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1
Read, Eliot Keith Curtis. "Investigating influenza A virus RNA trafficking." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609127.
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Xiao, Wu. "Intracellular trafficking of adeno-associated virus type 2." [Gainesville, Fla.] : University of Florida, 2002. http://purl.fcla.edu/fcla/etd/UFE1001195.
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Muenzner, Julia. "Viral subversion of host cell membrane trafficking." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267890.
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Enveloped viruses acquire their membrane coat from the plasma membrane or intracellular organelles and rely on cellular machinery to facilitate envelopment and egress of virus progeny. This thesis examines egress-related interactions between host cell factors and proteins of two different enveloped viruses: hepatitis D virus (HDV) and herpes simplex virus 1 (HSV-1). HDV is a small RNA virus causing fulminant hepatitis or severely aggravating cirrhosis and hepatocellular carcinoma. HSV-1 is a large DNA virus infecting epithelial and neuronal cells. Infection with HSV-1 not only triggers the development of recurring sores on oral or genital mucosa, but can also cause severe disease in neonates and immunocompromised patients. The interaction between the large antigen of HDV (HDAg-L) and the N-terminal domain (NTD) of clathrin, a protein crucial for endocytosis and intracellular vesicular trafficking, was examined by structural, biochemical and biophysical techniques. Co-crystal structures of NTD bound to HDAg-L peptides derived from different HDV genotypes revealed that HDV interacts with multiple binding sites on NTD promiscuously, prompting re-evaluation of the binding between cellular peptides and NTD. Surprisingly, co-crystal structures and pull-down capture assays showed that cellular peptides containing clathrin-binding motifs can also bind multiple sites on the surface of NTD simultaneously. In addition, the structures of viral and cellular peptides bound to NTD enabled the molecular characterization of the fourth peptide binding site on NTD, the “Royle box”, and led to the identification of a novel binding mode at the “arrestin box” peptide binding site on NTD. The work in this thesis therefore not only identifies the molecular basis of HDV:clathrin interactions, but also furthers our understanding of basic clathrin biology. Even though many HSV-1 proteins have been implicated in the envelopment and egress of viral particles, only few interactions between HSV-1 and cellular proteins promoting these processes have been described. Therefore, the HSV-1 proteins gE, UL21 and UL56 were selected and characterized bioinformatically and/or biochemically. Cellular proteins interacting with UL56 were identified by yeast two-hybrid screening and quantitative mass spectrometry. Co-immunoprecipitation and pull-down experiments confirmed the Golgi-trafficking protein GOPC, components of the mammalian trafficking protein particle complex, and the ubiquitin ligase NEDD4 as novel binding partners of UL56, thereby suggesting exciting new avenues for the investigation of cellular mechanisms contributing to HSV-1 envelopment and egress.
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Price, Philip John Ritchie. "Leukocyte trafficking during infection with modified vaccinia virus Ankara." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-173737.
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Berka, Ursula, Martin Volker Hamann, and Dirk Lindemann. "Early Events in Foamy Virus - Host Interaction and Intracellular Trafficking." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-127078.
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Here we review viral and cellular requirements for entry and intracellular trafficking of foamy viruses (FVs) resulting in integration of viral sequences into the host cell genome. The virus encoded glycoprotein harbors all essential viral determinants, which are involved in absorption to the host membrane and triggering the uptake of virus particles. However, only recently light was shed on some details of FV’s interaction with its host cell receptor(s). Latest studies indicate glycosaminoglycans of cellular proteoglycans, particularly heparan sulfate, to be of utmost importance. In a species-specific manner FVs encounter endogenous machineries of the target cell, which are in some cases exploited for fusion and further egress into the cytosol. Mostly triggered by pH-dependent endocytosis, viral and cellular membranes fuse and release naked FV capsids into the cytoplasm. Intact FV capsids are then shuttled along microtubules and are found to accumulate nearby the centrosome where they can remain in a latent state for extended time periods. Depending on the host cell cycle status, FV capsids finally disassemble and, by still poorly characterized mechanisms, the preintegration complex gets access to the host cell chromatin. Host cell mitosis finally allows for viral genome integration, ultimately starting a new round of viral replication.
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Lindemann, Dirk, Ursula Berka, and Martin Volker Hamann. "Early Events in Foamy Virus - Host Interaction and Intracellular Trafficking." MDPI, 2013. https://tud.qucosa.de/id/qucosa%3A28912.
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Here we review viral and cellular requirements for entry and intracellular trafficking of foamy viruses (FVs) resulting in integration of viral sequences into the host cell genome. The virus encoded glycoprotein harbors all essential viral determinants, which are involved in absorption to the host membrane and triggering the uptake of virus particles. However, only recently light was shed on some details of FV’s interaction with its host cell receptor(s). Latest studies indicate glycosaminoglycans of cellular proteoglycans, particularly heparan sulfate, to be of utmost importance. In a species-specific manner FVs encounter endogenous machineries of the target cell, which are in some cases exploited for fusion and further egress into the cytosol. Mostly triggered by pH-dependent endocytosis, viral and cellular membranes fuse and release naked FV capsids into the cytoplasm. Intact FV capsids are then shuttled along microtubules and are found to accumulate nearby the centrosome where they can remain in a latent state for extended time periods. Depending on the host cell cycle status, FV capsids finally disassemble and, by still poorly characterized mechanisms, the preintegration complex gets access to the host cell chromatin. Host cell mitosis finally allows for viral genome integration, ultimately starting a new round of viral replication.
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Lindemann, Dirk, Ursula Berka, and Martin Volker Hamann. "Early Events in Foamy Virus - Host Interaction and Intracellular Trafficking." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-178848.
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Here we review viral and cellular requirements for entry and intracellular trafficking of foamy viruses (FVs) resulting in integration of viral sequences into the host cell genome. The virus encoded glycoprotein harbors all essential viral determinants, which are involved in absorption to the host membrane and triggering the uptake of virus particles. However, only recently light was shed on some details of FV’s interaction with its host cell receptor(s). Latest studies indicate glycosaminoglycans of cellular proteoglycans, particularly heparan sulfate, to be of utmost importance. In a species-specific manner FVs encounter endogenous machineries of the target cell, which are in some cases exploited for fusion and further egress into the cytosol. Mostly triggered by pH-dependent endocytosis, viral and cellular membranes fuse and release naked FV capsids into the cytoplasm. Intact FV capsids are then shuttled along microtubules and are found to accumulate nearby the centrosome where they can remain in a latent state for extended time periods. Depending on the host cell cycle status, FV capsids finally disassemble and, by still poorly characterized mechanisms, the preintegration complex gets access to the host cell chromatin. Host cell mitosis finally allows for viral genome integration, ultimately starting a new round of viral replication.
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Gao, William Ning Da. "Viral and cellular proteins involved in vaccinia virus egress." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/280280.
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Vaccinia virus (VACV) is a large double-stranded DNA virus with a cytoplasmic site of replication. It has a complex life cycle that produces two distinct infectious virion forms, Intracellular Mature Virions (IMVs) and Extracellular Enveloped Virions (EEVs). The host cell microtubule trafficking machinery is hijacked by the virus at three distinct positions of the viral life cycle. After virus entry, the virus cores are transported to pre-nuclear sites where they form viral factories that ultimately produce fully functional and infectious IMVs. A small proportion of IMVs are further transported to sites of wrapping, where they are enveloped by a host-derived double membrane to form Intracellular Enveloped Virions (IEVs). The IEVs are then transported to the cell periphery to facilitate efficient viral spread. The viral proteins A36, F12 and E2 together with the kinesin-1 microtubule motor protein are thought to be involved in IEV egress from the site of wrapping to the cell periphery, although the exact mechanism of movement is unclear. Until recently, A36 was the only known protein to interact with the kinesin-1 motor through kinesin light chain (KLC), but F12 has also been shown to interact with KLC through E2. The precise mechanism of how the IEV interacts with and activates the kinesin-1 motor protein is unclear, and this study explores the interactions of IEV proteins with KLCs in detail, mapping interactions between KLC and A36 or F12/E2. A36, F12 and E2 also show no sequence or predicted structural homology to any other known proteins, and structural studies were performed in an attempt solve their 3D structure. The CRISPR-Cas9 targeted genome editing tool was also utilised to knockout different KLC isoforms in multiple cell lines to assess their contribution to IEV egress as well as cellular trafficking. These studies will provide insight into the mechanisms behind the spatial and temporal control of kinesin motor activity in the cell.
