Academic literature on the topic 'Virus overexpression'

ABSTRACT Retrovirus budding is greatly stimulated by the presence of Gag sequences known as late or L domains. The L domain of human immunodeficiency virus type 1 (HIV-1) maps to a highly conserved Pro-Thr-Ala-Pro (PTAP) sequence in the p6 domain of Gag. We and others recently observed that the p6 PTAP motif interacts with the cellular endosomal sorting protein TSG101. Consistent with a role for TSG101 in virus release, we demonstrated that overexpressing the N-terminal, Gag-binding domain of TSG101 (TSG-5′) suppresses HIV-1 budding by blocking L domain function. To elucidate the role of TSG101 in HIV-1 budding, we evaluated the significance of the binding between Gag and TSG-5′ on the inhibition of HIV-1 release. We observed that a mutation in TSG-5′ that disrupts the Gag/TSG101 interaction suppresses the ability of TSG-5′ to inhibit HIV-1 release. We also determined the effect of overexpressing a panel of truncated TSG101 derivatives and full-length TSG101 (TSG-F) on virus budding. Overexpressing TSG-F inhibits HIV-1 budding; however, the effect of TSG-F on virus release does not require Gag binding. Furthermore, overexpression of the C-terminal portion of TSG101 (TSG-3′) potently inhibits budding of not only HIV-1 but also murine leukemia virus. Confocal microscopy data indicate that TSG-F and TSG-3′ overexpression induces an aberrant endosome phenotype; this defect is dependent upon the C-terminal, Vps-28-binding domain of TSG101. We propose that TSG-5′ suppresses HIV-1 release by binding PTAP and blocking HIV-1 L domain function, whereas overexpressing TSG-F or TSG-3′ globally inhibits virus release by disrupting the cellular endosomal sorting machinery. These results highlight the importance of TSG101 and the endosomal sorting pathway in virus budding and suggest that inhibitors can be developed that, like TSG-5′, target HIV-1 without disrupting endosomal sorting.

ABSTRACT Sphingosine 1-phosphate (S1P)-metabolizing enzymes regulate the level of sphingolipids and have important biological functions. However, the effects of S1P-metabolizing enzymes on host defense against invading viruses remain unknown. In this study, we investigated the role of S1P-metabolizing enzymes in modulating cellular responses to influenza virus infection. Overexpression of S1P lyase (SPL), which induces the degradation of S1P, interfered with the amplification of infectious influenza virus. Accordingly, SPL-overexpressing cells were much more resistant than control cells to the cytopathic effects caused by influenza virus infection. SPL-mediated inhibition of virus-induced cell death was supported by impairment of the upregulation of the proapoptotic protein Bax, a critical factor for influenza virus cytopathogenicity. Importantly, influenza virus infection of SPL-overexpressing cells induced rapid activation of extracellular signal-regulated kinase (ERK) and STAT1 but not of p38 mitogen-activated protein kinase (MAPK), Akt, or c-Jun N-terminal kinase (JNK). Blockade of STAT1 expression or inhibition of Janus kinase (JAK) activity elevated the level of influenza virus replication in the cells, indicating that SPL protects cells from influenza virus via the activation of JAK/STAT signaling. In contrast to that of SPL, the overexpression of S1P-producing sphingosine kinase 1 heightened the cells' susceptibility to influenza virus infection, an effect that was reversed by the inhibition of its kinase activity, representing opposed enzymatic activity. These findings indicate that the modulation of S1P-metabolizing enzymes is crucial for controlling the host defense against infection with influenza virus. Thus, S1P-metabolizing enzymes are novel potential targets for the treatment of diseases caused by influenza virus infection.

