Untersuchungen zum intrazellulären Transportmechanismus von Marburgvirus Nukleokapsiden

Das Marburgvirus bildet zusammen mit dem Ebolavirus die Familie der Filoviridae. Beide Viren lösen beim Menschen und bei nicht-menschlichen Primaten ein starkes hämorrhagisches Fie-ber mit hoher Sterblichkeitsrate aus. Die Viren besitzen ein einzelsträngiges, nicht-segmentiertes RNA-Genom in negativ...

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Bibliographic Details
Main Author: Schudt, Gordian
Contributors: Becker, Stephan (Pof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2013
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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.