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1
Barkley, Russell. "Investigation of an Oncolytic MeV Cell-Cell Fusion Phenomenon Induced by an siRNA." Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/41531.
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Oncolytic measles virus is a promising cancer therapeutic in clinical trials which
possesses multiple characteristics that are advantageous over traditional therapies.
Currently, clinical oncolytic measles virus vectors are unmodified or express reporter
transgenes that benefit its therapeutic efficacy. The next phase in its development will
see genetically engineered vectors encoding transgenes that enhance its antineoplastic effects. To this end, preclinical research has focused on studying novel transgenes which favour viral replication, cytotoxicity, and the anti-cancer immune response. We sought to encode artificial micoRNAs targeting RIG-I as a strategy to interfere with innate immunity. Silencing RIG-I with multiple siRNAs yielded one which promotes measles virus syncytia formation through a mechanism that appears to be independent of RIG-I. The mechanism caused by the siRNA leads to enhanced measles virus cell-cell fusion and has peculiar characteristics which are not fully understood.
2
Garg, Himanshu. "Feline Immunodeficiency Virus (FIV) Envelope Glycoprotein-Mediated Cell Fusion and Apoptosis." NCSU, 2003. http://www.lib.ncsu.edu/theses/available/etd-11042003-141554/.
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Feline Immunodeficiency Virus (FIV) and Human Immunodeficiency Virus (HIV) are lentiviruses that are remarkable similar in their genomic organization, receptor usage and pathogenesis. Based on this FIV has evolved into a well-established small animal model for studying AIDS. FIV and HIV cause a progressive depletion of T cells via a still unknown mechanism though numerous studies support a role of membrane expressed HIV env glycoprotein in apoptotic killing of CD4+ T cells. HIV env glycoprotein is a heterodimer of surface expressed gp120 that binds to CD4 and a chemokine receptor and transmembrane gp41 that mediates fusion and syncytia formation. The role of the fusion process in HIV env-mediated apoptosis remains controversial even though evidence suggests that cytopathic effect of HIV is related to the fusogenic potential of env glycoprotein. Blocking HIV env receptor interactions either at the level of gp120 or gp41 blocks both syncytia formation and apoptosis. This suggests a crucial role for HIV gp41 in fusion, as well as apoptosis. The hydrophobic pre-transmembrane (pre-TM) region of HIV gp41 is important for membrane fusion and sequence analysis reveals a similar region in FIV gp41. The current study was undertaken to determine the role of different regions of FIV env in mediating fusion and apoptosis in bystander cells and to determine whether the two phenomena are related. FIV env interactions with target cells were blocked at the level of gp120 or gp41 and the effect of these on fusion and apoptosis studied. The role of FIV gp41 pre-TM region in fusion and apoptosis was also determined. Our findings support a role of FIV env in apoptotic loss of T cells and this phenomenon correlates with env-mediated fusion.
3
Bickerton, Erica Jane. "Cellular tropism and cell-to-cell fusion properties of the infectious bronchitis virus spike glycoprotein." Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/35165/.
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There are numerous vaccines available for the control of infectious bronchitis virus (IBV) in poultry, however protection is short-lived and poorly cross-protective between strains. The vaccines must currently be grown in embryonated eggs, a cumbersome and expensive process. The ability to grow vaccines on a cell-line such as Vero cells would be highly advantageous. The spike (S) glycoprotein of IBV is comprised of two subunits, S1 and S2, has a vital role in virulence in vivo and is responsible for cellular tropism in vitro. This project aims to identify the amino acids present in the S glycoprotein involved in determination of cellular tropism and cell-to-cell fusion. The IBV Beaudette strain is able to replicate in both primary chick kidney (CK) cells and Vero cells, whereas the IBV M41 strain replicates in primary cells only. Recombinant IBVs with chimaeric S genes were generated using a reverse genetics system with the genomic background of Beaudette and part of the S gene from M41. Their growth characteristics and cellular tropism were investigated. The S2 subunit of Beaudette was found to be sufficient to confer the ability to grow on Vero cells and swapping just three amino acids with corresponding ones from M41 was sufficient to remove the ability of the Beaudette S glycoprotein for growth on Vero cells. Beaudette was further adapted to syncytia formation on Vero cells by serial passage and isolates were sequenced to identify amino acid changes between parent and Vero-adapted viruses that are potentially involved in cell-to-cell fusion. Understanding the way in which IBV infects host cells is vital in order to rationally design better vaccination and treatment strategies and help to reduce the prevalence of IBV infection in poultry worldwide. Using the IBV reverse genetics system, we now have the potential to grow IBV vaccines on Vero cells.
4
Symeonides, Menelaos. "HIV-1-Induced Cell-Cell Fusion: Host Regulation And Consequences For Viral Spread." ScholarWorks @ UVM, 2016. https://scholarworks.uvm.edu/graddis/589.
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Human immunodeficiency virus type 1 (HIV-1) is a human retrovirus of the lentivirus subgroup which primarily infects T cells and macrophages, and causes acquired immune deficiency syndrome (AIDS). Since its emergence in the early 1980s, HIV-1 has caused a global pandemic which is still responsible for over one million deaths per year, primarily in sub-Saharan Africa.
HIV-1 has been the subject of intense study for over three decades, which has resulted not only in major advances in cell biology, but also in numerous drug treatments that effectively control the infection. However, cessation of treatment always results in reemergence of the infection due to the ability of HIV-1 (and other lentiviruses) to establish a persistent quiescent infection known as latency. The elimination of latently-infected cells is the primary goal of current research towards a cure for HIV-1, alongside efforts to develop vaccines, which have thus far been fruitless.
The spread of HIV-1 to susceptible target cells (which express the receptor CD4 and a co-receptor; CXCR4 or CCR5) can take place when antigen-presenting cells, such as dendritic cells, capture virus particles and then pass them on to target cells, without themselves becoming infected. Alternatively, productively infected T cells or macrophages can spread HIV-1 either by shedding virus particles to the milieu, which are then stochastically acquired by target cells, or through transient contacts between infected and uninfected cells known as virological synapses (VSs). VS-mediated cell-to-cell transmission is thought to be highly efficient due to the release of virus directly onto (or very near to) a target cell, and some evidence suggests that the VS is a privileged site which allows the virus to evade neutralizing antibodies and drugs. However, and most importantly, it is of central interest to us because the same transient cell adhesions that facilitate virus transfer can also result in the fusion of the two cells to form a syncytium, due to the presence of the viral fusogen Env and its receptor and co-receptor on either side of the VS. While T cell syncytia can be found in vivo, they remain small, and it appears that the majority of VSs resolve without fusion.
The regulation of HIV-1-induced cell-cell fusion and the fate of those syncytia are the focus of the work presented here. A family of host transmembrane proteins, the tetraspanins, which regulate cell-cell fusion in other contexts (e.g. the fusion of myoblasts to form and maintain myotubes), were found to inhibit HIV-1-induced cell-cell fusion. Our investigations have further characterized this regulation, concluding that tetraspanins allow cells to reach the fusion intermediate known as hemifusion before their ability to repress fusion takes effect. In parallel, because syncytia are nevertheless found both in infected individuals and in a humanized mouse model for HIV-1, we also became interested in whether small T cell-based syncytia were able to participate in HIV-1 spread by transmitting virus to target cells. Using a simple three dimensional in vitro culture system which closely recapitulates those in situ observations, we found that small syncytia can contact target cells and transmit virus without fusing with them. Overall, these studies further our understanding of HIV-1-induced syncytia and reveal a previously unrecognized role for these entities as active participants in HIV-1 spread.
