Die Phosphorylierung des Ebolavirus VP30 reguliert die virale Transkription und Replikation

Ebola virus is a negative strand RNA virus, belonging to the family Filoviridae together with the closely related Marburg virus. Since filoviruses cause outbreaks of a severe hemorrhagic fever with high mortality rates in humans and nonhuman primates in central Africa they are classified as BSL4 pathogens. Transcription and replication of the virus requires the viral nucleocapsid complex: the single stranded RNA genome in negative orientation which is surrounded by the nucleoprotein NP together with the viral polymerase complex consisting of L and VP35 as well as the fourth nucleocapsid protein VP30. VP30 represents an Ebola virus specific transcription factor which is essential for viral transcription but dispensable for viral replication. The activity of VP30 as transcriptional activator is regulated via its phosphorylation state: nonphosphorylated VP30 induces the initial steps of viral mRNA synthesis whereas phosphorylation of VP30 leads to a block in viral transcription. In the first part of the thesis we investigated the role of VP30 phosphorylation for the regulation of viral transcription / replication by using VP30 mutants mimicking the different phosphorylation states of the protein. We demonstrate that viral replication was favored when VP30 was phosphorylated whereas nonphosphorylated VP30 diminished replication. By analyzing the interaction of VP30 phosphorylation mutants with the other components of the nucleocapsid complex, we identified a novel VP30-VP35 interaction which was shown to be regulated via VP30 phosphorylation. We propose a model in which the phosphorylation of VP30 might interfere with the VP35 interaction leading to the dissociation of the phosphorylated VP30 from the transcriptase complex in order to promote viral replication by VP35, L and NP. Furthermore we could demonstrate that the dynamic phosphorylation of VP30 was essential for the initiation of primary transcription at very early stages of viral infection. We describe that a possible phosphorylation of VP30 serine residue 29 was sufficient for rendering VP30 transcriptional active in order to generate a recombinant Ebola virus with only one phosphate acceptor site within VP30. The generated virus showed no significant differences regarding morphology or growth kinetics compared with a wildtyp virus. Moreover, the generation of a viable recombinant EBOV with VP30 serine 30 as unique phosphate acceptor was not possible and attended by the development of compensatory mutations within the VP30 phosphorylation site indicating a high evolutionary pressure on this region. These results underline the significance of the VP30 phosphorylation for the viral life cycle: while the dynamic phosphorylation is an essential requirement for the earliest steps of viral life cycle, our study suggests further a modulating role of VP30 phosphorylation for the composition of the viral polymerase complex resulting in the transition from transcription to replication processes.