Chimeric poliovirus RNAs, possessing the 5 nontranslated region (NTR) of hepatitis C virus in place of the 5 NTR of poliovirus, were used to examine the role of the poliovirus 5 NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3 NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5-terminal cloverleaf and 3 NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA.The single-stranded positive-sense RNA genome of poliovirus functions sequentially as an mRNA for viral protein synthesis and then as a template for viral negative-strand RNA synthesis (33). Cytoplasmic extracts from HeLa cells support the sequential translation and replication of poliovirus RNA (5, 31). In the presence of 2 mM guanidine HCl, cell-free translation-replication reactions support the translation of viral RNA and the accumulation of viral preinitiation RNA replication complexes (4, 6). Guanidine HCl reversibly blocks the initiation of negative-strand RNA synthesis by interfering with the function of viral protein 2CATPase (3,38,40). When preinitiation RNA replication complexes are resuspended in reaction mixtures in the absence of guanidine HCl, the preinitiation RNA replication complexes synchronously initiate the synthesis of viral negative-strand RNA (6). Ribosomes translating the viral mRNA within preinitiation RNA replication complexes prevent the synthesis of negative-strand RNA (8,15). Thus, the conversion of viral ribonucleoprotein complexes into preinitiation RNA replication complexes is an important temporally regulated event in the replication of poliovirus RNA.Contemporary models of eukaryotic mRNA translation suggest that the 5Ј and 3Ј nontranslated regions (NTRs) of mRNAs communicate via RNA binding proteins bound to both termini of the mRNA (14,19,42). In particular, poly(A) binding protein bound to 3Ј-terminal poly(A) interacts with eukaryotic initiation factors eIF4G I and II anchored to the 5Ј NTR of mRNA, bringing the 5Ј and 3Ј NTRs into proximity (49). During the course of a poliovirus infection, viral protein 2APro mediates the cleavage of eIF4G I and II and poly(A) binding proteins (11,18,23,24). This leads to the shutoff of cap-dependent host protein synthesis and precludes the ability of eIF4G I and II and poly(A) binding protein to form proteinprotein bridges between the 5Ј and 3Ј termini of mRNAs, including poliovirus RNA. Therefo...