5
Leung, Sze-Yui Horasis, and 梁思睿. "Fibronectin: role in viral cell association, fusion and entry of influenza A virus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48329708.
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The influenza A viral hemagglutinin (HA) protein binds to sialic acid (SA) groups of cellular surface glycoproteins to achieve viral attachment and entry. The SA binding specificity of HA is one of the major determinants for controlling viral tropism and host specificity.
Fibronectin (FN) is a ubiquitinious glycoprotein secreted on cell surface, either circulating in plasma, or as one of the best characterized components of the extra cellular matrix. With its binding properties towards different types of molecules and pathogens, it has been utilized by different bacterial and viral pathogens for binding, entry, propagation and pathogenesis. The binding affinity and region of plasma FN to influenza A viral glycoprotein was identified in early 1980s. Evidence also suggests the binding is SA associated. FN associates with different viral pathogens. However, evidence of FN direct involvement in influenza A pathogenesis remains unknown.
The objective of this thesis is to test the involvement of cellular FN in influenza A viral infection. To perform the study, FN siRNA and anti-FN antibody were applied. This study demonstrated possible involvement of FN in the replication of human H1N1 and highly pathogenic avian H5N1 viruses. It also discovered that FN is very important for the replication of H1N1 virus, but not H5N1 virus. Interestingly, the result suggested that FN does not affect the initial virus-host binding, but it has an effect on post-attachment events.
Key amino acid positions controlling the SA binding specificity of seasonal human or avian influenza A viruses have been identified in the HA. In this thesis, reverse genetics and mutagenic work identified that viruses with a α2,3-linked SA (SA α2,3) binding preference were not inhibited by anti-FN antibody, while viruses with a α2,6-linked SA (SA α2,6) specificity were severely inhibited. This surprising finding of SA binding preference related FN involvement in post-attachment event led to the further investigation on the structural involvement of FN and viral entry pathway analysis.
The 9th and 10th of type III repeating units of FN form the cell-binding domain of the protein for cell attachment. From site specific antibody inhibitory studies, the cell binding region of FN near the synergy adhesion site(SAS) and Arg-Gly-Asp-Ser(RGDS) cell adhesion signal was identified to be important for the replication of viruses that have a α2,6 SA binding preference, but it was also found to be independent of α5β1 integrin receptor.
After attaching to a host cell, the virus was internalized in an endosome via clathrin- or caveolin- mediated endocytosis. By application of pathway inhibitors, the FN association with viral entry pathway was evaluated. Though this study failed to identify a single specific FN mediated viral entry pathway, this pathway study indicated the possibility of FN various involvement in influenza viral entry. The study indeed indicated that viruses have difference SA binding preferences are different in their choices in viral entry pathways.
This thesis did not only introduce cellular FN as a novel host factor, but also identified possible target and brought new light in the control of influenza A viral infection.<br>published_or_final_version<br>Public Health<br>Doctoral<br>Doctor of Philosophy
6
Wagenaar, Timothy Robert. "Regulation of infected cell fusion by the vaccinia virus A56 and K2 proteins." College Park, Md.: University of Maryland, 2008. http://hdl.handle.net/1903/8044.
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Thesis (Ph. D.) -- University of Maryland, College Park, 2008.<br>Thesis research directed by: Dept. of Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
7
Hummel, Kimberly Brown. "Alteration of the measles virus glycoproteins as a mechanism to reduce cell fusion during persistence." Diss., Georgia Institute of Technology, 1994. http://hdl.handle.net/1853/25597.
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Hutchinson, Lloyd M. "Glycoprotein K of herpes simplex virus (HSV), role in viral egress and HSV-induced cell-cell fusion." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0016/NQ30094.pdf.
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Al-Torki, Reem. "Mapping of B-cell epitopes on the fusion protein of human respiratory syncytial virus." Thesis, London School of Hygiene and Tropical Medicine (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415976.
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Marques, Sandra Eugénia Leite. "Expressão em Escherichia coli de antigénios do Cell fusing agent virus (Flaviviridae: Flavivirus) como proteína de fusão." Master's thesis, Faculdade de Ciências Médicas. UNL, 2012. http://hdl.handle.net/10362/8531.
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RESUMO: O Cell Fusing Agent Vírus (CFAV), considerado como o primeiro “flavivírus
específicos de insectos” (ISF), parece estar exclusivamente adaptado aos seus
hospedeiros, não replicando em células de vertebrados. Apesar de ter sido identificado
há mais de três décadas (1975), a verdade é que muito pouco se conhece sobre a sua
biologia. Dado o seu parentesco filogenético com alguns outros flavivírus encontrados
naturalmente em mosquitos de diferentes géneros colhidos em diferentes regiões do
globo, este vírus poderá ser usado como modelo para o estudo de ISF. No entanto,
necessitam do desenvolvimento de ferramentas básicas, tais como clones moleculares
ou baterias de soros contendo anticorpos que reconheçam uma ou mais proteínas
codificadas pelo genoma viral, produzidas, por exemplo, a partir de antigénios virais
produzidos de forma recombinante.
Com este trabalho pretendeu-se a optimização de protocolos que permitiram a
expressão e purificação parcial de quatro proteínas [duas proteínas estruturais (C e E) e duas não estruturais (NS3hel e NS5B)] do CFAV em E. coli, todas elas produzidas
como proteínas de fusão com “caudas” (tags) de hexahistidina nos seus extremos
carboxilo.
Para a expansão do CFAV foram utilizadas células Aedes albopictus (C6/36).
Após a realização da extracção do RNA viral e a obtenção de cDNA, procedeu-se
amplificação, por RT-PCR, das regiões codificantes das proteínas C, E, NS3hel e
NS5B, utilizando primers específicos. Os quatro fragmentos de DNA foram
independentemente inseridos no vector pJTE1.2/blunt usando E. coli NovaBlue como
hospedeira de clonagem e, posteriormente, inseridos em vectores de expressão pET-28b
e pET-29b usando E. coli BL21(DE3)pLysS e Rosetta(DE3)pLysS como hospedeiras de
expressão.
Após da indução, expressão e purificação das proteínas recombinantes C, E, NS3hel e NS5B, foi confirmada a autenticidade destas proteínas produzidas através do método Western Blot com um anticorpo anti-histidina. --------- ABSTRACT: The Cell Fusing Agent virus (CFAV) considered as the first "insect- specific
flavivirus" (ISF) and seems to be uniquely adapted to their hosts, not replicating in
vertebrate cells. Although it has been known for more than three decades (1975), the
truth is very little is known about its biology. Given its close phylogenetic relationship with other flavivirus naturally circulating in various genera of mosquitoes collected from different regions of the globe, this virus could be used as a model for the study of ISF. However, such studies require the development of experimental basic tools, such as molecular clones or serum batteries containing antibodies that recognize one or more proteins encoded by the viral genome, produced, for example, from viral antigens recombinant produced.
In this work, we carried out the optimization of protocols that allowed the
expression and partial purification of four proteins [two structural proteins (C and E)
and two nonstructural proteins (NS3hel and NS5B)] CFAV in E. coli as fusion protein
for c-terminal hexahistidine tags.
