Expression and biochemical and structural characterisation of the RNA dependent RNA polymerase of Lassa virus

Abstract

In this work the domain structure of the Lassa virus L protein was investigated. Potential inter-domainlinkers were predicted and functionally analyzed in the minireplicon system. Two sites were identified at which L protein could be physically separated without significant loss of function in the minireplicon system, indicating that the corresponding domains are able to trans-complement each other. Physical interaction of the N- and C-terminal domains was verified by co-immunoprecipitation and confocal immunofluorescence microscopy. The data indicate that the L protein is composed of at least three domains.In the second part full length L proteins of Lassa virus AV, Bantou and Mopeia virus should be expressed in three different expression systems. Furthermore, a polymerase assay should be established to allow biochemical characterisation of the polymerase activity of L protein. Expression of L protein was successfull in insect cells and HeLa cells, but not bacteria. The rekombinant proteins could be purified using either His- or Strep-Tag but it was not possible to detect polymerase activity