Functional features of the bovine enterovirus 5'-non-translated region

The bovine enterovirus (BEV) serotypes exhibit unique features of the non-translated regions (NTRs) which separate them from the other enteroviruses. Their most remarkable property is an additional genome region of 110 nt located between the 5'-cloverleaf and the internal ribosome entry site (IRES). This genome region has the potential to form an additional cloverleaf structure (domain I*) separated from the 5'-cloverleaf (domain I) by a small stem-loop (domain I**). Other characteristics involve the putative IRES domains III and VI. In order to investigate the features of the 5'-NTR, several full-length coxsackievirus B3 (CVB3) cDNA plasmids with hybrid 5'-NTRs were engineered. After exchange of the CVB3 cloverleaf with the BEV1 genome region representing both cloverleafs, a viable virus chimera was generated. Deletion of domain I** within the exchanged region also yielded viable virus albeit with reduced growth capacity. Deletion of sequences encoding either the first or the second BEV cloverleaf resulted in non-infectious constructs. Hybrid plasmids with exchanges of the IRES-encoding sequence or the complete 5'-NTR were non-infectious. Transfection experiments with SP6 transcripts containing 5'-NTRs fused to the luciferase message indicated that IRES-driven translation is enhanced by the presence of the CVB3 cloverleaf and both BEV1 cloverleaf structures, respectively. Deletion of either the first or the second BEV cloverleaf domain reduced but did not abolish enhanced luciferase expression. These results suggest that the substitution of two putative BEV cloverleaf structures for the putative coxsackieviral cloverleaf yields viable virus, while BEV sequences encoding the IRES fail to functionally replace CVB3 IRES-encoding sequences.