For the expansion of the CFAV we used Aedes albopictus (C6/36) cells. After completion of the viral RNA extraction and cDNA obtained, amplification of the coding
regions of the C, E, NS5B and NS3hel proteins was carried out by RT-PCR using
specific primers. The four DNA fragments were independently inserted into the vector
pJTE1.2/blunt using E. coli NovaBlue as cloning host and then inserted into expression vectors pET-28b and pET-29b using E. coli BL21(DE3)pLysS and Rosetta(DE3)pLysS
as expression host.
After induction, expression and purification of recombinant C, E, NS3hel and NS5B proteins Western Blot analyses with an anti-histidine antibody confirmed the
authenticity of these proteins produced.
11
Gerald, Schneikart. "Respiratory syncytial virus fusion protein-specific B cell repertoires induced by natural infection or vaccination." Doctoral thesis, Università di Siena, 2018. http://hdl.handle.net/11365/1050834.
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Respiratory syncytial virus (RSV) infections are the major contributor to acute lower respiratory syndrome in newborns. Infections generally result in hospitalization and sometimes in death. A vaccine is not available yet, despite decades of research. Vaccine development is hampered in consequence of a failed vaccine trial in the 1960s which entailed fatal outcomes. An alternative to direct vaccination of children is maternal vaccination for passive immunization of babies before birth. RSV infects every person repeatedly throughout life which implies that pregnant woman carry RSV-directed memory B cell (MBC) repertoires. A successful maternal vaccine would therefore elicit high titer RSV-neutralizing IgGs by reactivation of pre-existing MBCs which would protect newborns during the first months of life. RSV has two neutralizing antigens, of which the fusion protein (RSV F) is the most promising vaccine candidate. RSV F mediates the fusion process of viral and cellular membranes, wherefore it exists in a pre-fusion (pre F) or post-fusion (post F) conformation. Even though there is more interest in the pre F conformation as immunogen, the post F conformation may be equally considered as successful vaccine antigen. The pre F conformation is very metastable and readily switches to the highly stable post F conformation, which implies that a post F-based vaccine would be more cost-efficient to produce. More importantly, several neutralizing epitopes are preserved on the post F conformation and a substantial amount of RSV F-directed MBCs induced by natural infection are actually pre/post F cross-reactive. Since most of the pre/post F cross-reactive MBCs were previously shown to have higher affinities for the post F conformation, a post F-based vaccine may be ideal for reactivation and clonal expansion of MBCs which express neutralizing B cell receptors (BCRs). Every antigen, also RSV post F protein, has its own signatures within BCR repertoires because of preferential selection of BCR characteristics for B cell clonal expansions. In order to understand how RSV F protein shapes BCR repertoires, RSV F-directed BCRs were isolated from a healthy blood donor and three vaccinees who received an RSV post F vaccine. BCR repertoire analysis confirmed the assumption that the pre and post F protein have their own signatures within RSV F-directed BCR repertoires. The different characteristics indicated longer affinity maturation of pre F-reactive MBCs. Furthermore, estimation of clonal relatedness between the pre and post F-binding BCR repertoires from the healthy donor provided indications that a substantial number of the isolated BCRs are actually pre/post F cross-reactive, which confirmed a previous study. Analysis of the BCR repertoires isolated from the RSV post F-vaccinees showed that the vaccine induced a biased MBC response with preferential BCR characteristics. There were several implications that the post F-vaccine expanded primarily pre/post F cross-reactive MBCs. In contrast to the high variability of pre/post F cross-reactive BCRs induced by natural infection, the vaccine-induced MBC response indicated a skewed selection of VH4 gene family-encoded BCRs for clonal expansion and affinity maturation. More importantly, estimation of clonal relatedness revealed convergent MBC responses between the three analyzed subjects, while several MBC lineages shared stereotypic characteristics with pre F-binding BCRs or RSV-neutralizing antibodies. Some of the supposedly pre/post F cross-binding or neutralizing BCR sequences were expressed as mAbs and functionally characterized. RSV pre/post F cross-binding and neutralization activities could be demonstrated for all of the expressed mAbs. This project demonstrated the potential of ‘clonal’ grouping as novel reverse approach to screen BCR repertoires for functional antibodies.
12
Whyte, Paul. "Identification and characterisation of protective B cell epitopes on the fusion protein of respiratory syncytial virus." Thesis, University of Reading, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241353.
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Conner, Rebecca. "The Effects of Adjacent Cell Fusion and Ultraviolet Radiation Exposure on Viral Plaque Formation with Herpes Simplex Virus Type I." TopSCHOLAR®, 1986. http://digitalcommons.wku.edu/theses/1906.
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In mammalian cell-virus systems, it has been observed that damage caused by exposure of the cell to ultraviolet radiation (UV) will result in an increase in viral plaque development rate. This phenomenon is termed the Large Plaque Effect (LPE). Apparently, viral plaque development increases at a faster rate for Herpes Simplex Virus (HSV) when it is assayed on certain UV-irradiated mammalian cells. The consequence of this increase in plaque development rate is that viral plaques appear larger on irradiated monolayers of cells when compared to plaques that developed on unirradiated cellular monolayers.
The cause of the LPE is not yet understood. It is though that the enhancement of plaque development, due to UV damage, is a manifestation of the excision repair mechanisms operating on the cellular genome. It is known that agents that act like UV and inhibit DNA synthesis, such as hydroxyurea, caffeine, and the carcinogen N-acetoxy-2-acetylaminofluorene, can produce the LPE. Conversely, cyclohexamide, which inhibits de novo protein synthesis, can completely prevent the LPE caused by UV. There is also some evidence of a cellular membrane effect involved in generating a non-UV LPE as observed in work with dimethyl sulfoxide. In addition, certain syncytial mutants of HSV are known to enhance membrane fusion.
14
Matheny, Elizabeth Lane. "Contribution of the membrane-proximal region of the vesicular stomatitis virus gycoprotein to host cell entry and membrane fusion." View the abstract Download the full-text PDF version, 2009. http://etd.utmem.edu/ABSTRACTS/2009-043-Matheny-index.htm.
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Thesis (Ph.D.)--University of Tennessee Health Science Center, 2009.<br>Title from title page screen (viewed on February 3, 2010). Research advisor: Michael A. Whitt, Ph.D. Document formatted into pages (x, 91 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 81-90).
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Ke, Lijing. "Mechanism of anti-influenza virus activity of Maillard reaction products derived from Isatidis roots." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/6501.
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The cyto-protective compositions and effects of antiviral Maillard reaction products (MRPs) derived from roots of Isatis indigotica F. were examined using biochemical and biophysical methods. The Maillard reaction was identified as the main source of compounds with antiviral activity, an observation which has led to the proposal of a new class of active compounds that protect cells from influenza virus infection. In the roots, arginine and glucose were revealed to be the predominant reactants for the Maillard reaction. Significant anti-influenza virus effects were demonstrated in the RIE MRPs derived from the roots (RIE refers to the ‘radix Isatidis extracts’), and in Arg-Glc MRPs which are synthesised with arginine and glucose. Arg-Glc MRPs were confirmed as suitable models for the study of the antiviral effects of the root extracts. Furthermore, RIE MRPs and Arg-Glc MRPs were found to bind to the plasma membranes of erythrocytes and MDCK cells, and altered their properties. A novel antiviral mechanism was proposed: that MRPs achieve their cyto-protective effects by binding to the cell membrane rather than by direct action on viral particles. To validate the proposed mechanism, the interaction between MRPs and membrane lipids was investigated by biophysical experiments with phospholipids bilayers. Arg-Glc MRPs affected the rigidity of lipid packing in monolayers and bilayers, while RIE MRPs enhanced the fluidity. Both types of MRPs inserted into the hydrophobic core of bilayers, to differing extents, and induced the stabilisation or destabilisation of bilayers in a concentrationdependent manner. At certain concentrations, MRPs prevented the lamellar structure of bilayers from being destabilised by a viral fusion peptide, improved the lipid order and thereby inhibited cell-virus membrane fusion. The mechanism of the anti-influenza virus activity of RIE was therefore correlated to the interaction between MRPs and phospholipid bilayers, an integral component of the plasma membrane.
