Funktionelle Charakterisierung der cytoplasmatischen Domäne des Marburgvirus Oberflächenproteins GP

The family of Filoviridae comprises Marburg virus (MARV) and Ebola virus, which are the causative agents of fulminant haemorrhagic fevers in humans and non-human primates. Infection of target cells is mediated by binding of viral particles to their cellular receptor, followed by endocytosis, fusion of viral and endosomal membranes, release of the nucleocapsid into the cytoplasm and transcription and replication of the viral RNA. The viral glycoprotein GP, a classical type I transmembrane protein, is composed of a large ectodomain (220 kDa), a membrane-spanning and a very short cytoplasmic domain. The function of the ectodomain and to some extent also the transmembrane domain of MARV GP for the viral life cyle have been extensively addressed. On the other hand the role of the cytoplasmic domain is completely enigmatic, possibly because the 8 amino acid long domain does not comprise typical signal sequences that can affect intracellular transport or the assembly and release of viral particles. In the course of this study it was shown that the intracellular transport of MARV GP along the the classical secretory pathway to the plasma membrane is not influenced by the cytoplasmic domain. This finding was illustrated by the posttranslational modifications like N-glycosylation or oligomerization of GP, which are not significantly modified in the absence of the cytoplasmic domain (GPΔCD). With the help of a MARV-specific infectious virus-like particle (iVLP) assay, as a model system for naive MARV infection, the impact of the cytoplasmic tail of GP on the assembly, the release and the infectivity of MARV was investigated. Via a quantitative immuno electron microscopy it was demonstrated that the cytoplasmic domain of GP has neither an impact on the incorporation of GP into iVLPs nor on their filamentous morphology; however, released iVLPs showed a significantly reduced infectivity. The simultaneously occuring reduction in O-glycosylation of GPΔCD revealed a conformational modification of the ectodomain induced by the absence of the cytoplasmic domain. With the help of a newly established entry assay it was elucidated that GPΔCD induced a diminished infectivity of iVLPs as well as a reduced entry into target cells. We assume that the absence of the cytoplasmic tail of GP mediates a conformational change in the ectodomain (so called “inside-out signaling”), resulting in a decreased fusion activity of GP and a subsequent reduced infectivity.