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Haines, Felicity Jade. "Trafficking of GP64 and Virus Egress in AcNPV-infected insect cells." Thesis, Oxford Brookes University, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432736.
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Dudleenamjil, Enkhmart. "BPV Entry and Trafficking in EBTr Cells." BYU ScholarsArchive, 2009. https://scholarsarchive.byu.edu/etd/2301.
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Bovine Parvovirus (BPV) belongs to the genus Bocavirus, family Parvoviridae. BPV is the leading etiologic agent among the pathogens that cause primary gastroenteritis of cattle. Many of the intracellular events associated with virus replication are unknown. In this research project, we investigated BPV internalization into the host cell and trafficking in the cytosol. Preliminarily, EBTr cells had abundant clathrin, virus attached to purified clathrin, and EM micrographs revealed virus in endocytic vacuoles. Assays detecting virus infectivity (i.e. viral protein synthesis), virus production (completion of the replication cycle), and quantitative PCR (qPCR) to detect viral transcripts were used to evaluate virus uptake and subsequent trafficking events in the presence of selective inhibitors. Cell toxicity mediated by the drugs was evaluated by the MTT test. Virucidal effects of the drugs were assessed. A control virus was used to verify the inhibitor technology. Immunofluoresceinated virus particles were found in clathrin-rich early endosomes. Clathrin-mediated endocytosis (CME) was examined by clathrin polymerization inhibiting agent (chloropromazine), lysosomotropic agents (ammonium chloride and chloroquine), a vacuolar ATPase inhibitor (bafilomycin A1), and a blocker of transition between endosomes (brefeldin A). Caveosome pathway inhibitors included phorbol 12-myristate 13-acetate (a suppressor of caveolae formation), nystatin and methyl-beta-cyclodextrin (lipid raft blockers), and genistein (a tyrosine kinase phosphorylation inhibitor). Trafficking of BPV was investigated using specific inhibitors of proteasomal activity, actin-myosin function, and microtubule-dynein function. The proteasomal protease suppressor (lactacystin), and a proteasomal chymotrypsin inhibitor (epoxomicin) were used. The role of actin was probed by cytochlasin D, latrunculin A, and ML-7. The microtubule inhibitors nocodazole, vanadate, and EHNA were used to probe microtubule function. The inhibitors of CME reduced virus production and reduced infectivity, a result confirmed by qPCR. The blockers of caveolin-mediated entry did not interfere with virus production nor virus infectivity. Proteasome activity blockage did not affect the virus replication. But the virus cycle was affected by actin blockage and by microtubule blockage detected by qPCR. Taken together these data indicate that BPV uptake is mediated by clathrin coated pits and is acid-dependent. Further processing of BPV in the cytosol does not require proteasomal enzymes. Actin-associated vesicular transport appears to be essential to virus replication and trafficking to the nucleus appears to be mediated by microtubules.
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Lux, Kerstin. "Visualization of the intracellular trafficking of incoming Adeno-associated virus type 2." Diss., kostenfrei, 2006. http://edoc.ub.uni-muenchen.de/8690/.
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McDermott, Daniel Scott. "Priming and tracking the virus-specific T cell response." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/1882.
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CD4 and CD8 T cells play a vital role in mediating the clearance of viral pathogens following infection. Mice deficient- or depleted of their CD4 and/or CD8 T cells exhibit a diminished ability to control viral replication following infection and in some cases develop a persistent viral infection. CD8 T cells upregulate cytotoxic effector molecules such as granzyme B, Fas and TNF-related apoptosis-inducing ligand (TRAIL) that them to directly kill virus-infected cells. Following a systemic virus infection the CD8 T cell response is primed within secondary lymphoid organs, such as the spleen and lymph nodes (LNs). Although, it has been shown that the LNs are important for the generation of optimal CD8 T cell responses following systemic viral infections, the relative role of the spleen versus the LN in priming the CD8 T cell response is unknown. Studies in this thesis demonstrate that LNs, but not the spleen, are critical for the optimal generation of a CD8 T cell response following a systemic intraperitoneal (i.p.) lymphocytic choriomeningitis virus (LCMV) infection. Using adoptively transferred naïve LCMV-specific CD8 T cells, we demonstrate that the mediastinal LN (MedLN) serves as the initial draining LN and is responsible for priming the majority of the virus-specific CD8 T cell response following an i.p. LCMV infection. Moreover, the draining MedLN exhibits an increased frequency of CD62L- effector memory (TEM) CD8 T cells for up to 8 weeks following viral clearance. I demonstrate that the increased frequency of CD62L- TEM CD8 T cells is not due to residual viral antigen. Furthermore, a similar increase in CD62L- TEM CD8 T cells is found in the ipsilateral popliteal LN following a footpad LCMV infection. I demonstrate that the increased frequency of CD62L- TEM CD8 T cells in the draining LN is due to increased recruitment.
CD4 T cells promote the generation of both effector and memory CD8 T cells either indirectly through their CD40-CD40L-dependent maturation of dendritic cells or through the production of cytokines such as IL-2 and IFN-γ that directly interact with CD8 T cells. CD4 T cells are also critical for the generation of germinal center B cells and promote the differentiation of activated B cells into memory B cells and plasma B cells. However, CD4 T cells often recognize epitopes derived from a broad array of pathogen-encoded proteins, making it difficult to accurately quantify the magnitude of virus-specific CD4 T cell responses. Therefore, I evaluated a large panel of activation and/or memory markers to determine a combination that could be used to reliably identify antigen-specific CD4 T cells following viral infection. I show that the integrins CD11a and CD49d are upregulated in an antigen-dependent manner on virus-specific CD4 T cells following LCMV infection. Furthermore, memory LCMV-specific CD4 T cells retain their CD11ahiCD49d+ expression pattern. Using CD11a and CD49d as surrogate makers for antigen-specific CD4 T cells, I show that approximately 50% of the CD4 T cells following LCMV infection are virus-specific, indicating that the virus-specific CD4 T cell response is substantially larger than previously recognized. Furthermore, I demonstrate that CD11a and CD49d can be used to accurately track newly-activated CD4 T cells following a heterologous virus challenge.
In addition to LCMV, respiratory syncytial virus (RSV)-specific CD4 T cells are CD11ahiCD49d+. The two previously identified RSV CD4 T cell epitopes only account for ~3% of the CD11ahiCD49d+ CD4 T cell population during the peak of RSV infection, indicating that additional RSV-derived epitopes remain to be identified. Therefore, I used an overlapping peptide library spanning each of the RSV-derived proteins to identify novel RSV-specific CD4 and CD8 T cell epitopes. Using this approach, I identified 5 novel RSV-derived CD4 T cell epitopes and 4 novel CD8 T cell epitopes. Furthermore, I demonstrate that stimulation of CD4 T cells with 17-mer peptides results in over a 2-fold increase in the frequency of responding CD4 T cells as compared to stimulation with the commonly used 15-mer peptides. Collectively, the data shown here provides new insight into where and how the CD8 T cell response is initiated following a systemic virus infection, as well as provide a novel approach to track the endogenous CD4 T cell response following viral infections.
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Lau, Sheung-Yee Kathy. "The trafficking of viral and host membrane proteins during HSV-1 assembly." Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708835.
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McCrossan, Mari-Clare. "The effects of African swine fever virus on the integrity of the secretory pathway." Thesis, University of Sussex, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323056.
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Ontiveros, Steven J. "Intracellular trafficking of the hantaviral nucleocapsid protein and its function in modulation of immune signaling." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2009. https://www.mhsl.uab.edu/dt/2010r/ontiveros.pdf.
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Bériault, Véronique. "Elucidation of the roles of the human immunodeficiency virus type 1 RNA trafficking signals in the viral replication cycle." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=19453.