16
Boutin, Elodie. "Mécanisme d'inhibition de la fusion membranaire du virus de l'hépatite C par différents composés : l'arbidol, la silymarine et les molécules la composant." Thesis, Lyon 1, 2010. http://www.theses.fr/2010LYO10244.
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L'infection par le virus de l'hépatite C (VHC) est un problème de santé publique majeur car en absence de vaccin et de thérapie suffisamment efficace, l’infection peut dégénérer en carcinome hépatocellulaire. Il est alors important d’identifier de nouvelles cibles thérapeutiques et de développer de nouveaux antiviraux. Ainsi, nous avons étudié l'activité anti-VHC de différents composés : l'arbidol (Arb), la silymarine (SM) et les molécules la composant, notamment la silibinine (SbN). Ces composés ont l'avantage d'être déjà utilisés en médecine humaine depuis de nombreuses années et ont ainsi prouvé leur innocuité. Ils présentent un large spectre antiviral et inhibent plusieurs étapes du cycle viral, dont la fusion membranaire. Cette étape du cycle est intéressante à cibler car le virus serait bloqué précocement, avant de provoquer des dommages cellulaires.Nous avons approfondi notre connaissance du mécanisme d'inhibition de la fusion par Arb en montrant par différentes stratégies qu'il s'associe avec les phospholipides à l'interface membranaire et interagit avec des résidus aromatiques. Cela suggère que Arb pourrait former durant le processus de fusion un complexe entre glycoprotéine virale et membrane, permettant d'inhiber les changements conformationnels de la glycoprotéine, nécessaires à la fusion. De même SM et ses composés inhibent la fusion de pseudoparticules de HCV, probablement en stabilisant les membranes impliquées dans le processus. Enfin, nous avons observé une activité antivirale et anti-inflammatoire très différente entre deux formulations de SbN. Tous ces résultats sont discutés dans le contexte actuel d'un arsenal thérapeutique anti-HCV qui reste limité<br>Infection by the hepatitis C virus (HCV) is a major public health problem since the infection can lead to hepatocellular carcinoma in the current absence of vaccine and effective treatment. It is therefore important to identify new therapeutic targets and to develop novel antiviral drugs. Here we studied the anti-HCV activity of two compounds : arbidol (Arb), the herbal extract silymarin (SM) and molecules therein, including silibinin (SbN). These compounds are already in use in human medicine for several years and have proven safety. They display a broad antiviral spectrum and inhibit several steps of the virus life cycle, including membrane fusion. This step is very interesting to target, since the virus could be blocked upstream the cellular damages it could induce. Using different biophysical strategies, we showed that Arb associates with phospholipids at the membrane interface and interacts with aromatic residues. This suggests that Arb could form during the fusion process a complex between viral glycoprotein(s) and membrane, leading to the inhibition of the conformational changes within the glycoprotein that are required during the fusion process. SM and its components inhibit fusion of HCV pseudoparticles, probably by stabilizing the membranes involved in this process. Finally, we observed different antiviral and anti-inflammatory activities between two different formulations of SbN. Knowledge of these antiviral mechanisms should lead to innovative therapeutic strategies against HCV
17
Andersson, Christin. "Production and delivery of recombinant subunit vaccines." Doctoral thesis, KTH, Biotechnology, 2000. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3027.
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<p>Recombinant strategies are today dominating in thedevelopment of modern subunit vaccines. This thesis describesstrategies for the production and recovery of protein subunitimmunogens, and how genetic design of the expression vectorscan be used to adapt the immunogens for incorporation intoadjuvant systems. In addition, different strategies fordelivery of subunit vaccines by RNA or DNA immunization havebeen investigated.</p><p>Attempts to create general production strategies forrecombinant protein immunogens in such a way that these areadapted for association with an adjuvant formulation wereevaluated. Different hydrophobic amino acid sequences, beingeither theoretically designed or representing transmembraneregions of bacterial or viral origin, were fused on gene leveleither N-terminally or C-terminally to allow association withiscoms. In addition, affinity tags derived from<i>Staphylococcus aureus</i>protein A (SpA) or streptococcalprotein G (SpG), were incorporated to allow efficient recoveryby means of affinity chromatography. A malaria peptide, M5,derived from the central repeat region of the<i>Plasmodium falciparum</i>blood-stage antigen Pf155/RESA,served as model immunogen in these studies. Furthermore,strategies for<i>in vivo</i>or<i>in vitro</i>lipidation of recombinant immunogens for iscomincorporation were also investigated, with a model immunogendeltaSAG1 derived from<i>Toxoplasma gondii</i>. Both strategies were found to befunctional in that the produced and affinity purified fusionproteins indeed associated with iscoms. The iscoms werefurthermore capable of inducing antigen-specific antibodyresponses upon immunization of mice, and we thus believe thatthe presented strategies offer convenient methods for adjuvantassociation.</p><p>Recombinant production of a respiratory syncytial virus(RSV) candidate vaccine, BBG2Na, in baby hamster kidney(BHK-21) cells was investigated. Semliki Forest virus(SFV)-based expression vectors encoding both intracellular andsecreted forms of BBG2Na were constructed and found to befunctional. Efficient recovery of BBG2Na could be achieved bycombining serum-free production with a recovery strategy usinga product-specific affinity-column based on a combinatoriallyengineered SpA domain, with specific binding to the G proteinpart of the product.</p><p>Plasmid vectors encoding cytoplasmic or secreted variants ofBBG2Na, and employing the SFV replicase for self-amplification,was constructed and evaluated for DNA immunization against RSV.Both plasmid vectors were found to be functional in terms ofBBG2Na expression and localization. Upon intramuscularimmunization of mice, the plasmid vector encoding the secretedvariant of the antigen elicited significant anti-BBG2Na titersand demonstrated lung protective efficacy in mice. This studyclearly demonstrate that protective immune responses to RSV canbe elicited in mice by DNA immunization, and that differentialtargeting of the antigens expressed by nucleic acid vaccinationcould significantly influence the immunogenicity and protectiveefficacy.</p><p>We further evaluated DNA and RNA constructs based on the SFVreplicon in comparison with a conventional DNA plasmid forinduction of antibody responses against the<i>P. falciparum</i>Pf332-derived antigen EB200. In general,the antibody responses induced were relatively low, the highestresponses surprisingly obtained with the conventional DNAplasmid. Also recombinant SFV suicide particles inducedEB200-reactive antibodies. Importantly, all immunogens inducedan immunological memory, which could be efficiently activatedby a booster injection with EB200 protein.</p><p><b>Keywords</b>: Affibody, Affinity chromatography, Affinitypurification, DNA immunization, Expression plasmid, Fusionprotein, Hydrophobic tag, Iscoms, Lipid tagging, Malaria,Mammalian cell expression, Recombinant immunogen, RespiratorySyncytial Virus, Semliki Forest virus, Serum albumin,<i>Staphylococcus aureus</i>protein A, Subunit vaccine,<i>Toxoplasma gondii</i></p>
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Corey, Elizabeth Ann. "Characterization of the Relationship Between Measles Virus Fusion, Receptor Binding, and the Virus-Specific Interaction Between the Hemagglutinin and Fusion Glycoproteins: a Dissertation." eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/221.
