Charakterisierung von Oligomerisierungsdomänen des Marburg-Virus Nukleoprotein und deren funktionelle Bedeutung

The phosphorylated nucleocapsid protein NP is the major component of Marburg virus (MARV) nucleocapsid complex (NC), which further contains the viral structural proteins, VP35, VP30, L, and the genomic RNA. NP, VP35, and L are sufficient to mediate transcription and replication in a minigenome system. NP forms self aggregates and interacts with VP35, and VP30. Intracellularly, NP forms characteristic inclusion bodies that represent places where the helical nucleocapsids are accumulated. In the present thesis NP homo- and heterooligomerization domains were characterized and structural and functional properties were analyzed. Deletion mutants of NP revealed three domains which affected homooligomerization: aa 118-234 (N), aa 320-400 (CC), and aa 522-695 (C). The aa 320-400 displayed all features of two coiled coil motifs (C1: aa 320-50, C2: aa 371-400; CC). The intact two coiled coil motifs were essential for NP dependent viral transcription. The N- (aa 1-234) and C-terminus (aa 522-695) of NP were able to interact. Probably, interaction of N- and C-terminus takes places intramolecularly to regulate the activity of the other oligomerization domains. The shortest NP mutant that was able to form inclusion bodies covered the first 270 aa. It is assumed, that these 270 aa form helical structures which accumulate to inclusion bodies. Binding of VP35 on NP takes place in the N-terminal portion of NP (aa 1-390). The binding intensity to VP35 seems to be dependent on cooperation of various parts of NP. Essential but not sufficient for NP-VP35 interaction are two coiled coil motifs. Majority of phosphorylated serine and threonine residues is located between aa 390-480. In vitro studies revealed that both, homooligomerization and heterooligomerization with VP35 were dependent on phosphorylation state of a characteristic serine cluster (aa 450-455). The interaction with VP35 is inversely related to phosphorylation. Homooligomerization is favored by a moderate phosphorylation of the serine cluster whereas dephosphorylated or hyperphosphorylated serine cluster inhibits homooligomerzation. Phosphorylation of the serine cluster also regulated viral transcription. NP with de- or completely phosphorylated serine cluster was unable to mediate transcription. Only partially phosphorylated serine cluster supported viral transcription and replication.