5 ' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis

A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus rep lication, we determined how cloverleaf mutations affected the stability, tr anslation and replication of poliovirus RNA in HeLa S10 translation-replica tion reactions. Mutations within the cloverleaf destabilized viral RNA in t hese reactions. Adding a 5' 7-methyl guanosine cap fully restored the stabi lity of the mutant RNAs and had no effect on their translation. These resul ts indicate that the 5' cloverleaf normally protects uncapped poliovirus RN A from rapid degradation by cellular nucleases, Preinitiation RNA replicati on complexes formed with the capped mutant RNAs were used to measure negati ve-strand synthesis. Although the mutant RNAs were stable and functional mR NAs, they were not active templates for negative-strand RNA synthesis, Ther efore, the 5' cloverleaf is a multifunctional cis-acting replication elemen t required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to fo rm a circular ribonucleoprotein complex that regulates the stability, trans lation and replication of poliovirus RNA.