Rift Valley fever virus minigenome system for investigating the role of L protein residues in viral transcription and replication

Replicon systems are important molecular tools for investigating the function of virus proteins and regulatory elements involved in viral RNA synthesis. Various such systems were previously established for segmented negative strand viruses including the Rift Valley fever virus (RVFV). We have developed an ambisense minigenome system for RVFV with the specific aim to analyze the effects of L gene mutations on viral transcription versus replication. The S RNA segment with regulatory elements for ambisense gene expression served as backbone for the minigenome. Expression of the luciferase reporter gene allowed the overall activity of the RVFV replication complex to be assessed, while northern blot analysis enabled differentiation between synthesis of viral mRNA and replication intermediates. The functionality of the system was demonstrated by probing residues predictably involved in the active site of the cap-snatching endonuclease in the N-terminus of the L protein (D111, E125, and K143). Corresponding mutations led to a selective defect in the viral mRNA synthesis as described for other viruses of the Bunyavirales order. The analysis of further L gene mutants revealed an essential and specific role of a C-terminal region in the RVFV L protein (residues 1680–2068) in viral transcription. In summary, the established minigenome system is suitable for functional testing of the relevance of residues for viral transcription and replication and to validate hypotheses arising from structural or biochemical investigations of the RVFV replication complex. Application of the system to a small-scale mutagenesis screen disclosed a specific role of a C-terminal region in the RVFV L protein in mRNA synthesis.