Development of a novel reverse genetics system to assess the functional interplay of Lassa virus proteins

The Lassa virus (LASV) is an Old World arenavirus endemic to West Africa and causes hemorrhagic fever in humans. Since LASV is classified as biosafety level 4 pathogen, work with infectious LASV is restricted to high containment laboratories. Fortunately, life cycle modelling systems are a powerful tool to investigate highly pathogenic BSL4 viruses such as LASV under biosafety level 1 conditions. The simplest life cycle modelling system is a minigenome system, which uses a miniature version of the viral genome (called “minigenome”). This system can be used to investigate the processes of viral replication and transcription in eukaryotic cells. To examine the whole viral life cycle including budding and entry, minigenome systems can be further advanced into transcription and replication-competent virus-like particle (trVLP) assays. This type of system includes proteins needed for viral budding and entry, either supplied by expression plasmids or from the minigenome itself (“multicistronic trVLP assay”). In this thesis, both a minigenome and a trVLP system solely based on LASV components were successfully developed. Moreover, these life cycle modelling systems were then used to investigate conserved amino acid residues within the LASV matrix protein Z, which is essential for viral budding and inhibition of RNA synthesis. The mutation of the conserved amino acid residues leucine (L) at position 71, and prolin (P) at position 72 were associated with loss of proper RNA synthesis inhibition by Z. Furthermore, mutation of L71 and aspartic acid (D) at position 22 displayed abnormalities in the formation of infectious viral particles. In conclusion, two reverse genetics systems capable of overcoming biosafety level 4 restrictions were developed and used to investigate conserved amino acid residues within LASV Z in detail.