Vallbracht, Angelika
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Vallbracht, Angelika
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Vallbracht, Angelika
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Vallbracht, A.
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Item-typ:Veröffentlichung, Untersuchungen zur Blockade zellulärer antiviraler Mechanismen durch das Hepatitis A-Virus unter besonderer Berücksichtigung der Proteinkinase R und des Transkriptionsfaktors NF-kappaB(2003-12-17); ; ; Virus infection normally induces expression and secretion of type I interferon (IFN), leading to the establishment of an antiviral state in adjacent cells. In rhesus monkey kidney cells, hepatitis A virus (HAV) suppresses induction of IFN-b on the level of transcription, even after transfection with poly(IC), a dsRNA analogon. Furthermore, HAV is able to establish a persistent infection in cell culture and to suppress apoptosis. The objectives of this work were 1) to transfer the above results into a human cell culture system and 2) to examine possible cellular targets of HAV. Human MRC-5 fibroblasts were infected with HAV, and results show that IFN-b is not induced by infection and that after poly(IC) transfection HAV actively suppresses IFN-b production (determined by plaque reduction assay). Suppression of IFN-b expression occurs on the level of mRNA, as detected by RT-PCR suggesting one or more proteins of dsRNA-dependent signalling pathways to be affected by HAV. Protein kinase R (PKR) is involved in dsRNA-induced apoptosis and furthermore, PKR has been described to be involved in the activation of NF-kB, a transcription factor participating in induction of IFN-b. Autophosphorylation is a hallmark of PKR-activation and therefore, phosphorylation of PKR on Thr-451 was analysed by immunoblotting. In HAV-infected cells, PKR-phosphorylation appeared to be generally reduced, whereas a degradation of PKR could not be detected. When cellular localization of NF-kB was examined by immunofluorescence, poly(IC)-induced nuclear translocation of NF-kB was found to be unaffected and even enhanced by HAV, excluding a role for NF-kB in HAV-mediated suppression of IFN-b. However, HAV-induced activation of anti-apoptotic NF-kB may contribute to survival of HAV-infected cells. The suppression of IFN-b and the influence on PKR, NF-kB and cell vitality may allow HAV to establish a persistent infection in cell culture and might be a prerequisite for successful replication in vivo.Dissertation214 171 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Untersuchung der Rolle von IRF-7 bei der Suppression der Hämatopoese durch das Hepatitis A-Virus(2009-11-11); ; ; The interferon regulatory factor 7 (IRF-7) plays an important role in the monocyte (MO)-to-macrophage (MAC) differentiation. This work shows that hepatitis A virus (HAV) suppresses the activation of IRF-7 by blocking its phosphorylation and in the following its nuclear translocalisation. It is hypothesed that this ability of HAV causes the perturbance of the MO-to-MAC maturation observed in HAV infections.Dissertation236 111 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Entwicklung eines enzymatischen Testes zum Nachweis von Antikörpern gegen Lassavirus in menschlichen Seren unter Berücksichtigung verschiedener Lassavirus Serotypen(2006-02-02); ; Two highly sensitive and specific reverse enzyme- immunoassays were developed, to detect Lassa virus antibodies. IgG and IgM antibodies of the human sera were bound to microtiter plates coated either with Rheumatoid factor or with anti-human à µ-chain antibodies, respectively. For both tests a crude antigen extracted from Lassa virus infected tissue cultures cells was used. This antigen was in turn reacted with a labeled monoclonal antibody to Lassa virus nucleocapsid. The reverse Elisa turned out to detect IgG in serum samples of West African patients with recent Lassa fever and in 76 sera of healthy seropositive (IIF) West Africans with a sensitivity of 100. In 1031 controls samples of healthy Africans, who were negative for IgG and IgM antibodies by indirect immunofluorescence (IIF), also no IgM antibodies could by found by Elisa. Compared to IIF, the specificity of the reverse Elisa for IgG antibodies was only 95%. But upon retesting the 19 discrepant samples with three different Lassa virus antigens by