Die Rolle der Homooligomerisierung des Polymerasekofaktors VP35 im Vermehrungszyklus des Marburg-Virus

Das Marburg-Virus verursacht eine fieberhafte hämorrhagische Erkrankung bei menschlichen und nichtmenschlichen Primaten, die mit hohen Letalitätsraten einhergeht. Taxonomisch bildet das Marburg-Virus (MARV) zusammen mit dem Ebola-Virus (EBOV) die Familie der Filoviridae. Die Virionen sind aus einem...

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Bibliographic Details
Main Author: Möller, Peggy
Contributors: Klenk, Hans-Dieter (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2005
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Marburgvirus causes a fulminant hemorrhagic fever among humans and nonhuman primates with high fatality rates. Marburgvirus (MARV) and the closely related Ebolavirus (EBOV) together make up the family Filoviridae. The Marburgvirus particles are composed of a nucleocapsid complex, which is connected to the lipid envelope by a protein matrix. Nucleocapsids are constituted by the viral RNA genome and the four nucleocapsid proteins NP, VP35, VP30, and L, which are involved in the formation of different protein-protein complexes. The complex of VP35 and L represents the active RNA-dependent RNA polymerase, with VP35 serving as the polymerase cofactor. In the present study, we characterized the biological significance of homooligomerization of the polymerase cofactor VP35 for transcription and replication of viral RNA. Additionally, we focused on morphogenesis of new nucleocapsid complexes by investigation the interaction of VP35 and NP. A predicted coiled-coil domain between amino acids 70 and 120 of VP35 is essential and sufficient to mediate homooligomerization of the protein. Substitution of leucine residues 90 and 104 abolished (I) the probability to form coiled-coils, (II) homooligomerization, and (III) the function of VP35 in viral RNA synthesis. Furthermore it was found that homooligomerization of VP35 is a prerequisite for the binding of L. In contrast, inability to homooligomerize did not abolish the recruitment of VP35 into NP-induced inclusion bodies. These inclusions are presumed to represent centers of viral replication and formation of nucleocapsids. An N-terminal fragment of VP35 was found to keep NP in solution, which might be necessary to provide NP as substrate for encapsidation of newly synthesized RNA. In combination with the N-terminal binding site a second NP-binding site on VP35 was essential for the formation of typical inclusions. Homooligomers of VP35 were able to substitute a missing NP-NP self interaction site during formation of nucleocapsids. Together, the data presented that an N-terminal coiled-coil motif in VP35 is essential and sufficient for homooligomerization of the protein. While homooligomerization is dispensable for the recruitment of VP35 into the NP-induced inclusion bodies, it is essential for the function of the protein during replication and transcription since monomeric VP35 is not able to recruit the polymerase to the NP/RNA template.