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Two RNA trafficking cis sequences (A2RE-1 and A2RE-2) were identified in HIV-1 RNA. Their activity is assessed here during HIV-1 gene expression. Single point mutations in each A2RE reduced the levels of bound hnRNP A2, but also resulted in a marked accumulation of viral genomic RNA in the nucleus and a significant reduction in genomic RNA encapsidation in progeny virions, with the strongest phenotype observed for the A2RE-2 mutant. Immunofluorescence analyses revealed marked changes in viral gene expression patterns for pr55Gae and Vpr, as well as a significant reduction in Vpr incorporation levels. Viral infections were markedly compromised in the A2RE-2 mutant but this virus quickly reverted in culture. These data point to the importance of the HIV-1 A2RE determinants and the trans factor hnRNP A2 in the control HIV-1 gene expression patterns. This also provides further details on the implication of host factors in RNA trafficking during HIV-1 replication.
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Sieben, Christian. "Host cell invasion by influenza A virus." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16743.
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Influenzaviren müssen in die Wirtszelle aufgenommen werden, um dort ihr Genom freizusetzen und ihre Replikation mit Hilfe des Reproduktionsapparats der Zelle einzuleiten. Der komplexe Replikationszyklus der Influenza A Viren ist noch nicht vollständig verstanden. Er beginnt mit der Bindung des viralen Hämagglutinins (HA) an Sialinsäure (SA) auf der Zelloberfläche der Wirtszelle. In dieser Arbeit wurde die Virusbindung an Zellen mit unterschiedlicher Rezeptorkomposition verglichen. Dabei konnte gezeigt werden, dass für die zelluläre Spezifität die Präsentation des Rezeptors innerhalb der Plasmamembran der Zelle eine größere Rolle spielt als die Struktur des Rezeptorglykans selbst. Des Weiteren deuten die Beobachtung sehr kleiner Kräfte und ein stufenweises Öffnen von Bindungen auf eine multivalente Interaktion hin. Multivalenz wird oft in biologischen Bindungsprozessen beobachtet und kann Bindungskräfte enorm verstärken. Basierend auf diesen Ergebnissen wurden inhibitorische Nanopartikel entwickelt, die die natürliche Zelloberfläche als hochaffine Bindungsalternative imitieren. Verschiedenartige Nanopartikel wurden evaluiert und konnten die Virusaktivität um mehr als 80 % hemmen. Nach der Bindung wird das Virus durch Endozytose in die Zelle aufgenommen. Durch spezifische Virusmarkierung und gleichzeitiger Expression von zellulären Markerproteinen wurde der Transport einzelner Viren in lebenden Zellen verfolgt. Dabei konnte gezeigt werden, dass das Virus sowohl durch frühe, als auch durch späte Endosomen wandern muss, um sein Genom erfolgreich in das Zytoplasma zu entlassen. Außerdem verzögert das Virus die endosomale Ansäuerung um eine optimale Aufenthaltsdauer im Endosom und die lokalisierte Fusion in der Nähe des Zellkerns zu gewährleisten. Pharmakologisches Eingreifen in diese Prozesse konnte zudem weitere kritische Faktoren identifizieren, die die Effizienz der Virusinfektion stark beeinflussen.Influenza virus must enter a host cell to deliver its genome, use the cells reproductive machinery and eventually initiate its replication. The replication cycle of influenza A virus is very complex and still not fully understood. It generally starts with binding of the viral protein hemagglutinin (HA) to its cellular receptor sialic acid (SA). In this work, virus-cell attachment forces were investigated at the single molecule level using intact virus binding to living cells, a set-up that closely mimics the in vivo situation. Cells of different surface SA composition were compared. It could be shown that the unique presentation of the ligand within the cells plasma membrane, rather than the structure of the receptor-glycan itself, strongly affects cellular specificity. The low binding forces as well as the observation of stepwise unbinding events suggest a multivalent interaction type. Based on this finding, inhibitory nanoparticles mimicking the cell surface were constructed. Different particles were evaluated and shown to efficiently inhibit virus infection by ≥ 80 %. Since many molecular details of multivalent interactions remain poorly understood parameters such as ligand spacing and presentation were varied and revealed that the density of ligands as well as the interacting surface plays critical roles for virus inhibition. Upon attachment, the virus enters the cell by endocytosis. Virus trafficking was followed at the single-virus level in living cells. The kinetics of virus transport were visualized using fluorescent marker proteins in combination with specific virus labeling. It was found that the virus needs to progress through early and late endosomal compartments in order to efficiently uncoat and release its genome. Further, the virus delays the endosomal acidification to ensure optimal residence time and fusion in the region close to the host cell nucleus. Drug treatment furthermore unraveled critical factors influencing viral infection efficiency.
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Yoshida, Takeshi. "A CD63 Mutant Inhibits T-cell Tropic Human Immunodeficiency Virus Type 1 Entry by Disrupting CXCR4 Trafficking to the Plasma Membrane." Kyoto University, 2009. http://hdl.handle.net/2433/124283.
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Chaudhuri, Rittik. "The mechanism of HIV-1 Nef-mediated downregulation of CD4." Thesis, University of Cambridge, 2010. https://www.repository.cam.ac.uk/handle/1810/224775.
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Nef, an accessory protein of HIV-1, is a critical determinant of viral pathogenicity. The pathogenic effects of Nef are in large part dependent on its ability to decrease the amount of CD4 on the surface of infected cells. Early studies suggested that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein-trafficking machinery. However, the specific sorting pathway that Nef uses to modulate CD4 expression remained uncertain. According to one model, Nef was thought to interfere with the transport of newly synthesized CD4 from the TGN to the cell-surface. Another model claimed that Nef facilitated the removal of CD4 from the plasma membrane. The primary goal of this thesis was to determine which of these models was correct. To accomplish this objective, a novel Nef-CD4 system was developed in Drosophila S2 cells. Nef was not only able to downregulate human CD4 in S2 cells, but it did so in a manner that was phenotypically indistinguishable from its activity in human cells. An RNAi screen targeting protein-trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP-2 complex in the Nef-mediated downregulation of CD4. In contrast, depletion of the related AP-1 and AP-3 complexes, which direct transport from the TGN and endosomes, had no effect. The requirement for AP-2 was subsequently confirmed in a human cell line. Yeast three-hybrid and GST pull-down assays were then used to demonstrate a robust, direct interaction between Nef and AP-2. This interaction was found to depend on a [D/E]xxxL[L/I]-type dileucine motif, located in the C-terminal loop of Nef, that is essential for CD4 downregulation. While mapping the binding site of AP-2 on Nef, a second determinant of interaction in the C-terminal loop was identified. Mutation of this motif, which conforms to a consensus [D/E]D diacidic sequence, prevented Nef from binding to AP-2 and down-regulating CD4. However, the same mutations did not affect the ability of Nef to interact with either AP-1 or AP-3, providing further evidence that these complexes are not required for the modulation of CD4 expression. Additional experiments indicated that the Nef diacidic motif most likely binds to a basic patch on AP-2 α-adaptin that is not present in the homologous AP-1 γ and AP-3 δ subunits. As with the Nef diluecine and diacidic motifs, the α-adaptin basic patch was shown to be necessary for CD4 downregulation. Moreover, all three of these motifs were needed for the cooperative assembly of a CD4-Nef-AP-2 tripartite complex, which was observed here for the first time using a yeast four-hybrid system. The data in this thesis uniformly support an endocytic model of Nef-mediated CD4 downregulation. Indeed, there is now strong evidence that Nef simultaneously binds CD4 and AP-2, thereby connecting the receptor to the cellular endocytic machinery and promoting its rapid internalization from the plasma membrane. In addition, the identification of novel motifs required for this process has provided new insights on endocytosis, and may facilitate the development of pharmacological inhibitors of Nef function.
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Hull, Breana. "The Relationship between Sex Trafficking and Immune Function: The Role of Exposure to Abuse." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1523303474251169.
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Kaminsky, Paul Michael. "Focal adhesion kinase signaling regulates highly productive transduction of adeno-associated virus through integrin-mediated endocytosis." Thesis, The University of Iowa, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=3566662.
