Mutations in a conserved enteroviral RNA oligonucleotide sequence affect positive strand viral RNA synthesis

We showed earlier that a transition mutation U234C, located within the comp letely conserved 5 nucleotide (nt) tract 5'-CGUUA (nt232-236) in the 5' non -translated region (NTR) of the coxsackievirus B3 (CVB3) genome, attenuated CVB3 cardiovirulence in mice. To further explore the role of the sequence, we induced two single and one double transversion mutations in the conserv ed 5mer in a cardiovirulent CVB3 genome. The mutated sites partially or tot ally reverted to parental wild-type when progeny viruses were passaged at 3 7 degreesC, but remained stable when transfection and subsequent passages w ere performed at 33.5 degreesC. Viral replication in cell culture was atten uated at 37 degreesC or 39.5 degreesC relative to replication at 33.5 degre esC. While Western blot analysis demonstrated the level of protein translat ion consistent with virus replication, the ratios of positive to negative s trand viral RNA at 37 degreesC in murine cells demonstrated a 2-5 fold dimi nution from those measured at 33.5 degreesC. Mutant CVB3 strains failed to replicate productively when inoculated into mice. The biological data are c onsistent with an hypothesis that proposes a lesion with primary effects at the level of positive strand viral RNA synthesis that results in attenuati on of viral replication at physiologic temperature.