Histone deacetylases (HDACs) are vital epigenetic modifiers not only in regulating plant development but also in abiotic- and biotic-stress responses. Though to date, the functions of HD2C—an HD2-type HDAC—In plant development and abiotic stress have been intensively explored, its function in biotic stress remains unknown. In this study, we have identified HD2C as an interaction partner of the Cauliflower mosaic virus (CaMV) P6 protein. It functions as a positive regulator in defending against CaMV infection. The hd2c mutants show enhanced susceptibility to CaMV infection. In support, the accumulation of viral DNA, viral transcripts, and the deposition of histone acetylation on the viral minichromosomes are increased in hd2c mutants. P6 interferes with the interaction between HD2C and HDA6, and P6 overexpression lines have similar phenotypes with hd2c mutants. In further investigations, P6 overexpression lines, together with CaMV infection plants, are more sensitive to ABA and NaCl with a concomitant increasing expression of ABA/NaCl-regulated genes. Moreover, the global levels of histone acetylation are increased in P6 overexpression lines and CaMV infection plants. Collectively, our results suggest that P6 dysfunctions histone deacetylase HD2C by physical interaction to promote CaMV infection.

It is known that levels of the anti-apoptotic protein survivin are reduced during Murine norovirus MNV-1 and Feline calicivirus (FCV) infection as part of the apoptosis establishment required for virus release and propagation in the host. Recently, our group has reported that overexpression of survivin causes a reduction of FCV protein synthesis and viral progeny production, suggesting that survivin may affect early steps of the replicative cycle. Using immunofluorescence assays, we observed that overexpression of survivin, resulted in the reduction of FCV infection not only in transfected but also in the neighboring nontransfected CrFK cells, thus suggesting autocrine and paracrine protective effects. Cells treated with the supernatants collected from CrFK cells overexpressing survivin showed a reduction in FCV but not MNV-1 protein production and viral yield, suggesting that FCV binding and/or entry were specifically altered. The reduced ability of FCV to bind to the surface of the cells overexpressing survivin, or treated with the supernatants collected from these cells, correlate with the reduction in the cell surface of the FCV receptor, the feline junctional adhesion molecule (fJAM) 1, while no effect was observed in the cells transfected with the pAm-Cyan vector or in cells treated with the corresponding supernatants. Moreover, the overexpression of survivin affects neither Vaccinia virus (VACV) production in CrFK cells nor MNV-1 virus production in RAW 267.4 cells, indicating that the effect is specific for FCV. All of these results taken together indicate that cells that overexpress survivin, or cell treatment with the conditioned medium from these cells, results in the reduction of the fJAM-1 molecule and, therefore, a specific reduction in FCV entry and infection.

Various thioredoxin (Trx) proteins have been identified in plants. However, many of the physiological roles played by these proteins remain to be elucidated. We cloned a TRXh-like gene predicted to encode an h-type Trx in tobacco (Nicotiana tabacum) and designated it NtTRXh3, based on the biochemical activity of the NtTRXh3 protein. Overexpression of NtTRXh3 conferred resistance to Tobacco mosaic virus and Cucumber mosaic virus, both of which showed reduced multiplication and pathogenicity in NtTRXh3-overexpressing plants compared with controls. NtTRXh3 overexpression also enhanced tobacco resistance to oxidative stress induced by paraquat, an herbicide that inhibits the production of reducing equivalents by chloroplasts. The NtTRXh3 protein localized exclusively to chloroplasts in coordination with the maintenance of cellular reducing conditions, which accompanied an elevation in the glutathione/glutathione disulfide couple ratio. NtTRXh3 gene expression and NtTRXh3 protein production were necessary for these defensive responses, because they were all arrested when NtTRXh3 was silenced and the production of NtTRXh3 protein was abrogated. These results suggest that NtTRXh3 is involved in the resistance of tobacco to virus infection and abiotic oxidative stress.