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Recombinant adeno-associated virus (rAAV) is a widely used gene therapy vector. Although a wide range of rAAV serotypes can effectively enter most cell types, their transduction efficiencies (i.e., transgene expression) can vary widely depending on the target cell type. Integrins play important roles as co-receptors for rAAV infection, however, it remains unclear how integrin-dependent and -independent mechanisms of rAAV endocytosis influence the efficiency of intracellular virus processing and ultimately transgene expression. In this thesis, I examined the contribution of integrin-mediated endocytosis to transduction of fibroblasts by rAAV2. I found that promoting AAV2/integrin binding with Mn++ greatly enhanced (~17-fold) rAAV2 transduction independently of cell binding and endocytosis. Subcellular localization studies of rAAV2 demonstrated that integrin activation by Mn++ promoted AAV2 aggregation on α5 and β1 integrins and recruitment of the cytosolic integrin effector protein vinculin. Focal adhesion kinase (FAK), a down stream effector of integrin signals, was essential for AAV/integrin complex endocytosis and transduction, but not AAV2 recruitment to integrins. Recruitment of FAK to AAV2/integrin complexes was increased by transiently trapping the endocytic event at the plasma membrane by pharmacologic inhibition of dynasore. This also increased the size of AAV2 clusters found beneath the cell at FAK/integrin complexes resembling immature filopodia and caused a large, FAK-dependent (75-fold) increase in AAV2 transduction. These findings support a model whereby integrin activation at the cell surface can redirect rAAV2 toward a FAK-dependent entry pathway that is more productive for cellular transduction. This pathway appears to be conserved for other rAAV serotypes that contain a capsid integrin-binding domain (AAV1 and 6).
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Price, Philip John Ritchie [Verfasser], and Gerd [Akademischer Betreuer] Sutter. "Leukocyte trafficking during infection with modified vaccinia virus Ankara : the role of chemokine receptor 1 and complement activation / Philip John Ritchie Price. Betreuer: Gerd Sutter." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1058077538/34.
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Kim, Adonia Lee. "The Role of Adaptor Protein Complex-3 Delta-Mediated HIV-1 Gag Trafficking in HIV-1 Replication: A Dissertation." eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/612.
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The process of HIV-1 particle production is a multi-step process directed by the viral structural protein Gag. As Gag is the only viral protein required to form virus-like particles, it presents a viable target for anti-viral therapeutics of which there are currently none. Although the functions of Gag during the particle assembly process have been well characterized, one of the least known parts of the assembly process is how Gag is targeted to the site of virus assembly.
Two main virus assembly sites have been identified in cells that support HIV-1 replication: the plasma membrane or multivesicular bodies (MVBs). However the mechanism by which Gag is targeted to either of these sites remains unknown. The δ subunit of Adaptor Protein Complex 3 has previously been identified as a cellular co-factor for HIV-1 Gag and was reported to mediate Gag trafficking to MVBs, providing a mechanism for Gag targeting to this assembly site. Additionally, AP-3δ was reported to be required for HIV-1 production, suggesting that Gag to MVB targeting is also required for HIV-1 production.
The work presented in this thesis further investigates the role of AP-3δ in Gag trafficking to MVBs and its role in HIV-1 production in previously unexplored host environments. Through the use of RNA interference-mediated depletion of AP-3δ, we determined that AP-3δ is dispensible for virus replication in infected HeLa cells, chronically infected HeLa-LAV cells and infected primary human monocyte-derived macrophages. We concomitantly disrupted AP-3 function by disrupting its association with membranes and observed no effect on virus production. Collectively, these results demonstrate that AP-3δ is not required for HIV-1 replication. However, AP-3δ was demonstrated to be required for Gag targeting to MVBs thus presenting a new model for the function of AP-3δ in the context of HIV-1 replication.
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Li, Jianhui. "Cornichon Proteins: Unexpected Roles in Plant Pathogen Infection, ER Morphology Maintenance and Pollen Development." Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/77687.
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Cornichon (CNI) proteins are a conserved family of proteins among eukaryotes, from Erv14 in the yeast Saccharomyces cerevisiae to CNI homologs (CNIHs) in mammals and plants. Erv14 functions as a cargo receptor of coat protein complex II (COPII) for protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, en route to their final destinations. By interacting with specific cargo proteins, CNI proteins regulate key steps of embryo polarity in Drosophila, budding in yeast, and synaptic transmission in the mammalian brain. However, we have very limited understanding of plant CNIHs. Positive-strand RNA viruses assemble their viral replication complexes (VRCs) at specific host organelle membranes. With a better understanding of host factors involved in targeting viral replication proteins to the preferred organelles, we expect to block trafficking of viral replication proteins and thus, viral infection, by manipulating the required host proteins. Brome mosaic virus (BMV) is a model of positive-strand RNA viruses and its replication can be recapitulated in yeast. Importantly, BMV replication protein 1a is the only required viral protein to form VRCs at the perinuclear ER membrane in yeast. I demonstrate that Erv14 and COPII coat proteins are required for targeting BMV 1a to the perinuclear ER in yeast, suggesting a novel function of COPII vesicles in protein trafficking to the perinuclear ER membrane and in the BMV VRC formation. As for cellular functions, I show that plant CNIHs complement the defective distribution of BMV 1a in yeast mutant lacking Erv14. Taking advantage of Arabidopsis thaliana knockout mutants and knockdown of gene expression in Nicotiana benthamina, I also discover that CNIHs unexpectedly play crucial roles in pollen development, infection of a bacterial pathogen, and maintenance of ER tubules. I further confirm that CNI proteins are also required for maintaining ER tubules in yeast, suggesting a novel and conserved role in shaping ER morphology. Therefore, these findings indicate the functional diversity and redundancy of CNI proteins in key cellular processes and suggest a novel strategy to control plant pathogenic viruses and bacteria by manipulating plant CNIHs.Ph. D.
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Lê-Bury, Gabrielle. "Infection des macrophages par le VIH-1 : facteurs moléculaires impliqués dans la production virale et dans le développement de bactéries opportunistes The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking Pronounced stealth phenotype and differential pyroptosis induction by invasive Salmonella Typhimurium revealed by coinfection of human macrophages with HIV Role of Solute Carriers in efficient HIV-1 production by human macrophages." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCB094.
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Le Virus de l'Immunodéficience Humaine de type 1 (VIH-1) infecte les macrophages. Contrairement aux lymphocytes T CD4+, les macrophages résistent aux effets cytotoxiques du virus et représentent un réservoir pour ce pathogène. Dans ces cellules, le virus est produit et stocké dans un compartiment intracellulaire spécifique appelé VCC (Virus-Containing Compartment). Ce compartiment à pH neutre, transitoirement connecté à la membrane plasmique, reste cependant très peu caractérisé. Par ailleurs, le VIH-1 induit une perturbation des fonctions des macrophages, permettant ainsi le développement de bactéries opportunistes, telles que des souches particulières de Salmonella Typhimurium. Nous avons étudié en particulier des souches de Salmonella Typhimurium invasives non typhiques qui se sont développées, chez des patients séropositifs. Les objectifs de ma thèse ont donc été d'étudier, dans les macrophages primaires humains, les facteurs moléculaires impliqués dans la production du VIH-1 et le développement de souches invasives de Salmonella Typhimurium. Dans un premier temps, j'ai contribué à étudier les effets de l'infection par le VIH-1 sur les fonctions des macrophages. Leur fonction majeure est la phagocytose qui est un mécanisme de défense contre les pathogènes permettant leur internalisation et leur dégradation. Il avait déjà été montré au laboratoire que l'étape d'internalisation était en partie inhibée par le facteur de virulence Nef dans les macrophages infectés. Dans ce travail, nous avons montré que l'infection de ces cellules par le VIH-1 inhibe également la maturation des phagosomes, mais via la protéine virale Vpr. Nous avons aussi mis en évidence que le VIH-1 conduit le macrophage dans en état de pré-activation, mais empêche la cellule de répondre à un stimulus ultérieur comme une surinfection bactérienne. Dans un second temps, j'ai participé à l'étude des coinfections entre VIH-1 et les bactéries Salmonella Typhimurium invasives, qui ont émergé avec l'infection par le virus, en comparaison avec des souches de référence. Ce travail nous a permis de montrer que les bactéries, pour leur survie, n'exploitent pas le compartiment viral dans les macrophages co-infectés. J'ai ensuite observé que la souche invasive de Salmonella Typhimurium induit moins de mort cellulaire par pyroptose qu'une souche de référence. J'ai alors déterminé les voies de signalisation en amont de cette mort cellulaire qui est associée à un mécanisme inflammatoire. Ainsi, j'ai mis en évidence que la souche invasive de Salmonella détourne les mécanismes de pyroptose et survit mieux dans les macrophages, ce qui pourrait