Aufbau eines reversen Genetik-Systems für Lassa-Virus zur Untersuchung viraler Replikationsmechanismen

Abstract

Lassa virus belongs to the family of Arenaviridae and is the causative agent of a hemorrhagic fever in humans. Molecular mechanisms of genome replication and gene expression are largely unknown. To facilitate the functional analysis of this virus, a replicon system was developed. Using this system, the putative 19-nucleotide long promoter at the RNA termini and the RNA-dependent RNA polymerase domain (~500 amino acids) predicted within the L protein was characterised. For promoter analysis, each nucleotide of the 3' and 5' ends of the RNA genome analogue was changed into the 3 remaining residues by mutagenesis. Promoter activity was dependent on specific residues at both termini as well as specific base pairings predictably formed between the 3' and 5' termini, indicating that the functional promoter forms a 3'-5' duplex. The RNA polymerase domain was characterised by mapping essential amino acids. Potentially relevant residues were identified using secondary structure prediction and data from crystal structures of known RNA polymerases. A novel method was established for rapidly generating L protein mutants by PCR. Three types of mutations were generated and functionally tested: conservative changes in the predicted catalytic sites (motifs pre-A to E); a charged-to-alanin scan throughout the domain, and 2Xglycin insertions to search for loop regions (in total ~150 mutants). Northern blot analysis revealed one mutant that selectively impairs RNA transcription but not genome replication. Based on the data, a model of the RNA polymerase domain of Lassa virus is proposed. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.