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Measles (MV) virions, like those of other enveloped viruses, enter cells by fusing their lipid membranes with those of the target host cells. Additionally, infected tissues often possess giant multinucleate cells, known as syncytia, which are formed by fusion of infected cells with uninfected neighbors. Expression of both the MV attachment (H) and fusion (F) proteins is required for membrane fusion. MV H mediates receptor binding in order to bring the two membranes into close proximity prior to F activation and is thought to trigger F activation through a specific interaction between the two proteins.
Although measles H and F are efficiently transported to the cell surface when expressed independently, evidence has been reported in support of an intracellular interaction between the two proteins that can be detected using an ER co-retention approach. However, it was not determined if the putative co-retention was specific to the two measles glycoproteins, as is their ability to complement each other for efficient fusion promotion. Thus, in this thesis, the formation of an intracellular complex between MV H and F was re-examined. Consistent with the formation of an intracellular complex, cell surface expression and receptor binding of untagged wt MV H is slightly reduced by co-expression of an excess of ER-tagged MV F compared to co-expression with wt F. However, the reduction in surface expression is non-specific in that it can also be induced with heterologous proteins of NDV, which lack significant homology with those of MV. Although this approach did not detect a specific intracellular interaction between MV H and F, it cannot be ruled out that there is a weak association of the proteins that is undetectable by this method. This led to the use of an alternative approach to investigate the cellular site(s) of interaction between the measles H and F proteins.
Consistent with a cell surface interaction between MV H and F, the combination of surface biotinylation and co-immunoprecipitation detects formation of a virus-specific H-F complex. Approximately, 21% of the total amount of MV H at the cell surface can be captured with MV F using an antibody against the latter protein. Two complementary approaches were used to address the relationship between this cell surface interaction and receptor recognition by MV H. First, the proteins were co-immunoprecipitated from the surface of Chinese hamster ovary (CHO) cells, which do not express either MV receptor, CD46 or CD150. Similar levels of MV H can be co-immunoprecipitated with F from the surfaces of parental CHO cells and stably transfected cells that express, human CD46 (CHO-CD46), indicating that binding to CD46 is not the trigger for the H-F interaction. Second, MV H proteins, carrying mutations that dramatically reduce CD46 binding, were shown to co-immunoprecipitate efficiently with F from the surface of HeLa cells. Significantly, these results indicate that MV H and F interact in the absence of, and thus prior to, receptor binding. This is in direct contrast to the NDV HN-F cell surface interaction, which is thought to be triggered by receptor binding.
Identification of the domains of the para myxovirus attachment and fusion proteins that mediate membrane fusion activities is an essential part of understanding the mechanism of fusion. As a result of the H-F interaction prior to receptor binding, MV H attachment to its cellular receptor must result in conformational changes that trigger activation of the F protein. Site-directed mutagenesis analyses of two regions of MV H indicate that a HR domain in the stalk of the attachment protein is essential to the ability of H to activate F. However, either it is not the only region of H that interacts with F or it is indirectly involved in F activation because mutations in the HR do not disrupt MV H-F complex formation at the cell surface. Additionally, the functional interaction between MV H and F may be mediated, at least in part, by Loop 1 of the amino terminus of the C-rich region of the fusion protein. However, the exact role of this region of the F protein in fusion promotion remains to be determined. Importantly, the cell surface interaction between MV H and F proteins appears to be mediated by more that one region of each protein. In contrast to NDV, in no case has a definitive link between any single amino acid difference in MV H or F and an inability to form the cell surface H-F complex been established.
In conclusion, the data presented in this dissertation support a model of measles membrane fusion in which the Hand F proteins form a complex prior to receptor recognition. This complex may hold F in its meta-stable pre-fusion state until binding of H to receptors at the cell surface triggers dissociation of the complex, releasing F to assume its fusogenic form. Importantly, these data also indicate that, although paramyxoviruses may all use the same general process. for promotion of membrane fusion, the mechanism may vary in multiple aspects. A more complete understanding of the means by which measles promotes membrane fusion may direct the development of specific strategies aimed at interfering with the early stages of infection.
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Wychowski, Czeslaw. "Expression de la proteine de capside vp1 du poliovirus dans les bacteries et dans les cellules animales : identification d'un epitope de neutralisation et caracterisation de sequences indispensables a l'accumulation de proteines dans le noyau." Paris 7, 1987. http://www.theses.fr/1987PA077173.
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Tam, John. "Expression of the membrane fusion protein of measles virus in insect and mammalian cells using recombinant viruses." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=69521.
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The fusion (F) protein of measles virus is responsible for viral penetration at the plasma membrane and syncytia formation. In order to facilitate future structure/function studies, the F protein was expressed in insect cells using recombinant baculovirus, and in mammalian cells using both vaccinia and adenovirus recombinants. The baculovirus system exhibited the highest levels of recombinant protein expression, but the majority of the product was found to be in the form of detergent-insoluble precursors. Replacing the signal peptide of the F protein with insect-derived sequences did not enhance posttranslational processing. Also, expression of truncated, anchor-minus forms of the F protein did not result in improved solubility or secretion. The vaccinia virus recombinant VF exhibited higher levels of expression than measles virus-infected cells. However, the majority of the F protein was expressed as insoluble precursors. The biologically active portion, represented by the proteolytically cleaved F protein, was found to be soluble. The vP455 vaccinia virus recombinant and the AdF adenovirus recombinant expressed similar levels of processed F as in measles virus-infected cells. The products of these recombinants were found to be soluble. F protein expressed by vaccinia virus recombinants in primate and murine cells were observed to cause syncytia formation in the absence of measles virus hemagglutinin (H) co-expression. This H-independent fusion could not be inhibited by Z-D-Phe-L-Phe-Gly. When the adenovirus system was used, F protein could not facilitate fusion unless measles virus hemagglutinin was present as well.
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Krishna, Benjamin Anthony Cates. "Investigating and exploiting the latency-associated expression of the human cytomegalovirus gene US28 in early myeloid lineage cells." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/267737.
