Fig. 9: Functional relevance of the EvSR.

A Schematic representation of the genomic S segments of the recombinant RVFV strains rRVFV-vS-IGR-Ren (top) and rRVFV-vS-EvSR-Ren (bottom) with their predicted RNA structures underneath. The nucleotides that were changed in the various mutants are indicated by arrows (for details see Fig. S10). In the reporter S segment cartoons, the orange bar indicates the Renilla luciferase (Ren-Luc) gene and the red vertical line cartoon indicates the stop codon of the Ren-Luc gene. Symbols in the predicted structures are as in Fig. 8. B, C Multistep growth curves. BHK-21 cells were infected with the recombinant RVF viruses at an MOI of 0.01. At the indicated time points, infectious viruses in supernatants were titrated (B), and Ren-Luc activities in the cells measured (C). D tc-VLP donor cells. HEK293T cells were transfected with expression plasmids for RVFV L, N, and the GPs and either of the reporter S segments (including the EvSR mutants) depicted in (A). Ren-Luc reporter activity was measured at 72 h post transfection. E tc-VLP indicator cells. HEK293T cells, pretransfected with the RVFV L and N expression plasmid, were incubated with the indicated tc-VLP-containing supernatants, and Ren-Luc activity was measured 24 h later. In D and E all Ren-Luc activities are shown relative to the tc-VLPs with the IGR reporter segment which were set to 100%. In (B–E), mean values and SEM of 3 independent biological replicates with 3 technical replicates each are shown. Two-tailed unpaired t-tests (B, C) and one-way ANOVA with Dunnett’s correction for multiple comparisons (D, E) were used for statistical testing: P values above the significance threshold of 0.05 are indicated in the graphs.