Structural requirements of the higher order RNA kissing element in the enteroviral 3 ' UTR

The origin of replication (oriR) involved in the initiation of (-) strand e nterovirus RNA synthesis is a quasiglobular multi-domain RNA structure whic h is maintained by a tertiary kissing interaction. The kissing interaction is formed by base pairing of complementary sequences within the predominant hairpin-loop structures of the enteroviral 3' untranslated region, In this report, we have fully characterised the kissing interaction, Site-directed mutations which affected the different base pairs involved in the kissing interaction were generated in an infectious coxsackie B3 virus cDNA clone, The kissing interaction appeared to consist of 6 bp, Distortion of the inte raction by mispairing of each of the base pairs involved in this higher ord er RNA structure resulted in either temperature sensitive or lethal phenoty pes. The nucleotide constitution of the base which gaps the major groove of the kissing domain was not relevant for virus growth. The reciprocal excha nge of the complete sequence involved in the kissing resulted in a mutant v irus with wild type virus growth characteristics arguing that the base pair constitution is of less importance for the initiation of (-) strand RNA sy nthesis than the existence of the tertiary structure itself.