Der Einfluss der Phosphorylierung von Marburgvirus NP auf den viralen Lebenszyklus

Marburgvirus (MARV), Ebolavirus (EBOV) and Lloviuvirus (LLOV) form the family Filoviridae and possess a single-stranded RNA genome of negative polarity. Causing severe hemorrhagic fevers in humans and nonhuman primates, filoviruses are classified as BSL-4 pathogens. The nucleoprotein NP, together with the viral proteins VP30, VP35, VP24 and the polymerase L, form the nucleocapsid that encapsidates the viral RNA. Previous studies have shown that NP is phosphorylated at C-terminal serine and threonine residues. While in infected cells both phosphorylated and unphosphorylated NP can be detected, only the phosphorylated form is incorporated into released viral particles, suggesting a role of the phosphorylation in virogenesis. Seven phosphorylation regions have been identified in NP, one of those (phosphorylation region II) has already been studied more in detail. Phosphorylation regions VI and VII were studied in the present thesis, the function of the serine residues‘ phosphorylation in these regions was characterized. Using phosphomimetic mutants it could be shown that the phosphorylation of a single residue (S602) in phosphorylation region VI is sufficient for the conformational change of NP. NP that is phosphorylated at S602 is preferentially transported from the viral inclusion bodies that represent areas of viral replication and transcription to the plasma membrane when compared to the unphosphorylated form. Therefore, predominantly phosphorylated NP is available for the budding of new virions. The phosphorylation of the single serine (S619) in phosphorylation region VII was also studied by using phosphomimetic mutants: Replication and/or transcription are enhanced when S619 is unphosphorylated so that enough viral proteins and viral RNA are synthesized for the budding of new viral particles. Phosphorylation of S619 however is required for an efficient interaction with the nucleocapsid protein VP24 and therefore the formation of functionally active nucleocapsids. Taken together, the results presented in this thesis underline the importance of the phosphorylation of NP for the viral life cycle: The dynamic phosphorylation of NP conveys a broad spectrum of functions that are important in different stages of the viral life cycle.