Günther, Stephan
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Günther, Stephan
Official Name
Günther, Stephan
Alternative Name
Guenther, Stephan
ORCID
GND
7 results
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Item type:Publication, Aufbau von Replikonsystemen für Altwelt-Arenaviren und Herstellung rekombinanter Mopeia-Viren(2011-12-20); ; ; Lassa virus (LASV) causing hemorrhagic fever, Mopeia virus (MOPV) and lymphocytic choriomeningitis virus (LCMV) are the main representatives of the Old World arenaviruses. Minireplicon systems for MOPV and LCMV were established in analogy to the existing LASV system. The functional and physical integrity of replication complexes was tested by luciferase assay, Northern blotting, and coimmunoprecipitation studies. The minigenomes, NPs, and L proteins of LASV and MOPV could be exchanged without loss of function LASV and MOPV L proteins are functional also with LCMV NP, while the LCMV L protein requires homologous NP for activity. Analysis of LASV/LCMV NP chimeras identified a single LCMV-specific NP residue (Ile-53) and the C terminus of NP as being essential for LCMV L protein function. The defect of LASV and MOPV NP in supporting transcriptional activity of LCMV L protein was not caused by a defect in physical NP-L protein interaction. Furthermore the first reverse genetic system for production of infectious MOPV was established. Overall, new reverse genetic systems were established to study the replication cycle of Old World arenaviruses.doctoral thesis328 120 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Molekulare Diagnostik, Therapie und Epidemiologie von Erregern viraler hämorrhagischer Fieber unter besonderer Berücksichtigung des Lassa-Virus(2011-05-11); ; ; Viral hemorrhagic fevers are a severe problem for public health systems especially in remote tropical areas. Lassa fever, caused by Lassa virus, is endemic in West-Africa. Several hundred thousand cases occur every year. Laboratory diagnostics is essential to reliably diagnose the disease and differentiate it from other febrile infections. The first part of this work describes the development and evaluation of test-systems to detect Lassa virus RNA in clinical samples. Therapy of Lassa fever is limited to supportive measures and treatment with Ribavirin. Several mechanisms of action are discussed to be responsible for the inhibition of Lassa virus by Ribavirin in cell culture. The second part of this work shows that the inhibition of the inosine monophosphate dehydrogenase is not the responsible mechanism if action. In the last part, the molecular epidemiology of Lassa virus in West-Africa is discussed and both a geographic and a temporal analysis of Lassa virus evolution are presented.doctoral thesis224 143 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Strukturelle und funktionelle Charakterisierung des bunyaviralen L-Proteins(2018-05-24); ; ; The order Bunyavirales possesses the largest diversity of species and hosts among all RNA viruses and represents a major health concern not only for low income countries. Despite a rather simple molecular architecture with only a few proteins the different viruses replicate highly efficient in various host species. The L protein is the key player of viral replication and besides genome replication performs the specific and highly efficient cap-snatching mechanism to prime the translation of the viral genome which requires an endonuclease activity. This mechanism has most intensively been studied for the related influenza virus. So far there is evidence for cap-snatching endonucleases in the N-terminus of three bunyaviral L proteins which despite low sequence conservation show structural and functional homology to the influenza endonuclease. The CCHFV endonuclease has been recombinantly expressed and recovered in high purity. The protein shows structural homology in comparision to related nairoviruses but is larger in size compared to related bunyaviral families which can be attributed to two insertions in the sequence. The lack of in-vitro activity and a different coordination of manganese ions in the active site reveal closer relation to the endonuclease of the Arenaviridae family. The RVFV endonuclease has been identified in a recombinantly expressed N-terminal fragment of the L protein. The active site binds manganese ions and cleaves ssRNA in- vitro which is consistent with recently published data on related bunyaviral endonucleases and proves functional homology. Detailed structural data were not obtained due to poor protein quality. This thesis presents fundamental new insights in the cap-snatching endonucleases of two uncharacterized bunyaviral endonucleases which further underlines the structural and functional homology of the endonucleases and validates the conservation of the cap-snatching mechanism. The identified proteins are a valuable tool for high-throughput screening applications and thus the presented work provides an important progress in the future development of drugs to combat bunyaviral infectious diseases.doctoral thesis471 148 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Aufbau eines reversen Genetik-Systems für Lassa-Virus zur Untersuchung viraler Replikationsmechanismen(2006-12-20); ; ; Lassa virus belongs to the family of Arenaviridae and is the causative agent of a hemorrhagic fever in humans. Molecular mechanisms