Untersuchung differentieller Genexpression in Arenavirus-infizierten Zellen und Entwicklung einer therapeutischen siRNA gegen Arenaviren

Abstract

The pathogenesis of Lassa virus is poorly understood. Cellular mRNA levels in Lassa virus- and LCMV-infected HuH-7 cells were measured at the late phase of acute infection with two types of transcription array. Data analysis was based on statistical frameworks controlling the false-discovery rate. Data were considered relevant if they could be verified by both array systems and real-time RT-RCR. According to these criteria, there was no evidence for true changes in gene expression. Additionally the ability of siRNA as a therapeutic option in Lassa fever treatment was tested. Therefore conserved sequences of the Lassa virus genome were chosen as siRNA targets. Testing was performed using a Lassa virus minireplicon system and plasmids for CMV promoter-driven expression of Lassa virus mRNA analogues. The siRNA targeting the 5' end of NP mRNA led to 60-80% reduction of reporter gene activity (Renilla luciferase) in both systems. Vero cells transfected with NP-specific siRNA showed 60-90% reduction of infectious virus titre for the Lassa virus strains (NL, LIB-90, AV, CSF, NIG-10) and related arenaviruses (LCMV WE, LCMV Armstrong, Mopeia) included in the studies.