Fig 3.

VLP-based assay for determining the amino acids essential for transcription and replication. Shown are schematic outlines of experiments and data obtained using different L mutants. (a) Procedure used to generate LACV trVLPs. Donor cells were transfected with constructs for the minigenome system as described in the legend to Fig. 1 as well as the LACV envelope glycoproteins (GPs). A set of L mutants was used in parallel to wt L. As negative controls, L (column 1) or GPs (column 2) were omitted. After 48 h, cellular lysates and supernatants containing VLPs were harvested. (b) Minigenome system reporter activities were measured in cell lysates. (c) Outline of the VLP detection assay. Indicator cells expressing wt L and N were superinfected with trVLP supernatants. (d) Ren-Luc minigenome reporter activities of indicator cells as determined after 24 h of VLP infection. Activities of the cotransfected FF-Luc indicated comparable transfection efficiencies (data not shown). Mean values and standard deviations from three independent experiments are shown. SN, supernatant.