of genome replication and gene expression are largely unknown. To facilitate the functional analysis of this virus, a replicon system was developed. Using this system, the putative 19-nucleotide long promoter at the RNA termini and the RNA-dependent RNA polymerase domain (~500 amino acids) predicted within the L protein was characterised. For promoter analysis, each nucleotide of the 3' and 5' ends of the RNA genome analogue was changed into the 3 remaining residues by mutagenesis. Promoter activity was dependent on specific residues at both termini as well as specific base pairings predictably formed between the 3' and 5' termini, indicating that the functional promoter forms a 3'-5' duplex. The RNA polymerase domain was characterised by mapping essential amino acids. Potentially relevant residues were identified using secondary structure prediction and data from crystal structures of known RNA polymerases. A novel method was established for rapidly generating L protein mutants by PCR. Three types of mutations were generated and functionally tested: conservative changes in the predicted catalytic sites (motifs pre-A to E); a charged-to-alanin scan throughout the domain, and 2Xglycin insertions to search for loop regions (in total ~150 mutants). Northern blot analysis revealed one mutant that selectively impairs RNA transcription but not genome replication. Based on the data, a model of the RNA polymerase domain of Lassa virus is proposed. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.doctoral thesis263 210 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Wirkung von Cytokinen und cytokininduzierten Proteinen auf die Replikation von Lassa-Virus und Lymphocytärem Choriomeningitis Virus(2003-07-24); ; ; Lassa virus is thought to be resistent to the effects of interferon (IFN). This study demonstrates that Lassa virus replication is inhibited by IFN-alpha and IFN-gamma, but not tumor necosis factor alpha (TNF-alpha), in Huh7and Vero cells. However, the effects were orders of magnitudes smaller compared to vesicular stomatitis virus. Lassa virus AV, which had causeda fulminant Lassa fever, was somewhat more inhibited than Lassa virus NL and CSF, which had been associated with less severe disease. Mostimportantly, there were no clear differences in response to IFN-alpha, IFN-gamma, and TNF-alpha between the Lassa virus strains and the strains CH-5692, Armstrong, and WE of lymphocytic choriomeningitis virus (LCMV),a related Old World arenavirus which is less pathogenic to humans than Lassa virus. Using inducible overexpression cell lines, no evidence for a role of the IFN-stimulated proteins promyelocytic leukemia protein(PML) and Sp100 in the IFN-mediated inhibition of Lassa virus and LCMV replication was found. This and the previous finding that PML knockout facilitates LCMV replication in vitro and in vivo (M. Djavani et al., J.Virol. 75:6204-6208; W. V. Bonilla et al., J. Virol. 76:3810-3818) would fit with the hypothesis that PML is a mediator in the antiviral pathway rather than a direct effector protein. In conclusion, the data show thatLassa virus is susceptible to IFN-alpha and IFN-gamma which is of relevance for the design of antiviral strategies against Lassa fever, aiming at enhancing the IFN response. In contrast to influenza A viruses (S. H.Seo et al., Nat. Med. 8:950-954), there appears to be no correlation between the pathogenicity of Old World arenaviruses and their sensitivity to IFN.doctoral thesis241 132 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Etablierung und Charakterisierung von Mausmodellen für Lassa-Fieber(2011-12-20); ; ; In this study, transgenic HHD mice expressing human HLA A2.1 molecule instead of murine MHC-I molecules have been established as an animal model for Lassa virus (LASV) infection. It was found that HHD mice, in contrast to wild-type mice (C57BL/6), were susceptible for the LASV strain Ba366. LASV infection induced a severe illness with some fatalities. Genetic and functional T-cell depletions have shown that CD4 and CD8 positive T cells play a crucial role in pathogenesis in HHD mice. Immunological and histological findings indicate that the disease is associated with activation of macrophages, which is presumably T-cell dependent. Another murine animal model for LASV infection was established on the basis of interferon-alha/beta-receptor-deficient mice (A129). A129 mice were susceptible for all tested LASV strains and were affected by the infection. Biochemical, virological and histological analyzes showed similarities to Lassa fever disease. The suitability of A129 mouse model for antiviral compound screening was tested with ribavirin, the standard drug for human Lassa fever.doctoral thesis315 131 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Identifizierung unbekannter Funktionen im Lassa-Virus L-Protein(2010-07-01); ; ; The central domain of the 200-kDa Lassa virus L protein is a putative RNA-dependent RNA polymerase. N- and C-terminal domains may harbor enzymatic functions important for viral mRNA synthesis, including capping enzymes or cap-snatching endoribonucleases. In this work it was shown that the N-terminal region in L protein is important for mRNA, but not for genome synthesis. Bioinformatics and cell biological experiments lend support to the hypothesis that this region could be part of a cap-snatching enzyme.doctoral thesis246 141