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Human cytomegalovirus (HCMV) is a betaherpesvirus which establishes a lifelong persistent infection, underpinned by its ability to establish latent infection in early myeloid lineage cells, in the infected host. Although well controlled by a healthy immune system, HCMV causes pathological and life threatening disease in individuals with a compromised or immature immune response, which can come from primary HCMV infection or reactivation of latent infection. Although progress is being made in understanding the mechanisms by which HCMV maintains latency and reactivates, a better understanding is essential towards the aim of targeting and killing latently infected cells. In this thesis, I will present evidence that the HCMV-encoded chemokine receptor homologue US28, which is expressed during latent infection of CD14+ monocytes, is necessary for maintaining HCMV latency in these monocytes and, in the absence of US28 protein expression, HCMV undergoes lytic infection. US28 expression was found to attenuate cellular signalling pathways in latently infected cells; in particular, MAP kinase and NFκB. Interestingly, deletion of the US28 gene or inhibition of the US28 protein resulted in the expression of lytic antigens which allowed detection of infected monocytes by the immune system. This observation may lead to a potential new immunotherapeutic strategy against latent HCMV. Having demonstrated that US28 protein is expressed on the surface of latently infected monocytes, I tested whether a new fusion-toxin protein, called F49A-FTP, which binds US28 protein, could be used to target and kill latently infected cells. I developed a protocol for treating latently infected monocytes with F49A-FTP which resulted in a significant reduction in virus reactivation after monocyte differentiation to dendritic cells. I was also able to show that this treatment kills CD34+ progenitor cells, which were experimentally latently infected with HCMV, as well as latently infected monocytes from a healthy, seropositive blood donor. Finally, during my investigations into the role of US28 during HCMV latency, a mass spectrometry screen was performed to measure changes in cellular protein expression when US28 protein is expressed in isolation, in THP-1 monocyte-like cell line. This identified CTCF, a transcription factor which appears to be modified by US28 in THP-1 cells. I showed that CTCF has a repressive effect on the HCMV MIEP, and that CTCF likely plays a role in HCMV latency. In summary, this work provides insights into the role of US28 during HCMV latency, and proposes potential novel therapeutic strategies to kill latently infected cells.
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Krüger, Nadine [Verfasser]. "Interaction of bat-derived paramyxoviruses with chiropteran and non-chiropteran cells: Functional characterization of the African henipavirus and bat-derived mumps virus fusion and attachment glycoproteins / Nadine Krüger." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2014. http://d-nb.info/106520874X/34.
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Baidaliuk, Artem. "Interactions between the insect-specific flavivirus CFAV, its mosquito host aedes aegypti, and co-infecting arboviruses." Electronic Thesis or Diss., Sorbonne université, 2019. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2019SORUS494.pdf.
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Les virus de la dengue (DENV) et du Zika (ZIKV) sont des virus transmis par les moustiques qui causent de graves problèmes de santé publique à travers le monde. Ces virus transmis par des arthropodes (arbovirus) sont des virus à ARN du genre Flavivirus principalement transmis à l'homme par le moustique Aedes aegypti. De plus, les moustiques Ae. aegypti sont naturellement infectés par le cell-fusing agent virus (CFAV), le premier flavivirus spécifique d'insecte (FSI) ayant été décrit. L'évolution du CFAV, ses interactions avec les arbovirus chez les moustiques co-infectés et les réponses immunitaires des moustiques vis-à-vis du CFAV, sont mal connus. Ce travail doctoral a abordé plusieurs de ces aspects peu étudiés de la biologie du CFAV. Premièrement, de nouvelles séquences du génome complet du CFAV ont permis une analyse phylogénétique globale. Un manque de congruence phylogénétique entre le CFAV et Ae. aegypti indique que d’autres facteurs que la structure de la population hôte façonnent la diversité génétique du CFAV. Deuxièmement, dans des expériences de co-infection, le CFAV a inhibé la réplication du DENV et du ZIKV in vitro et leur dissémination in vivo. Ces résultats étayent l'hypothèse que les FSIs pourraient réduire la transmission des arbovirus dans la nature. Troisièmement, il a été montré qu’un élément viral endogène (EVE) dérivé du CFAV dans le génome d’Ae. aegypti régule la réplication du CFAV dans les ovaires via la voie des piARNs. Ce résultat suggère que les EVEs pourraient minimiser des coûts de reproduction associés à l'infection par des virus apparentés. Cette thèse a éclairé les interactions complexes entre le CFAV, Ae. aegypti et les arbovirus<br>Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne viruses that cause major public health problems worldwide. These arthropod-borne viruses (arboviruses) are RNA viruses of the Flavivirus genus that are primarily transmitted among humans by the mosquito Aedes aegypti. In addition, Ae. aegypti mosquitoes are naturally infected with cell-fusing agent virus (CFAV), the first-described insect-specific flavivirus (ISF). Little is known about CFAV evolution, interactions with arboviruses in coinfected mosquitoes, and mosquito immune responses to CFAV. This PhD work addressed such understudied aspects of CFAV biology. First, novel full-genome sequences of CFAV allowed a global phylogenetic analysis. A lack of phylogenetic congruence between CFAV and Ae. aegypti indicates that other factors than host population structure shape CFAV genetic diversity. Second, in coinfection experiments, CFAV inhibited DENV and ZIKV replication in vitro and their dissemination in vivo. These results support the hypothesis that ISFs may reduce arbovirus transmission in nature. Third, a CFAV-derived endogenous viral element (EVE) in the Ae. aegypti genome was found to regulate CFAV replication in the ovaries through the piRNA pathway. This finding suggests that EVEs may help to minimize reproductive costs associated with infection by cognate viruses. Overall, this PhD thesis shed light on the complex interactions between CFAV, Ae. aegypti, and arboviruses
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Tsai, Chang-wu, and 蔡倉吾. "Molecular Mechanisms of the Mouse Hepatitis Virus S Protein Involved in the Cell-Cell Fusion." Thesis, 1999. http://ndltd.ncl.edu.tw/handle/94630417674350045141.
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博士<br>國立臺灣大學<br>生化學研究所<br>87<br>Abstract
The spike (S) glycoprotein of mouse hepatitis virus (MHV) plays a major role in the viral pathogenesis. It is often processed into the N-terminal S1 and the C-terminal S2 subunits that were evident to be important for binding to cell receptor and inducing cell-cell fusion, respectively. As a consequence of cell-cell fusion, most of naturally occurring infections of MHV are associated with syncytia formation. So far, only MHV-2 was identified to be fusion-negative. In this study, the S gene of MHV-2 was molecularly cloned and the nucleotide sequence was determined. The MHV-2 S protein lacks a 12-amino-acid stretch in the S1 hypervariable region from amino acid residues 446 to 457 when compared to the fusion-positive strain MHV-JHM. In addition, there are three amino acid substitutions in the S2 subunit, Tyr-1144 to Asp, Glu-1165 to Asp and Arg-1209 to Lys. The cloned MHV-2 S protein exhibited fusion-negative property in DBT cells as the intrinsic viral protein. Furthermore, similar to the fusion-positive MHV-JHM strain, proteolytic cleavage activity was detected both in DBT cells infected with the fusion-negative MHV-2 and in the transfected cells that expressed the cloned MHV-2 S protein. Domain swapping experiments demonstrated that the 12-amino-acid stretch missing in the MHV-2 S1 subunit, but not the proteolytic cleavage site, was critical for the cell-fusion activity of MHV.
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Vaidya, Naveen K. "Membrane fusion between an influenza virus and a host cell : mathematical models /." 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR46017.
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Thesis (Ph.D.)--York University, 2008. Graduate Programme in Mathematics and Statistics.<br>Typescript. Includes bibliographical references (leaves 166-175). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR46017
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Liu, Kuang-Yang, and 劉光仰. "Effect of P20/GFP fusion on cell to cell movement of Bamboo mosaic virus satellite RNA." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/94102388791107864334.