IIF, 14 samples turned out to react positively (titer ≥20) with at least one antigen in IIF. When all 1107 West African serum samples were tested in the Elisa for IgG antibodies to the three different Lassa virus antigen, in about 30% strictly strain-specific reactions were seen.Dissertation275 96 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Inhibition zellulärer antiviraler Abwehrmechanismen durch das Hepatitis A-Virus - eine Analyse der beteiligten viralen Faktoren: Inhibition des IRF-3 -vermittelten Signalweges durch das Nichtstrukturprotein 2B des Hepatitis A-Virus(2007-12-19); ; ; Hepatitis A virus (HAV) antagonizes the innate immune response by inhibition of Newcastle disease virus (NDV)- or dsRNA-mediated interferon-beta (IFN-beta) gene expression. The aim of this dissertation was to investigate which viral properties or factors are involved in this suppression. It could be demonstrated that the nonstructural protein HAV-2B correlates with the ability of HAV to suppress the NDV- or dsRNA-mediated interferon-beta (IFN-beta) gene expression. Further investigations supported this finding that HAV-2B is involved in inhibition of activation of transcription factor IRF-3 by HAV. As IRF-3 is necessary for IFN-beta transcription, inhibition of this factor results in efficient suppression of IFN-beta synthesis. This ability might be of vital importance for HAV, which is an exceptionally slowly growing virus sensitive to IFN-beta, as it allows this virus to establish infection and maintain viral replication for a longer time.Dissertation221 115 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Analyse der Effekte des hepatotropen Cyanobakterientoxins Microcystin und des Hepatitis B- Virus- x Proteins auf die Zellzyklus- Regulation(2003-12-11); ; ; Epidemiological studies have suggested synergistic interactions between chronic hepatitis B virus (HBV) infection and microcystin (MCYST) exposure in the etiology of hepatocellular carcinoma (HCC). As a potent inhibitor of protein phosphatase 1 and 2A could probably induce the hyperphosphorylation of tumorsuppressor proteins, retinoblastoma protein and p53, including their associated cell cycle proteins. The MCYST- mediated hyper-phosphorylation of pRb and p53 in HepG2 cells was confirmed by protein analytical methods and functinal transfection analysis with a luciferase reportergen system. Hyperphosphorylation of tumor suppressor proteins, mediated through inhibition of protein phosphatase 1 and 2A are involved in the deregulation of the cell cycle and a biochemical alternativly pathway of tumor promotion. In addition to prove the possibility of MCYST- associated functional inactivation of p53 we performed apoptosis assays and to consider the influence of MCYST of the subcellular localization of p53 using a p53-GFP- transfection system. The human hepatitis B virus x-protein (HBx) is suspected to play a role in the hepatocarcinogenic process by virtue of its capacity to transactivate oncogenes and serveral other cellular genes. We demonstrated confirmed by transient transfection assays with diverse specific luciferase reportergen systems that HBx is able to stimulate the cell cycle progression and influence the checkpionts controll. Finally, with cotransfection analyses in HepG2 we showed that that MCYST and HBx in combination deregulate the activity of cell cycle proteins such as p53,pRb, E2F and c-myc. Taken together, our results indicate that MCYST and HBx in an initiated human hepatoma cell culture system (HepG2) alone and in combination affected the activity of essential regulators of the G1 phase of the cell cycle.These datas suggest a molecular mechanism by which MCYST and HBx are likely to contribute to viral carcinogenesis.Dissertation274 111 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Etablierung einer PCR-unabhängigen Methode zum Nachweis des Hepatitis C-Virus und Studien zur Entwicklung eines Replikationssystems(2003-12-18); ; ; In this study a method for the specific detection of hepatitis C virus (HCV) negative and positive strand RNA by the hybrid detection assay (HDA) was developed. The principle of this method is a hybridization of the viral RNA with a specific single-strand DNA probe and the detection of the RNA:DNA-Hybrid using an anti-RNA:DNA hybrid antibody. The HDA was optimized for sensitivity and strand specificity with