ABSTRACT Epstein-Barr virus nuclear antigen protein 3A (EBNA3A) is one of four EBNAs (EBNA-2, EBNALP, EBNA3A, and EBNA3C) through the cellular DNA sequence-specific transcription factor RBP-Jκ/CBF-1/CSL and are essential for conversion of primary B lymphocytes to lymphoblastoid cell lines (LCLs). In the present study, we investigated the effects of EBNA3A on EBNA2 activation of transcription in the IB4 LCL by conditionally overexpressing EBNA3A three- to fivefold. EBNA3A overexpression increased EBNA3A association with RBP-Jκ, did not change EBNA3C association with RBP-Jκ or EBNA or LMP1 expression, decreased EBNA2 association with RBP-Jκ, decreased c-myc expression, and caused G0/G1 growth arrest with prolonged viability. Expression of the fusion protein MycERTM in cells with conditional EBNA3A overexpression restored cell cycle progression and caused apoptosis. In contrast, MycER in the same cells without EBNA3A overexpression enhanced cell proliferation and did not increase apoptosis. These data indicate that EBNA3A overexpression inhibits protection from c-myc-induced apoptosis. In assays of EBNA2- and RBP-Jκ-dependent transcription, EBNA3A amino acids 1 to 386 were sufficient for repression equivalent to that by wild-type EBNA3A, amino acids 1 to 124 were unimportant, amino acids 1 to 277 were insufficient, and a triple alanine substitution within the EBNA3A core RBP-Jκ binding domain was a null mutation. In reverse genetic experiments with IB4 LCLs, the effects of conditional EBNA3A overexpression on c-myc expression and proliferation did not require amino acids 524 to 944 but did require amino acids 278 to 524 as well as wild-type sequence in the core RBP-Jκ binding domain. The dependence of EBNA3A effects on the core RBP-Jκ interaction domain and on the more C-terminal amino acids (amino acids 278 to 524) required for efficient RBP-Jκ association strongly implicates RBP-Jκ in c-myc promoter regulation.

ABSTRACT To explore mechanisms of entry for Ebola virus (EBOV) glycoprotein (GP) pseudotyped virions, we used comparative gene analysis to identify genes whose expression correlated with viral transduction. Candidate genes were identified by using EBOV GP pseudotyped virions to transduce human tumor cell lines that had previously been characterized by cDNA microarray. Transduction profiles for each of these cell lines were generated, and a significant positive correlation was observed between RhoC expression and permissivity for EBOV vector transduction. This correlation was not specific for EBOV vector alone as RhoC also correlated highly with transduction of vesicular stomatitis virus GP (VSVG) pseudotyped vector. Levels of RhoC protein in EBOV and VSV permissive and nonpermissive cells were consistent with the cDNA gene array findings. Additionally, vector transduction was elevated in cells that expressed high levels of endogenous RhoC but not RhoA. RhoB and RhoC overexpression significantly increased EBOV GP and VSVG pseudotyped vector transduction but had minimal effect on human immunodeficiency virus (HIV) GP pseudotyped HIV or adeno-associated virus 2 vector entry, indicating that not all virus uptake was enhanced by expression of these molecules. RhoB and RhoC overexpression also significantly enhanced VSV infection. Similarly, overexpression of RhoC led to a significant increase in fusion of EBOV virus-like particles. Finally, ectopic expression of RhoC resulted in increased nonspecific endocytosis of fluorescent dextran and in formation of increased actin stress fibers compared to RhoA-transfected cells, suggesting that RhoC is enhancing macropinocytosis. In total, our studies implicate RhoB and RhoC in enhanced productive entry of some pseudovirions and suggest the involvement of actin-mediated macropinocytosis as a mechanism of uptake of EBOV GP and VSVG pseudotyped viral particles.

Fusarium graminearum is a major fungal pathogen of wheat and other small grain cereal crops globally, causing Fusarium ear blight (FEB) disease. Like many other plant pathogens, F. graminearum is predicted to produce in planta secreted effector proteins that modulate plant metabolism to suppress or re-programme plant defences. Understanding the molecular functions of Fg effectors will help to elucidate the processes underlying wheat spike colonisation and fungal pathogenicity. With the aim of identifying Fg effector proteins that can suppress host plant defences, I selected using next generation sequencing and bioinformatic analysis, a set of small secreted proteins (SSP) to express in planta using the Barley stripe mosaic virus over-expression system (BSMV-VOX). I then tested whether expression of any of these SSPs enhanced Fg fungal infection of susceptible wheat spikes. Amongst the set of Fg SSP tested, FgSSP8, which encodes a ribonuclease protein, induced strong symptoms of necrosis in N. benthamiana leaves when infiltrated via the BSMV:FgSSP8. Three other genes tested (FgSSP7, FgSSP6 and FgSSP5) enhance FEB disease formation in the majority of the experiments when overexpressed in wheat ears prior to infecting with F. graminearum. FgSSP6 and FgSSP7 belong to the cerato-platanin protein (CPP) family. In several other plant pathogenic fungi, CPPs have been implicated in a number of virulence and plant protection mechanisms, including induction of host plant cell death, binding specific polymers and/or expansin-like activity. FgSSP5 encodes a protein that possesses the pfam domain RALF (Rapid alkalinization factor; PF05498.6). RALF domain-containing proteins are predominately found in plants and play a role in plant development regulating tissue expansion and/or negatively regulating pollen tube elongation. BLAST analyses identified RALF domain containing proteins in a restricted range of different pathogen species. Based on the VOX results and biochemical tests, our hypothesis is that pre-elevated cerato-platanins (FgSSP6 and FgSSP7) levels in the apoplast/surrounding the hyphae could initially shield the hyphae from detection by the plant, but late induce an intense defence response culminating in cell death to benefit the necrotrophic phase of Fg by increasing nutrient availability. FgSSP5 may be a specific virulence factor that manipulates a key plant process, by alkalinising the plant environment during infection, and using the same plant receptor repertoire used to recognise plant proteins. Once the mechanisms are further understood, these genes/proteins could potentially be novel intervention targets either for conventional chemistries and/or for methods such as host-induced gene silencing to achieve FEB disease and/or mycotoxin control. The characterisation of single and double gene deletion F. graminearum mutants is in progress.