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碩士<br>國立中興大學<br>生物科技學研究所<br>100<br>Satellite RNAs (satRNAs) are subviral agents which depend on helper viruses and host factors for replication, encapsidation and movement. Bamboo mosaic virus (BaMV), a member of the genus Potexvirus, is the only potexvirus that naturally associates with a satRNA, designated satBaMV, which encodes a 20 kDa RNA-binding protein (P20) involved in efficient long-distance movement and replication of satBaMV. The aims of this study were to investigate the abilities of other non-cognate viruses in supporting the replication of satBaMV and to explore the effects of P20 phosphorylaion on the movement of satBaMV. Dimeric satBaMV transgenic plant lines expressing P20 fused with green fluorescent protein (GFP) either at N- or C- terminus were generated. BaMV and two non-cognate viruses, Potato virus X (PVX) and Foxtail mosaic virus (FoMV), were inoculated onto the transgenic plant to monitor their abilities in supporting satBaMV. Through the examination of GFP expressions in the inoculated transgenic plants, it was confirmed that the non-cognate helper FoMV marginally supports the replication of satBaMV, whereas PVX does not. Plasmids harboring GFP fused to the N- or C-terminus of P20 protein with mutations on the serine at amino acid position 11 (S11) to inhibit or mimic the phosphorylation state of P20 were constructed, and there cell-to-cell movement ability were examined in Chenopodium quinoa by fluorescent microscopy. The results revealed that both mutations of P20 reduced the cell-to-cell movement of satBaMV, corroborating the previous finding that phosphorylation of S11 is important for the expression and movement of satBaMV.
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Mulampaka, Shiva Naresh. "Theoretical Studies of the Mechanisms of the Entry of Virus into Cells." Thesis, 2014. http://etd.iisc.ac.in/handle/2005/3082.
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Viruses cause human diseases by entering in to human cells. Many drugs have been developed that act at various stages of viral infection, but they fail due to their toxic side effects and high mutation rates of viruses. Recently, a new class of drugs called entry inhibitors has been developed which acts on the early stages of viral infection. These drugs have been developed by studying the entry process of viruses in to host cells. The success of these drugs, however, is still limited and research is being done to quantify the optimum dosage of these drugs and find new drugs targets.
We developed a mathematical model based on chemical reaction kinetics to estimate the threshold number of complexes between viral and target cell surface proteins necessary for HIV-1 entry into target cells. Our model quantitatively describes data of HIV entry in the presence of several entry inhibitors and presents an avenue for identifying optimal drug levels for restricting HIV entry.
Majority of viruses enter into host cells by either endocytosis of fusion. But when virus enters through endocytosis and when through fusion is still not clear. We developed a theory that predicts the virus entry pathway based on the underlying biophysical properties like membrane bending modulus, viral and cellular receptor concentration and the energy released by the formation of protein complexes. Through this theory of viruses we presented the entry of viruses through fusion or endocytosis on a phase diagram. We validated the phase diagram by comparing it with known pathways of existing viruses. This study may aid in unraveling the entry pathways of new viruses and may also help in identifying new drug targets.
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Mulampaka, Shiva Naresh. "Theoretical Studies of the Mechanisms of the Entry of Virus into Cells." Thesis, 2014. http://hdl.handle.net/2005/3082.
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Viruses cause human diseases by entering in to human cells. Many drugs have been developed that act at various stages of viral infection, but they fail due to their toxic side effects and high mutation rates of viruses. Recently, a new class of drugs called entry inhibitors has been developed which acts on the early stages of viral infection. These drugs have been developed by studying the entry process of viruses in to host cells. The success of these drugs, however, is still limited and research is being done to quantify the optimum dosage of these drugs and find new drugs targets.
We developed a mathematical model based on chemical reaction kinetics to estimate the threshold number of complexes between viral and target cell surface proteins necessary for HIV-1 entry into target cells. Our model quantitatively describes data of HIV entry in the presence of several entry inhibitors and presents an avenue for identifying optimal drug levels for restricting HIV entry.
Majority of viruses enter into host cells by either endocytosis of fusion. But when virus enters through endocytosis and when through fusion is still not clear. We developed a theory that predicts the virus entry pathway based on the underlying biophysical properties like membrane bending modulus, viral and cellular receptor concentration and the energy released by the formation of protein complexes. Through this theory of viruses we presented the entry of viruses through fusion or endocytosis on a phase diagram. We validated the phase diagram by comparing it with known pathways of existing viruses. This study may aid in unraveling the entry pathways of new viruses and may also help in identifying new drug targets.
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Tsai, Yu-Chen, and 蔡佑晨. "Expression of the Envelope Glycoprotein and Establishment of a Cell Fusion Assay of Dengue Virus." Thesis, 2001. http://ndltd.ncl.edu.tw/handle/95596026833282150742.
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碩士<br>國立臺灣大學<br>微生物學研究所<br>89<br>Dengue virus is a positive, single-stranded RNA enveloped flavivirus that is transmitted by mosquitoes, Aedes aegypti and Aedes albopictus. The virus is divided into four serotypes, DEN-1, DEN-2, DEN-3 and DEN-4.
In the process of virus entry, the envelope protein, E protein, plays an important role. It is a glycoprotein of 495 amino acids, with a transmembrane domain at the C-terminal. It is believed to form a stable, non-covalently linked homodimer. The extracellular domains of E glycoprotein are thought to interact with the host cell receptor.
The overall objective of this study is to investigate the dengue virus entry by establishing an E protein-mediated cell fusion assay.
In the first aim, we examined the expression of five DEN-2 E protein constructs, including pD2ME, pD2ME His, E His, pCB8D2J2 and pCB8D2J2VSV, in the mammalian cell line, 293 cells, to identify the ideal dengue E protein expression construct.
In the second aim, we developed a dengue cell fusion assay by cotransfecting NPCTW04 cells with pCB8D2J2VSV (from DEN-2, 16681 strain) and a reporter construct, pET-21a-GFP, followed by coculturing with target cells which were infected with recombinant vaccinia virus. Examination by fluorescent microscope revealed that NPCTW04 cell can fuse with BHK and K562 cells, two known dengue target cells, but not with H9 or Hut78 cells. The infectivity assay in different target cells revealed that DEN-2 (16681 strain) virus can replicate in BHK and K562 cells efficiently, but not in H9 or Hut78 cells. This finding indicated that our cell fusion assay correlated with cellular tropism of dengue virus.
In the third aim, we developed a real-time RT-PCR assay for quantification of DEN-2 and DEN-3 viruses, using the probe and primers targeting a highly conserved region in the capsid gene. This assay was used to study replication kinetics, in comparison with traditional plaque assay and immunofluorescence assay. It is a convenient, sensitive and accurate method of quantification, and has potential for future application.
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Ha, Michael Neul. "In Vitro and In Vivo Studies with Measles Virus and its Interaction with the Mouse Innate Immune System." Thesis, 2012. http://hdl.handle.net/1807/32725.
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Measles is one of the most contagious diseases known to mankind. Despite the availability of a safe and effective vaccine, approximately 164,000 measles-related deaths were recorded in 2008. The inherent restricted host tropism of MV means that the development of authentic rodent models will be a valuable research tool in testing new vaccines and antivirals. In addition to the receptor requirement, mouse innate immunity has been shown to inhibit MV growth. In this thesis, the contributions of several key components of the mouse innate immune system on the inhibition of MV replication were examined. The transcription factor interferon regulatory factor 3 (IRF3), which normally plays a key role in mediating innate immune signaling, contributed relatively little in inhibiting MV replication both in vitro and in vivo. In contrast, the JAK/STAT pathway and the double-stranded RNA inducible protein kinase, PKR, played more important roles in controlling virus replication.