expliquer la dissémination observée chez les patients. Enfin, j'ai initié l'étude de nouveaux facteurs cellulaires impliqués dans la production virale par les macrophages. À la suite d'une analyse transcriptomique sur des macrophages primaires humains infectés ou non par le VIH-1, nous avons identifié un nombre important de transporteurs membranaires appelés SLC (Solute Carrier) dont l'expression est modulée par l'infection. Après la sélection de candidats, j'ai pu mettre en évidence que certains de ces SLC étaient importants pour la production virale par les macrophages. En conclusion, l'ensemble de ces travaux contribue à définir comment le VIH-1 infecte les macrophages et diminue leurs fonctions d'activation et de clairance, et comment se développent des bactéries opportunistes pathogènesHuman Immunodeficiency Virus type 1 (HIV-1) infects macrophages. In contrast to CD4+ T cells, macrophages are resistant to the cytotoxic effects of the virus and represent a reservoir for the pathogen. In these cells, the new virions are produced and stored in a specific intracellular compartment called Virus-Containing Compartment (VCC). This non-acidic compartment, transiently connected to the plasma membrane, remains poorly characterized. In addition, HIV-1 induces an alteration of macrophage function, allowing the development of opportunistic bacteria, such as specific strains of Salmonella Typhimurium. In particular, we studied invasive non-typhoidal Salmonella Typhimurium (iNTS) strains that developed in HIV-positive patients. The aims of my thesis have been to identify the molecular factors involved in the production of HIV-1 in primary human macrophages and to study the development of the invasive strains of Salmonella Typhimurium. First, I participed in studying the effects of HIV-1 infection on macrophage function. Their main role is phagocytosis, which is a defense mechanism enabling internalization and degradation of pathogens. It has previously been shown in the host laboratory that in HIV-1 infected macrophages, the internalization step is partially inhibited by the virulence factor Nef. In this work, we have shown that the infection of these cells by HIV-1 also inhibits the maturation of phagosomes, in this case, via the viral protein Vpr. Further, we have demonstrated that HIV-1 leads to a pre-activation state of the macrophage, while preventing the cell from responding to subsequent stimuli, such as bacterial superinfection. Secondly, I studied the coinfections between HIV-1 and an invasive strain of Salmonella Typhimurium that was compared to reference strains. This work demonstrated that bacteria do not hijack the viral compartment for their survival in co-infected macrophages. Additionally, the invasive strain of Salmonella Typhimurium was observed to induce less cell death by pyroptosis than a reference strain. The signaling pathways upstream of this cell death were determined to be associated with an inflammatory mechanism. Hence, it was demonstrated that the invasive strain of Salmonella hijacks the mechanism of pyroptosis to survive in macrophages. This may explain the dissemination observed in patients. Finally, a study of new cellular factors involved in viral production in macrophages was conducted. Following a transcriptomic analysis of human primary macrophages infected, or not, with HIV-1, we identified a large number of membrane transporters called SLC (Solute Carrier) whose expression was modulated by the infection. After selecting some of the candidates for further study, I have demonstrated that some of these SLCs are important for viral production in macrophages. In conclusion, this work contributes to defining how HIV-1 infects macrophages and disturbs their activation and clearance functions, and how opportunistic pathogenic bacteria develop
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Loustalot, Fabien. "Study of CAR membrane dynamics in adenovirus infection and CAR endogenous role in healthy and diseased brain." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTT029.
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Les pathogènes neurotropiques représentent une banque d’outils biologique afin de cibler spécifiquement le système nerveux central (SNC), pour son étude mais aussi dans l’optique d’une thérapie. Parmi eux, l’adénovirus canin de type 2 (CAV-2) est un vecteur prometteur pour cibler le SNC. CAR a été principalement étudié en tant que récepteur viral. Cependant, plusieurs études montrent que CAR est essentiel dans le développement du cœur ainsi que du système lymphatique. De manière intéressante, CAR est fortement exprimé pendant le développement du SNC, suggérant un rôle dans l’établissement des réseaux neuronaux. Dans ce travail, nous avons confirmé que CAR est lié aux mécanismes d’endocytoses et au trafic intracellulaire. L’endocytose de CAR est ligand dépendant. La partie intracellulaire de CAR régule son endocytose. Nos données suggèrent que CAR est l’unique récepteur pour CAV-2. Le présent travail de recherche montre aussi que CAR ne semble pas participer à la formation du SNC. En revanche, au niveau du SNC mature, CAR est impliqué dans la plasticité synaptique, dans la neurogénèse adulte et participe à l’homéostasie des synapses, mécanismes impliqués dans les processus mnésiquesThe coxsackievirus and adenovirus receptor (CAR) is a single-pass transmembrane protein belonging to the CTX subfamily of the immunoglobulin superfamily. CAR has been extensively studied as a viral receptor for coxsackie B viruses and some adenoviruses (AdVs). CAR is essential for the development of the cardiovascular and lymphatic system. Interestingly, CAR is highly expressed in the developing brain and has been hypothesized to regulate the establishment of the neuronal networks. In my PhD work, I showed that CAR can be link to the endocytic pathways and intracellular trafficking. CAR endocytosis is ligand-dependent and is regulated by CAR intracellular domain (ICD), suggesting strongly that CAR is most likely the unique receptor for canine adenovirus type 2 (CAV-2). Moreover, we demonstrated that CAR depletion in the developing brain did not significantly perturb brain development. In the healthy adult brain, CAR is relatively abundant and we demonstrated that CAR loss of function affected hippocampal plasticity, adult neurogenesis and synapse homeostasis, which affect cognition
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Perrier, Anabelle. "Etude du trafic intracellulaire de la protéine M du coronavirus du syndrome respiratoire du Moyen-Orient (MERS-CoV)." Thesis, Lille 2, 2019. http://www.theses.fr/2019LIL2S018.
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Les coronavirus sont une famille de virus émergents, comme l’ont montré les émergences récentes des coronavirus SARS-CoV (Severe acute respiratory syndrome coronavirus) et MERS-CoV (Middle-East respiratory syndrome coronavirus), pathogènes pour l’homme. Nous ne disposons ni d’antiviraux spécifiques ni de vaccins pour lutter contre les coronavirus.Des quatre protéines structurales du virus, la protéine M est le moteur de l’assemblage viral. Exprimée seule en cellules, elle peut dépasser le site d’assemblage dans la voie de sécrétion. Nous avons confirmé la localisation de MERS-M dans le TGN(trans-golgi network) et identifié deux signaux dans son domaine C-terminal impliqués dans son trafic : un signal DxE d’export du réticulum, et un signal KxGxYR de rétention dans le TGN. Le signal DxE avait déjà été identifié sur une autre protéine virale, tandis que le signal KxGxYR est un nouveau motif. Pour confirmer son rôle nous avons construit des chimères entre MERS-M et la protéine M du coronavirus IBV (Infectious bronchitis virus), localisée dans le compartiment intermédiaire entre le RE et le Golgi (ERGIC), et démontré que pour les deux protéines le domaine C-terminal est déterminant pour leur localisation spécifique.Un second projet de caractérisation de l’activité antivirale de la digoxigénine contre le HCoV-229E a été initié. Nous avons démontré que cette drogue inhibe le virus à une étape post-entrée, avec une concentration inhibitrice médiane (IC50) de 250nM, et qu’elle n’est pas toxique pour les cellules à cette concentration. Elle inhibe aussi l’infection par le virus de l’hépatite C, et elle semble cibler une étape précoce de la réplication des virus RNA (+)Coronaviruses are an important family of emerging pathogens, as shown by therecent emergence of pathogenic SARS-CoV (Severe acute respiratory syndromecoronavirus) and MERS-CoV (Middle-East respiratory syndrome coronavirus) in the lasttwo decades. There are still some knowledge gaps concerning the biology ofcoronaviruses and we do not have any specific treatment or vaccine.Among the four structural proteins of the virus, the M protein is considered to bethe motor of viral assembly. Expressed alone in cells, M proteins can go beyond theassembly site of the virus (Endoplasmic reticulum-Golgi intermediate compartment,ERGIC) in the secretory pathway. We confirmed MERS-M localization in the Trans-Golginetwork (TGN) and identified two signals involved in its intracellular trafficking in its Cterminaldomain: a DxE ER export signal, and a KxGxYR TGN retention signal. The DxEsignal was already identified on another viral protein, whereas the KxGxYR signal is anew motif. To confirm the role of KxGxYR signal in TGN retention, we constructedchimeras between MERS-M and the protein M of the Infectious bronchitis virus (IBV),located in the ERGIC. Our results suggest that for both MERS-M and IBV-M the Cterminaldomain is determinant for the specific localization of the proteins.We also initiated a project on the characterization of the antiviral activity ofdigoxigenin against HCoV-229E. Our results demonstrated that it inhibits HCoV-229E ata post-entry step, with an IC50 of 250nM, and that it is not toxic at this concentration.Digoxigenin also inhibits hepatitis C virus (HCV) and likely has an effect on an early stepof replication of RNA (+) viruses
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Rossi, Axel. "Intracellular fate of AAV particles in human Dendritic Cell and impact on Gene Transfer." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSEN028.
