A predicted secondary structural domain within the internal ribosome entry site of echovirus 12 mediates a cell-type-specific block to viral replication

The enterovirus 5′ nontranslated region (NTR) contains an internal ribosome entry site (IRES), which facilitates translation initiation of the viral open reading frame in a 5′ (m⁷GpppN) cap-independent manner, anal cis-acting signals for positive-strand RNA replication. For several enteroviruses, the 5′ NTR has been shown to determine the virulence phenotype. We have constructed a chimera consisting of the putative IRES element from the Travis strain of echovirus 12 (ECV12), a wild-type, relatively nonvirulent human enterovirus, exchanged with the homologous region of a full-length infectious clone of coxsackievirus B3 (CBV3). The resulting chimera, known as ECV12(5′NTR)CBV3, replicates similarly to CBV3 in human anal simian cell lines yet, unlike CBV3, is completely restricted for growth on two primary murine cell lines at 37°C. By utilizing a reverse-genetics approach, the growth restriction phenotype was localized to the predicted stem-loop II within the IRES of ECV12. In addition, a revertant of ECV12(5′NTR)CBV3 was isolated which possessed three transition mutations anal had restored capability for replication in the utilized murine cell lines. Assays for cardiovirulence indicated that the ECV12 IRES is responsible for a noncardiovirulent phenotype in a murine model for acute myocarditis. The results indicate that the 5′ NTRs of ECV12 anal CBV3 exhibit variable intracellular requirements for function anal serve as secondary determinants of tissue or species tropism.