The resurgence of measles in areas where the virus was once thought to be eradicated makes the development of anti-MV treatments essential. Concurrent to the development of an animal model to better study its pathogenesis, we wanted to look at the effect of MV inhibitors on its replication. The MV fusion inhibitor, carbobenzoxy-D-phenylalanine-L-phenylalanine-glycine (ZfFG), was developed in the past to study fusion; however, its mechanism of action has not yet been elucidated. To examine this, spontaneous ZfFG-resistant mutants were generated and characterized. Mutations were found in the HRB region of the fusion (F) protein, and when these were modeled using published paramyxovirus F crystal structures, data suggested that ZfFG targeted a small pocket present between the head and stalk regions of its pre-fusion conformation.
An authentic mouse model of measles developed from findings in this study may allow for in vivo efficacy testing of ZfFG in the future.
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Koslová, Anna. "Replikační bloky viru Rousova sarkomu v savčích buňkách." Doctoral thesis, 2017. http://www.nusl.cz/ntk/nusl-370879.
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One of the important tasks of virology and immunology is to explore the species- and cell-barriers preventing virus horizontal transmission and reveal the ways how viruses overcome these barriers and "adapt" to different species. This work is based on a well- established retroviral model - avian Rous sarcoma virus (RSV) and studies virus replication blocks in mammalian cells at both pre- and post-integration level. Interaction of the viral envelope glycoprotein (Env) with a specific cellular receptor mediates virus entry into cells. Although mammalian orthologues of specific chicken receptors do not support RSV entry, it was observed that some RSV strains are able to enter mammalian cells. Several RSV-transformed rodent cells lines were described and analysis of provirus H20- RSV in one these cells lines (hamster H-20 tumor cell line) showed multiple mutations including two crucial amino acid substitutions in different regions of Env. Substitutions D32G and L378S confer virus transmission to hamster, human and also chicken cells lacking the appropriate receptor. Altered conformation of H20-RSV Env is similar to a receptor-primed (activated) state of Env. This observation indicates that virus can circumvent the need of original cell receptor because of spontaneous Env activation caused by single...
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Štafl, Kryštof. "Molekulární mechanismy buněčné nepermisivity vůči viru Rousova sarkomu." Master's thesis, 2017. http://www.nusl.cz/ntk/nusl-355717.
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Most viruses can infect only a reduced range of organisms and an effective replication is possible only in selected hosts. These hosts are called permissive for the virus. Molecular principles of a nonpermissiveness and viral mechanisms of overcoming replication obstacles are still not clearly elucidated. This thesis discusses the molecular causes of the cellular nonpermissiveness against a model retrovirus - Rous sarcoma virus. The research is conducted on duck cells which are semipermis- sive to the subgroup C of Rous sarcoma virus. The virus can enter those cells, but it is not able to produce enough infectious viral progeny. Two blocks of the viral replication cycle in the duck cells are described in the thesis. The first one is the probably not optimal cellular receptor recognition. The second one is in the late phase of the replication cycle when the viral proteins are synthesized. The amount of the envelope glyco- protein coding mRNA is reduced due to the altered splicing ratios, and the virions produced from the duck cells are less infectious. This block is recessive and can be partially omitted by cell fusions with permissive chicken cells; therefore, the block is not caused by specific restriction fac- tors in sensu stricto. Additionally, the influence of mutations in duck adapted Rous...
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Tseng, Ying Hsin, and 曾縈馨. "Tumorigenicity of a hepatitis B virus core gene deletion mutant encoding a core-polymerase fusion protein in hepatoma cells." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/42078938731125928982.
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碩士<br>長庚大學<br>生物醫學研究所<br>100<br>Hepatitis B virus (HBV) is one of the most important human pathogens in chronic infectious diseases. Chronic hepatitis B virus infection can lead to chronic active hepatitis and liver cirrhosis, which further results in emergence of hepatocellular carcinoma. Naturally occurring mutations in HBV have been found in patients with acute or chronic HBV infection, wherein some mutants develop in certain stages of chronic HBV infection. Of these HBV mutations, core-gene-defective mutants have been found in some special patient groups. Our present study aims to investigate the prevalence of core-gene-defective HBV mutants in different patient groups as well as the possible pathological effect of the mutants in chronic HBV infection. Firstly, we collected serum samples from patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. To understand the prevalence of core-gene-defective HBV mutants in these three groups of patients, HBV DNA was extracted for PCR amplification of the core genes followed by nucleotide sequencing. We have found a core-gene-defective mutant which causes polymerase overexpression. This type of mutants was presence in patients with hepatocellular carcinoma, but not in patients with chronic hepatitis or liver cirrhosis. Cell growth assay revealed enhanced cell proliferation in hepatoma cells expressing the mutant protein. A lower percentage of apoptotic cells was found in hepatoma cells carrying the mutant HBV, compared to the wild type. Tumorigenecity experiments showed that hepatoma cells carrying the mutant HBV more easily developed xenograft tumors in nude mice. Further studied revealed that the mutant protein physically interacted with miR-10b and miR-183, the miRNAs targeting at many growth enhancing genes.
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Sawatsky, Bevan. "Functional characterization of the attachment glycoprotein of Nipah virus: role in fusion, inhibition of henipavirus infection, generation of chimeric proteins, and assembly of chimeric viruses." 2007. http://hdl.handle.net/1993/2809.
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Nipah virus (NiV) and Hendra virus (HeV) have been identified as the causes of
outbreaks of fatal meningitis, encephalitis, and respiratory disease in Australia,
Malaysia, Bangladesh, and India from 1994 until 2004. In order to accommodate
the unique genomic characteristics of NiV and HeV, a new genus within the
family Paramyxoviridae was created, named Henipavirus. NiV encodes two
surface glycoproteins: the attachment glycoprotein (G) binds to the cellular
receptor for the virus, while the fusion glycoprotein (F) mediates membrane
fusion between the virus and cell membranes. Expression of F and G in the same
cell results in cell-cell fusion in transfected cell monolayers, while expression of F
and G on their own in cell monolayers does not result in fusion. Co-culture of
singly-transfected F and G cells also does not result in fusion. Expression of NiV
G in transgenic CRFK cells results in resistance to NiV- and HeV-induced
cytopathic effect. Additionally, neither NiV nor HeV nucleic acid could be
detected in CRFK-NiV G that had been exposed to NiV or HeV. NiV G
expression also prevents NiV F+NiV G-mediated cell-cell fusion, but does not
affect cell surface expression of either virus receptor, ephrin-B2 and ephrin-B3.
Chimeric glycoproteins derived from NiV G and CDV H were constructed and
characterized. None of the chimeric glycoproteins were able to fuse when coexpressed
with either NiV F or CDV F. Only one of the chimeric glycoproteins (H145/G458) was detected on the cell surface by immunofluorescence assay (IFA).
None of the chimeric glycoproteins altered cell surface expression levels of
ephrin-B2 and ephrin-B3. Finally, recombinant NiV genomes (rNiV and rNiV
eGFPG) were constructed, as well as chimeric CDV genomes with NiV ORF
substitutions (rCDV eGFPH NiVFG and rCDV eGFPH NiVMFG). The only
chimeric virus that was generated, rCDV eGFPH NiVFG, was assessed for its
release from infected cells. rCDV eGFPH NiVFG was poorly released from
infected cells without a freeze-thaw cycle, but was also found to induce the cellsurface
down-regulation of the viral receptors ephrin-B2 and ephrin-B3.<br>October 2007