Untersuchungen zum intrazellulären Transportmechanismus von Marburgvirus Nukleokapsiden
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Philipps-Universität Marburg
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Abstract
Marburgvirus together with Ebolavirus constitute the family of Filoviridae. Both viruses possess a non-segmented single strand RNA genome in negative orientation and therefore belong to the order Mononegavirales. Marburgvirus causes severe hemorrhagic fever with high case fatality rates in humans and non-human primates. Seven genes are encoded by the viral ge-nome, five of the gene products, the nucleoprotein NP, the viral proteins VP24, VP30 and VP35 together with the polymerase L form the nucleocapsid. The nucleocapsid is surrounded by the matrix protein VP40 which connects the nucleocapsid with the lipid bilayer in which the glyco-protein GP is inserted. Viral replication takes place in the cytoplasm. This doctoral thesis inves-tigated the intracellular transport of nucleocapsids. Analyses were based on molecular biologi-cal and imaging techniques, mainly live-cell imaging, accompanied by confocal microscopy and scanning electron microscopy. To generate a fluorescently labeled Marburgvirus, a reverse genetic system was used to integrate an additional open reading frame into the viral genome, coding for a red fluorescently labeled VP40 (RFP-VP40). The constructed genome was used to rescue a recombinant Marburg virus (rMARVRFP-VP40) which expressed the fluorescent VP40. Expressing a green fluorescent VP30 (VP30GFP) in cells infected with rMARVRFP-VP40 both, the nucleocapsid and the matrix protein could be visualized in order to study their transport and interaction. By dual-color imaging, virally-induced inclusions were identified as the origin of new nucleocapsids that were transported to the plasma membrane. Although VP40 was re-cruited into inclusions, no detectable amount of VP40 was detected in freshly released nucle-ocapsids. Once released into the cytoplasm, nucleocapsids were transported in a seemingly random actin-based long-distance transport at speeds between 200 and 500 nm/s. Microtu-bules were not essential for the intracellular movement of Marburg virus nucleocapsids. Once nucleocapsids reached the cell periphery, movement slows down to ± 100 nm/s. At the plasma membrane, nucleocapsid became associated with the matrix protein which was a prerequisite for the subsequent exit process. Interaction of the matrix protein with the nucleocapsids is supported by Tyrosine phosphorylation of VP40 and also enables the recruitment of nucle-ocapsids into filopodia. Inside filopodia nucleocapsids were cotransported with an actin-based motor protein, Myosin 10.
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Created: 2013Issued: 2013-12-10Updated: 2013-12-10
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
LebendzellmikroskopieLife-cell microscopyVirologieactinAktinGFPvirologyGFPcytosceleton
DFG-subjects
MikroskopieVP30VP40Marburgvirus
DDC-Numbers
610
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Schudt, Gordian (1045774677): Untersuchungen zum intrazellulären Transportmechanismus von Marburgvirus Nukleokapsiden. : Philipps-Universität Marburg 2013-12-10. DOI: https://doi.org/10.17192/z2013.0706.
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This item has been published with the following license: In Copyright