Thèse doctorat : Neurosciences : Strasbourg 1 : 2007. Thèse doctorat : Neurosciences : Freiburg - Allemagne : 2007.
Thèse soutenue en co-tutelle. Titre provenant de l'écran-titre. Bibliogr. p. 204-228.

Quelques régions stables ont été découvertes sur les gp d’env du VIH-1 contre lesquelles des patients produisent des anticorps neutralisants. Les épitopes les plus prometteurs se trouvent dans la MPER et sont probablement exposés durant la fusion. Alors que les peptides isolés à partir de cette région ne sont pas parvenus à induire une réaction immunogène neutralisante, des études antérieures suggèrent que la membrane lipidique joue un rôle dans la structuration des antigènes et dans la réponse immunogénique.C’est pourquoi nous étudions la structure de ces épitopes. Cela nécessite leur surexpression, leur purification et leur reconstitution dans des liposomes. Une étude de CD montre qu’ils pourraient changer de conformation, cela sera confirmé par RMN. En outre, leur immunogénicité sera vérifiée par vaccination des souris. En plus, nous trouvons que le cholestérol peut modifier l’orientation des peptides englobant le motif CRAC de la gp41
A few stable regions have been discovered on the HIV-1 env gp against which some patients produce neutralizing antibodies. The most promising ones are located in the MPER and are probably exposed transiently during the fusion. Whereas the peptides isolated from this region failed to induce immunogenic response, previous studies suggest the lipid membrane plays a role in antigens structure and in the immunogenic response.That is why we investigate the structure of these épitopes in membrane models. This requires the production of these épitopes by bacterial overexpression, their purification and their reconstitution in liposomes. A CD study shows that they could undergo a conformational change; this will be confirmed by NMR. Also their immunogenicity will be checked by mice immunization. In addition, we find that cholesterol could change the orientation of peptides encompassing a gp41 CRAC motif

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.

碩士
中興大學
生物化學研究所
94
The triple gene block (TGB) of Bamboo mosaic virus (BaMV) encodes three movement proteins, TGBp1, TGBp2 and TGBp3, which are involved in cell-to-cell movement of viral RNA. However, detailed mechanism of the three TGB proteins to assist viral movement remains to be uncovered. The purpose of this study is to analyze the biochemical properties of TGBp3 as well as the possibility of interaction between TGBp3 and other viral movement components. Amino acid sequence analysis of TGBp3 revealed that it is a 6 kDa protein with an N-terminal hydrophobic transmembrane segment and a hydrophilic C-terminal region. Moreover, about 25% of the amino acid codons in TGBp3 are rarely used by E. coli. Therefore, E. coli Rosetta (DE3) / pLysSRARE carrying plasmid-borne tRNA complementary to the rare codons was used as host for TGBp3 expression. In addition, mutant TGBp3 with the rare codons in the whole coding sequence replaced with the frequently used ones was created. The overexpressed TGBp3 was purified by TALON immobilized metal affinity chromatography and a subsequent gel purification protocol. The identity of the purified protein was confirmed by N-terminal sequencing to be TGBp3. To confirm that TGBp3 is really an integral membrane protein, a micelle-vesicle transition theory was adopted to reconstitute TGBp3-containing proteoliposomes. Ficoll gradient centrifugation of the reconstituted sample revealed that TGBp3 was able to co-migrate with phospholipid, suggesting that it is membrane associated. Chemical treatments of TGBp3-containing liposomes revealed that most of TGBp3 is integrated in lipid-bilayer. In the future, the membrane topology of TGBp3 and the interaction between TGBp3 and other viral movement components will be studied.

