FIG 1.

Influence of L protein mutations on viral RNA synthesis as tested in the Lassa virus replicon system. Transcriptional activity of L protein mutants was measured via Ren-Luc reporter gene expression. The Ren-Luc activity is shown in the bar graphs (mean and range [n = 2 independent transfection experiments, panel B] or standard deviation [n ≥ 3 independent transfection experiments, panels A, C, D, and E] of standardized relative light units [sRLU] as a percentage of the wild type). Synthesis of antigenome and Ren-Luc mRNA was evaluated by Northern blotting using a radiolabeled riboprobe hybridizing to the Ren-Luc gene. A defective L protein with a mutation in the catalytic site of the RdRp served as a negative control (neg.). The methylene blue-stained 28S rRNA is shown as a marker for gel loading and RNA transfer. Immunoblot analysis of FLAG-tagged L protein mutants is shown for selected experiments. (A) Conserved aromatic residues (Phe, Tyr, Trp, His) along the L protein sequence, with the exception of the N-terminal endonuclease and the central RdRp domain, were exchanged with alanine. Mutants with an mRNA-defective phenotype are marked with an asterisk. (B) Aromatic residues (Phe, Tyr, Trp) as well as positively (Lys, Arg) and negatively (Asp, Glu) charged residues in the C terminus of L protein were exchanged with alanine. An mRNA-defective phenotype is marked with an asterisk. (C) Aromatic residues Phe-1979, Phe-2071, Trp-2173, and Tyr-2179 were exchanged with Ala, Leu, Ser, Asp, Trp, Phe, and/or Tyr. (D) Positively charged residues Arg-2018, Arg-2200, and Arg-2204 were exchanged with Ala, Leu, Ser, Asp, and Lys. (E) Double and triple mutation of Phe-1979, Phe-2071, Trp-2173, and Tyr-2179.