different amounts of synthetic RNA templates and reached a detection limit between 20 and 50 amol.Another intention of this work was the establishment of an efficient cell culture system for HCV. Various cell lines were infected with HCV and searched for the presence of HCV-RNA. Although negative strand RNA was detected by a Tth-based reverse-transcriptase polymerase chain reaction (RT-PCR) in U937- and FRhK4-cells, the development of an efficient in vitro system has been hampered.Nevertheless, this study provides evidence that the RNase L-Inhibitor (RLI) seems to support HCV-replication in vitro.Dissertation235 137 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Untersuchungen zur Suppression der IFN-ß-Induktion durch Proteine des Hepatitis A-Virus, unter besonderer Berücksichtigung des HAV/2B-ProteinsHepatitis A virus antagonizes the innate immune response by inhibition of IRF-3 activation which results in efficient suppression of interferon-β synthesis. This study showed that this ability of the virus is mediated by the viral non-structural proteins 2B and 3ABC. While the processing intermediate 3ABC targets MAVS, 2B might interfere with IRF-3 activation in an indirect manner by associating with intracellular membranes. Deletion of the C-terminal transmembrane domain of 2B results in a loss of function concerning the suppression of IRF 3 activation. In addition to suppression of IRF-3 activation, HAV partially down regulates protein expression which is mediated by the viral non-structural protein 2C. This function of 2C probably depends on its membrane association, too. In the second part of this work could be shown that the suppression of IRF-3 activation and down regulation of protein expression is not due to an adaptation of HAV to growth in cultivated cells because proteins of wildtype viruses show these properties also. These results imply that HAV is able to inhibit IRF-3 activation and partially down regulates protein expression in vivo.Dissertation249 288 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Untersuchungen zu Interaktionen des Hepatitis A-Virus mit zellulären antiviralen MechanismenHepatitis A virus (HAV) infection in vitro results in a persistent noncytopathic infection during which the viral replication is downregulated. This adjustment of HAV replication to a moderate level may contribute to the development and maintenance of persistence in cell culture and the propagation of HAV in vivo. In order to investigate the underlying mechanism, different cellular antiviral systems were examined. A possible replication control via HAV dependent induction of MxA or ISG20 gene expression could be excluded, whereas an investigation of the 2',5'-oligoadenylate synthetase/RNase L system showed obscure results. HAV upregulates RNase L expression at later stages of infection, resulting in an enhanced potential to activate RNase L in persistently infected cells. However, replication kinetics during differential expression of RNase L showed no impact on HAV titre, whereas the establishment of HAV infection in individual cells was partly blocked by RNase L overexpression.Dissertation319 137 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Untersuchungen zur Suppression der durch doppelsträngige RNA induzierten IFN-beta-Expression durch das Hepatitis A-Virus: Inhibition der RIG-I- und TLR3-vermittelten Signalwegezur Aktivierung des Transkriptionsfaktors IRF-3(2006-02-22); ; ; The subject of this dissertation is the suppression of interferon-beta induction by hepatitis A virus (HAV), which is mediated by inhibition of activation of transcription factor IRF-3. In a first approach to localize the cellular target of HAV in the two dsRNA-induced signaling pathways leading to the activation of IRF-3, which are mediated by RIG-I and TLR3, respectively, nuclear translocation of IRF-3 was analyzed by using GFP-IRF-3, and poly(IC) transfection or NDV infection as activating stimuli. HAV efficiently suppressed transloaction of IRF-3, so the upstream cytoplasmic signaling pathway components were examined by reporter assays after overexpression, monitoring IFN-beta-enhancer and IRF-3-activation. It is demonstrated that HAV blocks the RIG-I-mediated pathway upstream of the IRF-3 kinases IKKe and TBK1 to block IFN-beta induction, and that HAV negatively affects the TLR3 pathway as well.Dissertation227 234