碩士
國立中興大學
農業生物科技學研究所
85
The product of open-reading frame 1(ORF1) of bamboo mosaicvirus(BaMV) has been postulated to be a replicase that is involved in the replication of viral genome and in the capping reaction of the positive strand RNA according to the analysis of nucleotide sequence. In this study, the corresponding cDNA was inserted into pET-32a or pET29a and transformed intoEscherichia coli strain BL21(DE3). The recombinant viral protein, fused at N-terminus with thioredoxin , was over-expressed under the induction of isopropyl b-D-thiogalactopyranoside, and partial purified by differential S*Tag affinity chromatography or glycerol gradient centrifugation. The purified enzyme was able to produce P32-labeled RNA fragments upon the addition of particular RNA substrates. Among the RNA molecules tested in this study, such reaction was observed when the substrate was 3*-terrminal fragment of positive strand or negative strand RNA of BaMV. The sensitivity of the P32-labeled RNA products to nuclease S1 digestion suggests that they were arise from the incorporation of a-P32-GTP or a-P32-UTP moiety at 3*-end of RNA substrates. Neither guanylyltransferase or methyltransferase was observed in this study.

Vaccinia virus plays an important role in human medicine and molecular biology ever since the 18th century after E. Jenner discovered its value as a vaccination virus against smallpox. After the successful eradication of smallpox, vaccinia virus, apart from its use as a vaccine carrier, is today mainly used as a viral vector in molecular biology and increasingly in cancer therapy. The capability to specifically target and destroy cancer cells makes it a perfect agent for oncolytic virotherapy. Furthermore, the virus can easily be modified by inserting genes encoding therapeutic or diagnostic proteins to be expressed within the tumor. The emphasis in this study was the diagnosis of tumors using different vaccinia virus strains. Viruses with metal-accumulating capabilities for tumor detection via MRI technology were generated and tested for their usefulness in cell culture and in vivo. The virus strains GLV-1h131, GLV-1h132, and GLV-1h133 carry the gene encoding the two subunits of the iron storage protein ferritin under the control of three different promoters. GLV-1h110, GLV-1h111, and GLV-1h112 encode the bacterial iron storage protein bacterioferritin, whereas GLV-1h113 encodes the codon-optimized version of bacterioferritin for more efficient expression in human cells. GLV-1h22 contains the transferrin receptor gene, which plays an important role in iron uptake, and GLV-1h114 and GLV-1h115 contain the murine transferrin receptor gene. For possibly better iron uptake the virus strains GLV-1h154, GLV-1h155, GLV-1h156, and GLV-1h157 were generated, each with a version of a ferritin gene and a transferrin receptor gene. GLV-1h154 carries the genes that encode bacterioferritin and human transferrin receptor, GLV-1h155 the human ferritin H-chain gene and the human transferrin receptor gene. GLV-1h156 and GLV-1h157 infected cells both express the mouse transferrin receptor and bacterioferritin or human ferritin H-chain, respectively. The virus strains GLV-1h186 and GLV-1h187 were generated to contain a mutated form of the ferritin light chain, which was shown to result in iron overload and the wildtype light chain gene, respectively. The gene encoding the Divalent Metal Transporter 1, which is a major protein in the uptake of iron, was inserted in the virus strain GLV-1h102. The virus strain GLV-1h184 contains the magA gene of the magnetotactic bacterium Magnetospirillum magnetotacticum, which produces magnetic nanoparticles for orientation in the earth’s magnetic field. Initially the infection and replication capability of all the virus strains were analyzed and compared to that of the parental virus strain GLV-1h68, revealing that all the viruses were able to infect cells of the human cancer cell lines A549 and GI-101A. All constructs exhibited a course of infection comparable to that of GLV-1h68. Next, to investigate the expression of the foreign proteins in GI-101A and A549 cells with protein analytical methods, SDS-gelelectrophoresis, Western blots and ELISAs were performed. The proteins, which were expressed under the control of the strong promoters, could be detected using these methods. To be able to successfully detect the protein expression of MagA and DMT1, which were expressed under the control of the weak promoter, the more sensitive method RT-PCR was used to at least confirm the transcription of the inserted genes. The determination of the iron content in infected GI-101A and A549 cells showed that infection with all used virus strains led to iron accumulation in comparison to uninfected cells, even infection with the parental virus strain GLV-1h68. The synthetic phytochelatin EC20 was also shown to enhance the accumulation of different heavy metals in bacterial cultures. In vivo experiments with A549 tumor-bearing athymic nude mice revealed that 24 days post infection virus particles were found mainly in the tumor. The virus-mediated expression of recombinant proteins in the tumors was detected successfully by Western blot. Iron accumulation in tumor lysates was investigated by using the ferrozine assay and led to the result that GLV-1h68-infected tumors had the highest iron content. Histological stainings confirmed the finding that iron accumulation was not a direct result of the insertion of genes encoding iron-accumulating proteins in the virus genome. Furthermore virus-injected tumorous mice were analyzed using MRI technology. Two different measurements were performed, the first scan being done with a seven Tesla small animal scanner seven days post infection whereas the second scan was performed using a three Tesla human scanner 21 days after virus injection. Tumors of mice injected with the virus strains GLV-1h113 and GLV-1h184 were shown to exhibit shortened T2 and T2* relaxation times, which indicates enhanced iron accumulation. In conclusion, the experiments in this study suggest that the bacterioferritin-encoding virus strain GLV-1h113 and the magA-encoding virus strain GLV-1h184 are promising candidates to be used for cancer imaging after further analyzation and optimization
