in one step with immobilized metal ions (Ni+2). However, the chimeric erVP2H particles have a slightly smaller particulate size, approximately, 15 nm. The solubility of erVP2H is extremely low in the strain of BL21(DE3)pLysS and it was expressed with a limited amount in the strain of BL21(DE3)CodonPlus-RP. Besides, two C terminal deletion mutant proteins of VP2, 1167H and 1197H protein also failed to form particles. Thus, the erVP2 protein was selected to express in a laboratory-scale fermentor. In conclusion, the most significant finding in this work is that both of the expressed erVP2 and erVP2H proteins can form a particulate structure, which is not found previously and is believed to induce a strong immunological response in a vaccinated chicken.

博士<br>國立陽明大學<br>生化暨分子生物研究所<br>96<br>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.

碩士<br>國立中興大學<br>生物科技學研究所<br>103<br>Foot-and-mouth disease virus (FMDV) is the causative agent of the acute and highly contagious foot and mouth disease (FMD). The symptoms are blisters on the skin of foot and mouth. FMD affects the developing of livestock industry significantly and causes the economic loss around the world. Conventional FMD vaccines are based on the chemically inactivated virus, which induce neutralizing antibodies, control disease and protect from FMD infection. However, the disadvantage of incompletely inactivated vaccine would contain live viral residues causing outbreak of disease. Hence, developing a safe, valid and inexpensive FMD subunit vaccine is urgent. FMDV VP1 is the major epitope, which can be recognized by immune system and elicit the immunogenic response while infected with virus. Escherichia coli is one of the most extensive protein expression system for its advantages of fast growth, easily manipulated, high protein yields and low cost. In the previous study, we found that purified wild type Bamboo mosaic virus (BaMV) coat protein (CP) from E. coli could self-assembled into virus-like particles (VLPs) in 10 mM 2-(N-morpholino) ethanesulfonic acid buffer (MES buffer, pH 6.0). The VLPs are about 500nm in length. In this study, we generated pET expression plasmids designated as pBVP1 97 Nd35mCP and pBVP1 Manisa Nd35mCP by replacing 35 amino acids of BaMV CP at N-terminal with 37 amino acids of VP1 epitopes of FMDV 97 or Manisa subtype. The recombinant fusion proteins expressed in E. coli produced in high-yield and in a soluble form. The recombinant proteins were purified by Phenyl、DEAE、S200 column consecutively. In order to test whether in vitro expressed BVP1 could self-assemble into VLPs in the absence of BaMV genome, the recombinant proteins were incubated in different pH, ionic strength buffer conditions and in the different temperature. The TEM results revealed that BVP1 formed into VLPs. Compare to the filamentous VP1 Nd35mCP purified from plants, both VP1 97 mCP and VP1 Manisa mCP formed about 100 nm~200 nm rod-shaped VLP. The mentioning VLPs as a safe and enhanced immunogenic subunit vaccine to protect pigs from FMD will be tested in the future.

博士<br>國立中山大學<br>海洋資源學系研究所<br>96<br>Piscine nodaviruses are members of genus Betanodavirus, which infect more than 30 species of fish and cause massive mortality in larvae and juveniles. The infection causes great economic losses to aquaculture and sea-ranching. To study the dissociation and reassembly of betanodavirus, virus-like particles (VLPs) of dragon grouper nervous necrosis virus (DGNNV) were used. The experiments with calcium-chelating or reducing/oxidizing reagents elicited that the DGNNV VLPs required only calcium for particle assembly. With the recombinant VLPs, site-directed mutagenesis can be employed to investigate the roles of calcium-binding ligands in particle formation. In the mutational analysis of DxxDxD that is putatively involved in the coordination of calcium ions, the results showed that the D133N mutation significantly disrupted the assembly of VLPs while D130N and D135N mutants produced heterogeneous particles with broken shapes. The thermal stability of the VLP-forming fractions demonstrated that VLPs of D135N mutant were stable at a temperature of 85°C, which is slightly higher than that for wild-type, whereas VLPs of D130N mutant could not tolerate the thermal effects at a temperature higher than 60°C. It is deduced that three aspartate residues of the motif DxxDxD are all important for the efficient formation of DGNNV VLPs and, among them, the DxxD provides a more stable coordinate of calcium-ligand than DxD.

