Funktionelle Charakterisierung der Glykoproteine eines neu entdeckten afrikanischen Henipavirus

Henipaviruses are highly-pathogenic paramyxoviruses with today only two known representatives, that can cause infections in humans and livestock. Hendra virus occurs in australia and causes mainly respiratory infections in horses. Nipah virus derives from south-east-asia, where it regularly causes smaller outbreaks of severe encephalitis in humans. In general henipavirus infections are zoonotic infections with fruitbats of the species Pteropus as natural reservoir. Since some years it was belived that the distribution of the viruses is hampered to south-east-asia and australia. In 2009 the isolation of henipavirus-like rna-sequences out of a bat of the order Eidolon helvum from africa succeeded. One of this newly discovered african henipaviruses, GH-M74a, could be fully sequenced and cloned. Due to the lack of isolation of replicative viruses out of bats, the evoluation of the zoonotic potential is based on functional analyses of individual proteins in comparison to the proteins of humanpathogenic henipaviruses. Because of their importance for a sucsessfull infection of a new host cell and virus spread from cell to cell the two glycoproteins, the receptor-binding G and the fusion causing F protein of the newly identified GH-M74a (M74) should be functionally characterized in this work. Therefor the M74 glycoproteins where cloned into the expression vector pCAGGS. To facilitate their detection they were marked with an HA- or Flag-tag. With the help of indirect immunofluorescence analyses it was first proven that all cloned constructs are expressed. The ability of the two M74-glycoproteins to cause cell-to-cell-fusion when coexpressed was limited to very little cell lines. A fusion assay with heterotypic NiV-G or F-protein revealed that the main reason for the little fusion activity lays within the F-protein. Following western blot analyses showed a comparable total expression to NiV-F but a much more inefficient proteolytic activation and the fusion active form was hardly detectable on the cell surface. Coimmunoflourescence with NiV-F showed clear differences in the intracellular distribution of the two proteins. In addition tot hat, inhibitor studies indicate the endocytosis-dependend cleavage of the M74-F but revealed that M74-F is not cleaved by the NiV-F activating proteases cathepsin B or L. This is a clear sign for a different intracellular transport. To clarify if this modified transport and the reduced surface expression results from disrinct protein domains, M74-F chimeric, deletion and point mutants where generated in the second part of my work. The fact that no introduced minor or mayor mutation could restore the function leads to the guess that in contrast tot he other human pathogenic henipaviruses the reduced surface expression and fusion activity of the M74-F protein is a conserved intrinsic feature. Such a reduced bioactivity of a fusion protein could be a benefit for a bat-henipavirus. It could limit the spread in the reservoir host what causes persistant infection and the „survival“ of the virus. Such a reduced activity could in addition highly limit the zoonotic potential.