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Les vecteurs viraux dérivés du virus adéno-associé (AAV) apparaissent depuis deux décennies, comme des outils efficaces pour le transfert de gène in vivo. Cependant, malgré une faible immunogénicité et une absence de toxicité in vivo, leur optimisation requiert encore un effort important vers une meilleure compréhension de leur biologie et, en particulier, de leur interaction avec le système immunitaire. Au cours de ce travail de thèse, nous avons utilisé une méthode de sélection dirigée in vitro dans le but d’obtenir un variant de capside capable de transduire efficacement un type cellulaire non-permissif aux vecteurs AAV : les cellules dendritiques (DC). En effet, ces cellules jouent un rôle primordial dans l’établissement de la réponse immunitaire et, par conséquent, dans la persistance de l’expression du transgène in vivo. Cette technologie, très répandue dans la communauté AAV, a permis de sélectionner un variant de capside aux propriétés très intéressantes. La mutation sélectionnée, caractérisée in vitro comme induisant une instabilité de la capside, a permis d’identifier et de surmonter un point de blocage majeur dans le processus de transduction des DC par les vecteurs AAV consistant dans l’étape de décapsidation du génome du vecteur dans le noyau cellulaire. De manière intéressante, le variant obtenu exhibe un avantage en terme de transduction non seulement dans les DC mais aussi dans différents modèles de cellules primaires humaines (e.g. HUVEC) ou animales (OBC), peu ou pas permissive à l’AAV. De plus, des expériences de transfert de gène in vivo réalisées dans un modèle murin, indiquent que le variant sélectionné conduit à une meilleure expression du transgène, possiblement due à la mise en place d’un processus de tolérisation. Les propriétés remarquables de ce variant de capside, font de lui un candidat intéressant pour des applications médicalesVectors derived from the Adeno-associated virus (AAV) have emerged as an efficient system for in vivo gene transfer. However, despite their low immunogenicity and good tolerance in vivo, a better characterization of the host-AAV interaction is required to be able to fully exploit AAV’s potential fora gene therapy or gene vaccination. In this PhD project, we have used an in vitro directed evolution strategy to select an AAV capsid variant able to transduce human dendritic cell (DC), a non-permissive cell type which plays a critical role in the initiation of immune responses and, consequently, on the persistence of the expression of transgene in vivo. This procedure allowed us to identify an AAV variant characterized by a decreased stability of the capsid in vitro. The use of this mutant as a vector to transduce human DC resulted in an improved uncoating of the vector genome in the cell nucleus, thus identifying this step as major barrier toward DC transduction. Interestingly, the selected variant also displayed an increased transduction efficiency not only in DC but also in different primary human and animal cell types, poorly or non-permissive to AAV. Finally, when injected in mice, this AAV variant resulted in a higher expression of the transgene, associated to a low level of immune responses, suggesting the induction of tolerant state. The remarkable features suggest that our selected variant capsid is a promising candidate for medical applications
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Agrawal, Amit. "Nanoparticle Probes for Ultrasensitive Biological Detection and Motor Protein Tracking inside Living Cells." Diss., Georgia Institute of Technology, 2006. http://hdl.handle.net/1853/19798.
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Semiconductor quantum dots (QDs) have emerged as a new class of fluorescent probes and labeling agents for biological samples. QDs are bright, highly photostable and allow simultaneous excitation of multiple emissions. Owing to these properties, QDs hold exceptional promise in enabling intracellular biochemical studies and diagnosis with unprecedented sensitivity and accuracy. However, use of QD probes inside living cells remains a challenge due to difficulties in delivery of nanoparticles without causing aggregation and imaging single nanoparticles inside living cells. In this dissertation, a systematic approach to deliver, image and locate single QDs inside living cells is presented and the properties of molecular motor protein driven QD transport are studied. First, spectroscopic and imaging methods capable of differentiating single nanoparticles from the aggregates were developed. These technologies were validated by differentiating surface protein expression on viral particles and by enabling rapid counting of single biomolecules. Second, controlled delivery of single QDs into living cells is demonstrated. A surprising finding is that single QDs associate non-specifically with the dynein motor protein complex and are transported to the microtubule organizing center. Accurate localization and tracking of QDs inside cell cytoplasm revealed multiple dynein motor protein attachment resulting in increased velocity of the QDs. Further, spectrin molecule which is known to recruit dynein motor protein complex to phospholipid micelles was found to associate with the QDs. These results may serve as a benchmark for developing new QD surface coatings suitable for intracellular applications. Since, nanoparticles are similar in size to viral pathogens; better understanding of nanoparticle-cell interactions should also help engineer nanoparticle models to study virus-host cell interactions. (Contains AVI format multimedia files)
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Cardoso, Ricardo de Souza. "Participação de proteínas da via secretória no tráfego e montagem do vírus sincicial respiratório." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17136/tde-21072016-162417/.
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O vírus sincicial respiratório humano (HRSV) é o mais frequente agente patogênico da família Paramyxoviridae. Apesar de sua grande importância e impacto em saúde pública, alguns aspectos demandam elucidação. Entre eles, estão os mecanismos de tráfego intracelular de proteínas virais para o sitio de montagem. Baseado nisso, fizemos um estudo de imunofluorescência tentando contribuir para o entendimento da participação da via secretória no tráfego de proteínas estruturais de HRSV que não são glicosiladas: proteínas de matriz (M) e de nucleocapsídeo (N). Pudemos observar que essas proteínas seguem rota similar àquelas que são glicosiladas no Golgi, como a proteína de fusão (F). Ademais, as proteínas M e N, além de colocalizarem com proteínas celulares da via secretória, tais como trans-Golgi network-46 (TGN46) e sorting nexin-2 (SNX2), também influem no recrutamento de proteínas celulares para os corpos de inclusão virais, como mostrado no caso da proteína Glut1. Os dados indicam que proteínas M e N de HRSV seguem pela via endocítica inicial, acumulam-se em corpos de inclusão que seriam fábricas virais e, no caso de TGN46, podem ser incorporadas aos vírus em brotamentoHuman respiratory syncytial virus (HRSV) is the most relevant cause of respiratory infection in children worldwide. Despite its importance in public health, some aspects of the mechanisms of the trafficking of viral structural proteins remain unclear. In the present study, immunofluorescence was used to understand how the virus matrix (M) and nucleocapsid (N) proteins, which are non-glycosylated , are addressed to inclusion bodies in Hep-2 cells (MOI=3). M and N proteins followed similar intracellular trafficking routes as compared to the glycosylated fusion (F) viral protein. Moreover, M and N proteins colocalized with two key elements of the secretory pathway: trans-Golgi network- 46 (TGN46) and sorting nexin-2 (SNX2). Viral proteins M and N appear to be involved in the recruitment of cell proteins at the formation of virus inclusion bodies, as shown for Glucose Transporter Type 1 (Glut1). The data suggest that HRSV M and N proteins follow the secretory pathway, initiating in early endosomes, as indicated by the co-localization with TGN46 and SNX2. In addition, these host cell proteins accumulate in inclusion bodies that are viral factories, and can be part of budding viral progeny. Therefore, HRSV M and N proteins, even though they are not glycosylated, take advantage of the secretory pathway to reach virus inclusion bodies. Confocal images suggest that SNX2, which is known for its membrane-deforming properties, could play a pivotal role in HRSV budding
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Tavares, Lucas Alves. "O envolvimento da proteína adaptadora 1 (AP-1) no mecanismo de regulação negativa do receptor CD4 por Nef de HIV-1." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17136/tde-06012017-113215/.