Das Vaccinia Virus spielt in der Humanmedizin und Molekularbiologie eine wichtige Rolle seit E. Jenner im 18. Jahrhundert seinen Nutzen als Impfvirus entdeckt hat. Nach der erfolgreichen Ausrottung der Pocken, wird das Vaccinia Virus heutzutage neben der Anwendung als Impfstoffträger hauptsächlich als viraler Vektor in der Molekularbiologie und in zunehmendem Maße in der Krebstherapie verwendet. Die Fähigkeit Krebszellen gezielt zu zerstören, macht es zu einem perfekten Wirkstoff für die onkolytische Virotherapie. Des Weiteren kann das Virus durch das Inserieren von Genen, die für therapeutische oder diagnostische Proteine kodieren, und im Tumor exprimiert werden, modifiziert werden. Der Schwerpunkt dieser Arbeit war die Tumordiagnose mit Hilfe verschiedener Vaccinia Virusstämme. Viren mit der Fähigkeit, Metalle anzureichern wurden zur Tumordetektion mittels Kernspintomographie hergestellt und auf ihre Nutzbarkeit in Zellkultur und in vivo getestet. Die Virusstämme GLV-1h132, GLV-1h132 und GLV-1h133 tragen das Gen, welches für die zwei Untereinheiten des Eisenspeicherproteins Ferritin kodieren unter der Kontrolle von drei verschiedenen Promotoren. GLV-1h110, GLV-1h111, und GLV-1h112 tragen das Gen, welches für das bakterielle Eisenspeicherprotein Bacterioferritin kodiert, wohingegen das inserierte Gen in GLV-1h113 für die codon-optimierte Version dieses Proteins kodiert, die eine effizientere Expression in humanen Zellen ermöglichen soll. GLV-1h22 beinhaltet das Transferrin-Rezeptor-Gen, welches eine wichtige Rolle in der Eisenaufnahme spielt, und GLV-1h114 und GLV-1h115 beinhalten das murine Transferrin-Rezeptor-Gen. Für eine möglicherweise bessere Eisenaufnahme wurden die Virusstämme GLV-1h154, GLV-1h155, GLV-1h156 und GLV-1h157 mit je einer Version eines Ferritin-Gens und eines Transferrin-Rezeptor-Gens generiert. GLV-1h154 trägt die Gene, die für Bacterioferritin und den humanen Transferrin Rezeptor kodieren, GLV-1h155 trägt die Gene für die humane Ferritin H-Untereinheit und den humanen Transferrin Rezeptor. Zellen, die mit GLV-1h156 und GLV-1h157 infiziert wurden, exprimierten den Maus-Transferrin-Rezeptor und Bacterioferritin beziehungsweise die humane Ferritin-H-Untereinheit. Die Virusstämme GLV-1h186 und GLV-1h187 wurden mit einer mutierten Form der leichten Untereinheit von Ferritin, für die eine Überladung mit Eisen gezeigt wurde, beziehungsweise mit der leichten Untereinheit des wildtypischen Gens ausgestattet. Das Gen, das für den Divalenten Metal Transporter 1 kodiert, welches ein bedeutendes Protein für die Aufnahme von Eisen darstellt, wurde in den Virusstamm GLV-1h102 inseriert. Der Virusstamm GLV-1h184 trägt das magA Gen des magnetotaktischen Bakteriums Magnetospirillum magnetotacticum, welches magnetische Nanopartikel zur Orientierung im Erdmagnetfeld produziert. Zunächst wurde die Infektions- und Replikationsfähigkeit aller Viren analysiert und mit der des Ausgangsstammes GLV-1h68 verglichen, was zeigte, dass alle Viren in der Lage waren humane Krebszellen der Zelllinien GI-101A und A549 zu infizieren. Alle Konstrukte zeigten einen vergleichbaren Infektionsverlauf zu GLV-1h68. Als nächstes, um die Expression der fremden Proteine in GI-101A und A549 Zellen zu untersuchen, wurden SDS-Gelelektrophorese, Western Blots und ELISAs durchgeführt. Die Proteine, welche unter der Kontrolle von starken Promotoren exprimiert wurden, konnten mit diesen Methoden detektiert werden. Um die Expression von MagA und DMT1 zu detektieren, welche unter der Kontrolle des schwachen Promotors exprimiert wurden, wurde die sensitivere Methode RT-PCR angewendet, mit der zumindest die Transkription der Gene nachgewiesen werden konnte. Die Bestimmung des Eisengehaltes in infizierten GI-101A und A549 Zellen zeigte, dass die Infektion mit allen Viren im Vergleich zu uninfizierten Zellen zu einer Eisenanreicherung führte, sogar die Infektion mit dem Ausgangsstamm GLV-1h68. Für das synthetische Phytochelatin EC20 wurde auch eine Anhäufung von verschiedenen Schwermetallen in Bakterienkulturen gezeigt. In vivo Experimente mit A549 tumor-tragenden athymischen Nacktmäusen ergaben, dass Viruspartikel 24 Tage nach der Infektion hauptsächlich im Tumor gefunden wurden. Die von den Viren vermittelte Expression der rekombinanten Proteine in den Tumoren wurde erfolgreich mit Hilfe von Western Blots detektiert. Die Eisenansammlung in Tumorlysaten wurde mit dem Ferrozine Assay untersucht und führte zu dem Ergebnis, dass Tumore, die mit GLV-1h68 infiziert wurden, den höchsten Eisengehalt vorwiesen. Histologische Färbungen bestätigten die Erkenntnis, dass die Eisenansammlung nicht ein direktes Resultat der Insertion von eisenansammelnden Genen in das Virusgenom war. Darüberhinaus wurden tumortragende Mäuse, denen Virus injiziert wurde, mittels Kernspintomographie analysiert. Zwei verschiedene Messungen wurden durchgeführt, wobei die erste Messung sieben Tage nach Virusinjektion mit einem sieben Tesla Kleintier-Scanner durchgeführt wurde und die zweite Messung mit einem humanen drei Tesla Scanner 21 Tage nach Virusinjektion. Tumore von Mäusen, die mit den Virusstämmen GLV-1h113 und GLV-1h184 injiziert wurden, zeigten verkürzte T2- und T2*-Relaxationszeiten, was auf eine verbesserte Eisenakkumulation hinweist. Zusammenfassend deuten die Experimente dieser Studie darauf hin, dass der Virusstamm GLV-1h113, welcher für Bacterioferritin kodiert, und der Virusstamm GLV-1h184, welcher für MagA kodiert, nach weiterer Untersuchung und Optimierung vielversprechende Kandidaten für die Krebs Bildgebung sind

