The introduction of mutations in the wild type coxsackievirus B3 (CVB3) IRES RNA leads to different levels of in vitro reduced replicative and translation efficiencies

Fig 1. Structure and sequence of domain V of CVB3 IRES. Position of introduced mutations (1–6) on IRES domain V producing CVB3 mutants (M1 to M6) are indicated.

Fig 2. One-step growth cycle of CVB3 wild type and M1-M6 mutant viruses in HeLa cells. Cells were infected at a MOI of 10 TCID50/cell at 37°C and 5% CO2. Samples were analysed for infectious virus particle formation at the indicated times by determining titers using Reed and Munch method on HeLa cell monolayers harvested by freezing at specific times shown. Experiments were performed in triplicate. p.i., post infection. The data shown are the mean +/- SD from three replicated experiments (n = 3). P< 0.05 compared vs control, Student’s t test.

Fig 3. (A) Polyprotein products of in vitro translation of wild type and M1-M6 mutants of CVB3 in cell-free reaction mixtures supplemented with decreasing S10 HeLa cell extracts (20% to 0%). Transcript RNAs derived from wild-type CVB3 and its mutants were translated and analyzed in HeLa cell extracts as described in material and methods. (B) In vitro translation efficiencies of CVB3 wild type and M1-M6 mutants. Densitometric quantifications of synthetic wild type and mutant polyproteins revealed on dried SDS-polyacrylamide gel. The percentages indicated are those of S10 extract. The data shown are the mean +/- SD from three replicated experiments (n = 3). P< 0.05 compared vs control, Student’s t test.

Fig 4. Histology of hearts and pancreas of Swiss mice at day 10 after oral inoculation with CVB3 wild type and mutant viruses as described previously [26]. Shown are murine heart (a) and pancreas (b) tissues stained with hematoxylin and eosin from mice inoculated with CVB3 wild type, (c, d) heart and pancreas from a control uninfected mice and (e, f) heart and pancreas from mice inoculated with M3 virus (all 4 mutant viruses induced a similar lack of pathology in mice, S1 Fig). Lymphocyte infiltrations are shown in each organ tissues. Experiments were performed on three mice for each mutant and for each day post inoculation.