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O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal.The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.
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Chu, Li-Wei, and 瞿立威. "Visualization of virus trafficking and intracellular processing using single-virus tracking." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/63hjca.
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博士國立陽明大學
生醫光電研究所
105
Viral life cycles involve with a series of complicated interactions of the virus with host cells from proteins, structures, and machineries. The development of antiviral drug needs to understand these complicated mechanisms of virus infection. Unfortunately, the several critical molecular mechanisms of virus are poorly understood. Single-virus tracking is a real-time imaging technique that can monitor successfully individual virus trafficking behavior and kinetic processing in live cell. Super-resolution microscopy contributes to visualize the details of virus particles and subcellular structures. In our studies, super-resolution microscopy facilitated us to differentiate the multiport pathways of Dengue virus (DENV) entry. Using single-virus tracking demonstrated that DENV traffics from endosomes to autophagosomes subsequently. In addition, we established that pH sensor conjugating-DENV and FRET labeled-DENV particles were used to visualize acidification and viral fusion in autophagosomes, respectively. It implied DENV takes the autophagic pathway to bypass cellular defense system and proceeds to the membrane fusion to release RNA genome for replication. On the other hand, the single-virus tracking approach was employed to investigate the trafficking routes of Vaccinia virus particles in HeLa cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm.
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Chang, Chih-Hao, and 張志豪. "Long-distance trafficking of Bamboo mosaic virus satellite RNA in plant." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/3aczvd.
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Chen, Tzu-Jung, and 陳姿融. "Investigation of the intracellular trafficking pathway of vaccinia virus in HeLa cells." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/51213580262036727129.
Full textAbstract:
碩士國立陽明大學
生命科學暨基因體科學研究所
99
Vaccinia virus is the prototypic member of the Orthopoxvirus genus and has a wide host range in vitro and in vivo. Vaccinia virus contains an envelope and enters cells through either endocytosis or plasma membrane fusion. We previously showed that the WR strain of vaccinia virus entered HeLa cells through fluid-phase endocytosis process that depends on cellular proteins dynamin and vaccinia virus penentration factor (VPEF) but not clathrin nor caveolae. However, after being uptake into cells, the intracellular trafficking route of the internalized vaccinia virus remains unclear. The goal of my thesis is to elucidate the trafficking process of internalized vaccinia mature virus inside the cells until membrane fusion that releases viral cores in cytoplasm. Here, we utilized two sets of experimental designs to address this issue. First we monitored mCherry-labeled virus particles uptake process in order to identify virus transport from PI3P-enriched macropinosomes to Rab5+endosome and other intracellular compartments prior to membrane fusion. Furthermore, using functional interference assays, we perturbed different intracellular vesicle trafficking processes in order to delineate the intracellular trafficking pathway of the internalized virions prior to membrane fusion. The results showed that vaccinia virus was internalized and transported to Rab5+ early endosomes but not Rab7+ late endosomes. Instead, Rab11+ recycling endosomes appeared involved in virus trafficking in cells. Future work will be performed to verify the role of retromer complexes in virus endocytic entry process and to investigate how rab11 and retromer complex interact to regulate vaccinia virus trafficking.
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Schenkel, Alan Rowe. "Lymphocyte trafficking and population dynamics after Simian Immunodeficiency Virus infection of Rhesus Macaques." 1998. http://catalog.hathitrust.org/api/volumes/oclc/42430536.html.
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Lux, Kerstin [Verfasser]. "Visualization of the intracellular trafficking of incoming adeno-associated virus type 2 / Kerstin Lux." 2006. http://d-nb.info/989558703/34.
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Difato, Francesco. "Optical microscopy to study the role of cytoskeleton in cell locomotion and virus trafficking." Doctoral thesis, 2008. http://www.nusl.cz/ntk/nusl-274188.
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3. General conclusions 150 The interest in optical microscopy is constanly growing, mainly because of its unique features in examining biological systems in four dimensions (x-y-z-t)1 . The work presented here was focused on biological applications of optical microscopy by exploring and improving the spatial and temporal resolution performances and by futher developing optical tools for manipulating biological samples. First, I studied the resolution performances of the system in the three dimensional space and I contributed in improving the experimental spatial resolution of microscope by applying deconvolution. In this respect, theoretical modelling can characterize the image formation process of the microscope, but only experimental measurement of the PSF can quantify the limitations of the real system. Indeed, experimental PSF presents shape assymetry due to spherical aberrations introduced by optical elements, while theoretical PSF is symmetric and account only for the resolution limits of an ideal imaging system. The disadvantage of experimental PSF is that could be corrupted by noise, otherwise deconvolution with the theoretical PSF offer only a qualitative improvement of the image, because the introduced artefacts cannot be quantified. Deconvolution of the acquired data with experimental PSF...
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Scheifele, Lisa Z. "From the Ribosome to the membrane subcellular trafficking of the Rous sacrcoma virus Gag polyprotein /." 2004. http://etda.libraries.psu.edu/theses/approved/WorldWideIndex/ETD-468/index.html.
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Cheng, Chung-Ting, and 鄭全廷. "Using tunable-lens to reveal three-dimension intracellular trafficking of single-virus particle in living cells." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/xe5ceh.
Full textAbstract:
碩士國立陽明大學
生醫光電研究所
106
Single-particle tracking (SPT) is of growing importance in the biophysical community. SPT allows researchers to follow the fate of individual virus particles, to probe dynamic interactions between viruses and cellular machinery. In the past, our lab’s publication also use epi fluorescence microscopy to observe two-dimensional tracking images. Reveals the relationship between dengue virus and autophagosome in the early stage of infection. However, intracellular is a three-dimensional space, and it is necessary to develop three-dimensional real-time single particle tracking. In order to upgrade the system to the 3D level, we have used an electrically tunable lens that can quickly change the imaging plane. The electrically tunable lens is mounted under the objective lens to synchronize the excitation light and the radiation energy by ETL regulation. And write the LabView program synchronization CMOS, shutter and electronically controlled variable lens connection. After measuring the z-axis of the dried fluorescent beads, it is known that the z-axis distance varies by about 0.13 μm per 1 mA of the ETL. During the one hour of tracking the dried fluorescent beads, the z-axis position and the fluorescence intensity of the fluorescent beads remained unchanged, indicating the stability of the system. In addition, the system effectively tracks the movement of the fluorescent beads as the dried fluorescent beads move over the z-axis over time. And we used dengue virus to infect Huh7.5 cells expressing GFP-LC3, and tracked the movement of the organelles and virus particles in living cells. In summary, the system achieves a z-axis resolution of at least 10nm and a time resolution of less than 1 second, and enables image and tracking of live cells and virus particles.
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(8199633), Thu M. Cao. "Structure- Function Studies Of Flavivirus Non-Structural Protein1." Thesis, 2020.
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Flaviviruses is a genus within the family Flaviviridae. The genus consists of more than 70 viruses, including important threatening human pathogens such as dengue virus (DENV), West Nile virus (WNV), and Zika virus (ZIKV). These viruses are causative agents for a range of mild to lethal diseases and there are currently no US- licensed therapeutic treatments for infection. The virus genome is a positive-sense, single-stranded RNA, encoding ten viral proteins. Of the ten flavivirus proteins, Non- Structural protein 1 (NS1) remains the most elusive in terms of its functions. To date NS1 has been linked to disease pathology and progression and plays roles in virus replication and assembly. However, little is understood how NS1 orchestrates these functions and how NS1 from different viruses function distinctively from one another. Moreover, flavivirus NS1 has a peculiar ability to associate with lipid membranes. During the life cycle of NS1, the protein travels through the classical secretory path- way, similar to infectious virus particles, and is secreted into the extracellular space as mostly hexameric oligomers containing a lipid core. How the protein binds to lipids and whether such lipid binding is important for NS1 functions and overall flavivirus pathology remain unknown. Using structure-based mutagenesis, we found a group of mutants on WNV NS1, which particularly altered the viral specific infectivity but maintained wild-type level of virus replication. Purified mutated virus particles revealed that the specific infectivity alteration was not because of the particle but interaction of the virus particles and NS1 mutated proteins. Here we demonstrated that specific residues on NS1 were responsible for distinctly roles in NS1 functions and the virus specific infectivity was regulated by NS1 protein. In other structure-base study, we focused on the membrane association ability of NS1. All structure-predicted regions on NS1 were examined for its contribution for the membrane/lipid binding function. This interaction was required for NS1 biology activities including intracel- lular trafficking, oligomerization, and endocytosis. The lipidomes from deletion of each membrane association region revealed differences in lipid classes binding to each region and the composition flexiblity of the lipid cargo of NS1 hexamer.