博士<br>國立中興大學<br>生物科技學研究所<br>92<br>VP2 is a major structural protein of infectious bursal disease virus(IBDV). It has been demonstrated as the major host-protective immunogen of IBDV and contains the antigenic regions responsible for eliciting the virus neutralizing antibodies in the host. In this study, the precursor protein (VPX) of infectious bursal disease virus (IBDV) host immunogen VP2 protein was expressed in insect cells, including Sf9 and Hi-5 cells, to examine its regenerated particle types and the immunogenicity induced by those particles. Since the expressed protein, VPXH, was engineered a His-tag, consisting of six histidine residues, at their C-terminal end. When expressed in Hi-5 cells, rVPXH was efficiently processed at its C-terminus by cellular proteases to yield VP2-like proteins whose molecular weight was similar to that of VP2. However, proteolytical processing of VPXH in Sf9 cells was hampered. The expressed rVPXH was purified using immobilized metal-ion affinity chromatography (IMAC). Under TEM observation of Ni-NTA purified VPXH, at least three architectures of particles were observed, including the tubular structure and two of spherical structure of isometric particle structure and a new one of icosahedral particles, with a size of approximately 20-25nm and 30-35 nm in diameter, respectively. After separation of rVPXH formatted particles, chromatographic results indicate that the expressed rVPXH protein and very few of VP2-like protein formed isometric particle structure and very few of twisted tubular structure, as well as icosahedral particles formed by the degraded products of rVPXH protein, VP2-like protein. Finally, we also demonstrated that when susceptible chickens were vaccinated with the IMAC-purified rVPXH protein (40 g/bird), virus-neutralizing antibodies were induced. This indicated that those particles are highly immunogenic. Based on our results, we found that Hi-5 harbors excellent ability of proteolytic cleavage of VPX. Therefore, this study our effort also to investigate the proteolytic processing of IBDV polyprotein in insect cell. When IBDV polyprotein was expressed in insect cells, higher productivity of IBDV VLP was observed in Hi-5 cells. Moreover, the accumulation of matured VP2 and VLPs assembly was exhibited rather efficiently than in Sf9. In addition to IBDV-like particles assembled in Hi-5 cells, some of particulated subviral particles with 23 nm in diameter were also assembled. Chromatographic results show that the IBDV subviral particle was formed by VP2 protein. When a higher multiplicity of infection(MOI) strategy was used, accumulation of VP2 protein is more significantly. The excess VP2 protein resulted in formation of subviral particles. However, at low MOI, the relative productivity of IBDV VLP and subviral particles increased in batch culture. Our results demonstrate that Hi5, harboring excellent ability of proteolytic cleavage of recombinant protein, the efficiency of IBDV-like particles production is superior to Sf9 culture. These finding therefore may provide a methodological improvement using Hi-5 cells for optimal production of IBDV VLP as an effective IBDV vaccine against infectious bursal disease or as for crystallization to study IBDV structure.

碩士<br>國立清華大學<br>分子與細胞生物研究所<br>92<br>ABSTRACT Baculovirus has been employed as an efficient gene delivery vector into mammalian cells for a variety of purposes. In this study, we further expanded the applications of baculovirus as a tool to study the assembly of hepatitis delta virus-like particle. To this end, two recombinant baculoviruses were constructed to express large hepatitis delta antigen and hepatitis B surface antigen under the regulation of mammalian promoters. Simple and efficient gene transduction into hepatoma cell lines (80-90% as determined by flow cytometry) and high level transgene expression (in the order of microgram per million cells) were achieved by incubating the cells with unconcentrated virus supplemented with Dulbecco’s phosphate-buffered saline at 25 �aC for 6 h. Quantitative real-time PCR (Q-PCR) analyses quantitatively revealed that baculovirus transduction was more efficient than plasmid transfection with respect to DNA uptake and DNA transport to the nucleus, even for cells grown to near confluency. HDV antigen expression was enhanced by EGTA pretreatment and addition of sodium butyrate in culture medium and prolonged by superinfection. L-HDAg was correctly isoprenylated and localized to nucleus of the transduced cells as the authentic protein. Upon co-transduction, HDV-like particle could be assembled and secreted into medium, which were further purified from the culture medium by isopycnic ultracentrifugation and visualized by electron microscopy. This study demonstrated that baculovirus transduction is a suitable method alternative to plasmid transfection for efficient formation of HDV-like particles. This system may be also a useful tool in investigating cellular processes involved in HDV assembly and in producing large amount of HDV-like particles in bioreactor for medical research.