Strukturelle und funktionelle Charakterisierung des bunyaviralen L-Proteins

The order Bunyavirales possesses the largest diversity of species and hosts among all RNA viruses and represents a major health concern not only for low income countries. Despite a rather simple molecular architecture with only a few proteins the different viruses replicate highly efficient in various host species. The L protein is the key player of viral replication and besides genome replication performs the specific and highly efficient cap-snatching mechanism to prime the translation of the viral genome which requires an endonuclease activity. This mechanism has most intensively been studied for the related influenza virus. So far there is evidence for cap-snatching endonucleases in the N-terminus of three bunyaviral L proteins which despite low sequence conservation show structural and functional homology to the influenza endonuclease. The CCHFV endonuclease has been recombinantly expressed and recovered in high purity. The protein shows structural homology in comparision to related nairoviruses but is larger in size compared to related bunyaviral families which can be attributed to two insertions in the sequence. The lack of in-vitro activity and a different coordination of manganese ions in the active site reveal closer relation to the endonuclease of the Arenaviridae family. The RVFV endonuclease has been identified in a recombinantly expressed N-terminal fragment of the L protein. The active site binds manganese ions and cleaves ssRNA in- vitro which is consistent with recently published data on related bunyaviral endonucleases and proves functional homology. Detailed structural data were not obtained due to poor protein quality. This thesis presents fundamental new insights in the cap-snatching endonucleases of two uncharacterized bunyaviral endonucleases which further underlines the structural and functional homology of the endonucleases and validates the conservation of the cap-snatching mechanism. The identified proteins are a valuable tool for high-throughput screening applications and thus the presented work provides an important progress in the future development of drugs to combat bunyaviral infectious diseases.