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Baird, Nicholas Lindsay Wills John W. "Acyl determinants of the Herpes simplex virus type 1 UL11 tegument protein required for membrane trafficking and virion envelopment." 2009. http://etda.libraries.psu.edu/theses/approved/PSUonlyIndex/ETD-3682/index.html.
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Liu, Hao-Ping, and 劉浩屏. "Prenylated Rab acceptor 1 promotes the intracellular trafficking and NF-kappaB signaling of Epstein-Barr virus-encoded latent membrane protein 1." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/84338281528989187834.
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博士國立陽明大學
微生物及免疫學研究所
94
Latent membrane protein 1 (LMP1), which is an Epstein-Barr virus (EBV)-encoded oncoprotein, induces NF-kappaB signaling by mimicking the tumor necrosis factor receptor (TNFR). Unlike TNFR-based signaling, however, LMP1 signals primarily from intracellular compartments in a ligand-independent manner. In this study, we seek to further elucidate the mechanism underlying ligand-independent LMP1 signaling by exploring the cellular factors mediating the intracellular trafficking of LMP1. Using a yeast-two-hybrid screen, we identify a new LMP1-interacting molecule, prenylated Rab acceptor 1 (PRA1), which is involved in intracellular LMP1 trafficking and LMP1-induced NF-kappaB signaling. Co-precipitation analyses demonstrated that LMP1 interacts with PRA1 for the first time through LMP1’s transmembrane domain, in particular the membrane-spanning segments 3-6. The fluorescence resonance energy transfer (FRET) microcopy and advanced bioluminescence resonance energy transfer (BRET2) assay revealed that LMP1 specifically interacts with PRA1 at the Golgi apparatus in living cells. The immunofluorescence microscopy further showed that co-localization of LMP1 and PRA1 to the Golgi is sensitive to nocodazole and brefeldin A. Co-expression of a PRA1 export mutant or knockdown of PRA1 led to redistribution of LMP1 and its associated signaling molecules from the Golgi to the ER and subsequent impairment of LMP1-induced NF-kappaB activation. Importantly, the PRA1 knockdown-associated impairment of LMP1 signaling could be restored by reconstitution of the wild-type but not the mutant PRA1. Moreover, neither CD40- and TNFR1-mediated NF-kappaB signaling nor the functional integrity of the Golgi apparatus was affected by PRA1 knockdown, indicating that PRA1 selectively mediates the trafficking and signaling of LMP1. Finally, targeting of the LMP1 C-terminus to the Golgi by fusion with a palmitoylation sequence derived from GAP-43 was insufficient to trigger LMP1-induced NF-kappaB activation unless the LMP1 derivative was able to interact with PPA1, implicating that PRA1 is not merely an interacting-transporter for LMP1, but is crucial for LMP1-mediated signaling. Collectively, these novel findings provide important new insights into LMP1, and identify an unexpected new role for PRA1 in cellular signaling.
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Huang, Ying-Ping, and 黃盈屏. "The study on the viral RNA intracellular trafficking and the involvement of autophagy pathway in the infection of Bamboo mosaic virus in Nicotiana benthamiana." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/8kjk74.
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博士國立中興大學
生物科技學研究所
103
The cDNA-amplified fragment length polymorphism technique was applied to isolate the differentially expressed genes during Bamboo mosaic virus (BaMV), a single-stranded positive sense RNA virus, infection on Nicotiana benthamiana plants. One of the upregulated genes was cloned and predicted to contain a TBC domain designated as NbRabGAP1 (Rab GTPase activation protein 1). No significant difference was observed in BaMV accumulation in the NbRabGAP1-knockdown and the control protoplasts. However, BaMV accumulation was 50% and 2% in the inoculated and systemic leaves, respectively, of the knockdown plants compared to those of the control plants. By measuring the spreading area of BaMV infection foci in the inoculated leaves, we found that BaMV moved less efficiently in the NbRabGAP1-knockdown plants than in the control plants. Transient Over-expression of the wild type NbRabGAP1, but not the disable NbRabGAP1 activity mutant, significantly increaseincreases BaMV accumulation in N. benthamiana. These results suggest that NbRabGAP1 with a functional Rab-GAP activity is involved in virus movement. Since chloroplast pPhosphoglycerate kKinase (PGK) was reported to interact with the very 3’ end of BaMV RNA, it displayed a high possibility of viral RNA localized at chloroplasts. To observe the localization of viral RNA in infected cells, we employed the interaction of NLS-GFP-MS2 (phage MS2 coat protein) with the modified BaMV RNA containing the MS2 coat protein binding sequence in live plant cells. In the experiment, we have constructed the eight repeats of MS2 binding sequence at the intergenic region of between BaMV replication gene and the movement protein gene. Furthermore, we have reconstructed the fluorescence fusion protein to display a stronger nucleus location signaling (MS2-NLS-GFP was changed to NLS-MS2-OFP) in cells. Using confocal microscopy, we observed that the genomic RNA of BaMV was localized at the chloroplasts of the infected cells. Furthermore, we can also detect the minus-strand RNA in the isolated chloroplasts from BaMV infected leaves. Taken together, these results suggested that BaMV genomic RNA targeting to the chloroplasts as a replicate site is the most recommended. Autophagy plays an important role in nutrient recycling for plant homeostasis while plants suffer the abiotic or biotic stresses including the response to the pathogen infection. In this study, we would like to investigate the effect of autophagy on the infection cycle of BaMV. Initially, we examined the relative gene expression of some autophagy-associated genes (ATGs) and found out that these genes were upregulated post BaMV infection. The coat protein and viral RNA accumulation levels of BaMV were reduced when the cells were treated with the autophagy inhibitors, wortmannin and 3-MAMethyladenine. Nevertheless, the coat protein accumulation of PVXPotato virus X, CMVucumber mosaic virus and TMVobacco mosaic virus was also decreased when treated with wortmannin but not with 3-MA, an inhibitor through blocking the classIII PI3Ks which was critical during the late stage of vesicle expansion. Furthermore, we also found that the coat protein and viral RNA accumulation levels of BaMV were reduced in the NbATG8-silenced protoplasts and plants. As well, the accumulation of BaMV coat protein was increased in NbATG8-transiently expressed protoplasts. Altogether, these results suggest that BaMV utilizes the autophagosome for replication in some way albeit the overall mechanism of the interaction between autophagosome and virus was still unclear.
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Žíla, Vojtěch. "Myší polyomavirus: Role buněčného cytoskeletu v endozomálním transportu viru a vlastnosti minoritních kapsidových proteinů." Doctoral thesis, 2014. http://www.nusl.cz/ntk/nusl-338093.
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Mouse polyomavirus (MPyV) is a non-enveloped DNA tumor virus, which replicates in the host cell nucleus. MPyV enters cells by receptor-mediated endocytosis and its subsequent transport towards the nucleus requires acidic environment of endosomes and intact microtubules, which are important for virus delivery to endoplasmic reticulum (ER). In ER, capsid disassembly and uncoating of viral genome take place. The mechanism of subsequent translocation of viral genome from ER into nucleoplasm is still only poorly understood process with predicted involvement of cellular factors and viral minor capsid proteins VP2 and VP3. Once the genome appears in the nucleus, early viral antigens are produced and mediate suitable environment for replication of viral genomes. After replication of viral DNA and morphogenesis of virions, virus progeny is released from the cells during its lysis. The research presented in the first part of thesis focused on intracellular transport of MPyV and involvement of cytoskeletal networks during virus delivery to the ER. In particular, we investigated still unclear role of microtubules during virus trafficking in endosomes, and involvement of microtubular motors. We found that MPyV trafficking leading to productive infection does not require the function of kinesin-1 and kinesin-2,...