Coxsackievirus B3 (CVB3) causes viral myocarditis and can ultimately result in dilated cardiomyopathy. There is no vaccine available for clinical use. Translation initiation of CVB3 RNA is directed by an internal ribosome entry site within the 5'-untranslated region. We have previously described that Sabin3-like mutation (U-473 to C) introduced in CVB3 genome led to a defective mutant with a serious reduction in translation efficiency. In the present study, we analyzed, in vitro, the effect of the Sabin3-like mutation on the binding affinity of RNA domain V to some standard translation initiation factors: eIF4G, eIF3b, and eIF4B by filter-binding assays and UV-crosslink assays. We have demonstrated that this single-nucleotide exchange impairs the binding affinity of these cellular factors within the mutant RNA. These data indicate how this decisive Sabin3-like mutation mediates viral translation attenuation. Taken together, these findings strongly suggest that the mutant strain could be considered a candidate for an attenuated CVB3 vaccine.