博士
國立成功大學
基礎醫學研究所
96
Dengue virus infection may cause self-limited dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). The viral load is the key point of the pathogenesis of dengue virus infection. Moreover, vascular leakage, thrombocytopenia and hemoconcentration are the hallmarks of dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Permeability aberration may contribute to the plasma leakage, which is most likely affected by certain soluble mediators. Cytokines may play an important role in the vascular leakage. Autophagy, a cellular response against stresses, functions to recycle protein and organelles. However, the roles of autophagy in disease pathogenesis and microbial infection remain unclear. Several viral infections could induce autophagy and replicate using the autophagosome. We found that dengue virus-2 (DV2) infection can induce punctate GFP-microtubule-associated protein light chain (GFP-LC3) and increased expression of the proteolytic derivate LC-II in DV2 infected cells. DV2 infection induced autophagosome formation revealed by immuno-gold labeling of LC3 protein under the transsimission electron microscopy. The mTOR/p70S6K signaling pathway may be involved in DV2-induced autophagy. Further study reveals that DV2 infection-induced autophagy can enhance the replication of the virus in the infected cells. Furthermore, high levels of monocyte chemoattractant protein-1 (MCP-1) were detected in the plasma of DHF patients. DV2 infection can induce MCP-1 production in monocytes, but not in hepatoma and endothelium cells. The production of MCP-1 is dengue virus-dose dependent. Exposure of human umbilical vein endothelial cells (HUVEC) to recombinant human MCP-1 (rhMCP-1) or the cultured supernatant of DV2-infected human monocytes increased vascular permeability of HUVECs. Neutralizing monoclonal antibody of MCP-1 partially reduced vascular permeability. The distribution of the tight junction protein ZO-1 on the cellular membrane of HUVEC was disrupted either by pretreatment of recombinant MCP-1 or the conditioned medium from DV2-infected monocytes. All together, we unravel that dengue virus infection can induce autophagosome formation and increase viral replication. Moreover, increased viral load sustains the overexpression of MCP-1, which further increases the degree of the vascular permeability. Our findings open a new direction toward understanding the pathogenesis of DHF.

