Die Funktion der Coiled-Coil-Domäne im Nukleoprotein des Ebolavirus

Ebola virus and Marburg virus make up the family Filoviridae. Due to their singlestranded RNA genome in negative orientation, they belong to the order Mononegavirales. Filoviruses cause a severe hemorragic fever in humans and non-human primates with fatality rates up to 90%. Because of the severe illness, and the fact that there is no specific treatment or vaccination, they are classified as BSL-4 pathogen. The Nucleocapsid is built by the non-segmented RNA-genome, the nucleoprotein NP, the viral polymerase L, the polymerase-cofactor VP35 and VP30. Subject of this study is a coiled coil domain within NP and its influence on various functions of NP. First the role of the coiled coil domain in the interaction of NP with VP30 and VP35 was investigated by immunofluorescence analysis. NP-mutants without coiled coil 3D-structure or without coiled coil domain were still able to interact with VP35, but not with VP30. This could be due to two binding domains on NP for VP35. Binding of VP30 to NP is either mediated directly by the coiled coil or requires a 3D confirmation determined by the NP coiled coil. Furthermore, this study is focussing on the influence of the coiled coil domain on NPNP interaction and self assembly of NP. By immunofluorescence analysis it could be proofed that the formation of NP inclusion bodies (inclusions) depends on a functional coiled coil. Knock-out mutants could still be recruited to inclusions build by NPwt, suggesting that the loss of the coiled coil domain only causes a partly loss of the ability for self-interaction. With an complementation assay it could be shown that the coiled coil domain is sufficient to mediate NP-NP interactions. NP forms helices that are supposed to be the framework of the nucleocapsids. Analysis of NP-helices by electron microscopy suggested that the ability to form helices also depends on an intact coiled coil domain or the presence of NPwt. NP mutants alone were not able to assemble to helices. Oligomerization mediated by the coiled coil domain or an interaction with RNA influenced by this domain could be reasons for these findings. The last part of the study is dedicated to the influence of the NP coiled coil domain on viral transcription. In an iVLP assay a dominant negative effect of NP with deleted or mutated coiled coil domain could be observed. This in probably due to a defect of NP with deleted or mutated coiled coil in encapsidation the virus sepecific minigenome that serves as template for the viral polymerase complex which is also indicated by the altered NP helices formation.