Charakterisierung der Rolle des endosomalen Recycling-Systems für den intrazellulären Transport des Marburg Virus Glykoproteins

Das Marburg Virus (MARV), das zur Familie der Filoviridae gezählt wird, ist ein Erreger schwerer Fieber mit Letalitätsraten von bis zu 90 %. Das Glykoprotein GP stellt das einzige Oberflächenprotein des MARV dar und daher ist sein korrekter Transport zu den „Budding-Sites“ an der Plasmamembran sowie...

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Bibliographic Details
Main Author: Halwe, Sandro
Contributors: Becker, Stephan (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:German
Published: Philipps-Universität Marburg 2019
Medizin
Subjects:
GP
Online Access:PDF Full Text
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Table of Contents: Marburg virus (MARV), belonging to the family Filoviridae, is the causative agent of severe fever with lethality rates of up to 90%. The glycoprotein GP represents the only surface protein of MARV and thus its proper transport to the budding-sites at the plasma membrane and subsequent incorporation into virus particles is essential for the viral infection cycle. Regarding delivery to the cell surface the current model presumes trafficking of GP via the classical secretory pathway involving endoplasmic reticulum, Golgi apparatus and trans-Golgi-network. However, neither the exact transport steps between the Golgi apparatus and the plasma membrane nor host factors regulating trafficking of GP are known. Therefore, the aim of this study was to characterize the post-Golgi transport of MARV-GP and to identify potential subcellular compartments and host factors mediating trafficking of GP to the cell surface in general and to the VP40-enriched budding-sites in particular. In the first part of this thesis, it was shown by confocal microscopy that MARV-GP colocalizes with marker proteins of recycling endosomes (Transferrin receptor, GFP-Rab11) in transiently transfected as well as in MARV-infected cells. Since a re-endocytosis from the plasma membrane could be excluded, these results were a hint for a role of the endosomal recycling system during biosynthetic transport of GP between the Golgi apparatus and the cell surface. In the second part, it was demonstrated that functional ablation of the endosomal recycling system by overexpressing a dominant-negative mutant of Myosin Vb (Myosin Vb Tail) leads to an aberrant distribution of GP which as a consequence accumulates in or near perinuclear Myosin Vb Tail aggregates. By blocking GP exit from the Golgi apparatus, it was further shown that GP enters the Myosin Vb-positive compartment after leaving the Golgi. Furthermore, the interference with GP trafficking by dominant-negative Myosin Vb Tail significantly reduced GP levels at the cell surface in general and targeting of GP to VP40-enriched sites at the plasma membrane in particular. In contrast, distribution of MARV matrix protein VP40 as well as of MARV-NP, the major component of viral nucleocapsids, were not affected by the expression of Myosin Vb Tail pointing towards the use of distinct trafficking pathways by MARV-GP and the other viral structural proteins. Surprisingly, inhibition of Rab11, a major Myosin Vb regulator, by overexpression of dominant-negative or constitutively active mutants or siRNA-mediated knockdown did not perturb GP distribution or surface transport indicating that Rab11 is not essential for MARV-GP transport. In sum, these results suggest that after leaving the Golgi apparatus MARV-GP transits the Myosin Vb-associated endosmal recycling system. A proper transit through the recycling system is critical for GP transport to the cell surface as well as for its targeting to plasma membrane regions enriched in the matrix protein VP40 and therefore an important step during the formation of the MARV envelope.