The landmark paper discussed in this chapter is ‘Gene therapy for pain: Results of a Phase I clinical trial’, published by Fink et al. in 2011. In this study, the first of its kind, researchers studied the efficacy and safety of a modified herpes simplex virus (HSV) vector used to deliver PENK, which encodes proenkephalin, which is cleaved into the enkephalin peptides Met-enkephalin and Leu-enkephalin, which induce analgesia by acting on opioid receptors. The development of the HSV vector was based in part on results studies in which adenovirus, adeno-associated virus, or non-viral vectors were used to overexpress genes. Overexpression of a variety of large molecules leads to a reduction in pain-related behaviour in animals. Gene therapy in the treatment of chronic pain seems to offer a promising alternative to systemic or highly invasive therapies. However, additional research is needed to determine the safety, effectiveness, and cost-efficiency of this approach.

Japanese Encephalitis Virus (JEV) is a mosquito borne flavivirus infection. Transmission of JEV starts with the infected mosquito bite where human dermis layer act as the primary site of infection. Once JEV makes its entry into blood, it infects monocytes wherein the viral replication peaks up without any cell death and results in production of TNF-α. One of the most characteristics pathogenesis of JEV is the breaching of blood brain barrier (BBB). JEV propagation occurs in neurons that results in neuronal cell death as well as dissemination of virus into astrocytes and microglia leading to overexpression of proinflammatory cytokines. JEV infection results in host cells mediated secretion of various types of cytokines including type-1 IFN along with TNF-α and IFN-γ. Molecule like nitrous oxide (NO) exhibits antiviral activities against JEV infection and helps in inhibiting the viral replication by blocking protein synthesis and viral RNA and also in virus infected cells clearance. In addition, the antibody can also acts an opsonizing agent in order to facilitate the phagocytosis of viral particles, which is mediated by Fc or C3 receptor. This chapter focuses on the crucial mechanism of JEV induced pathogenesis including neuropathogenesis viral clearance mechanisms and immune escape strategies.