The cre(2C) hairpin is a cis-acting replication element in poliovirus RNA and serves as a template for the synthesis of VPgpUpU. We investigated the role of the cre(2C) hairpin on VPgpUpU synthesis and viral RNA replication in preinitiation RNA replication complexes isolated from HeLa S10 translation-RNA replication reactions. cre(2C) hairpin mutations that block VPgpUpU synthesis in reconstituted assays with purified VPg and poliovirus polymerase were also found to completely inhibit VPgpUpU synthesis in preinitiation replication complexes. Surprisingly, blocking VPgpUpU synthesis by mutating the cre(2C) hairpin had no significant effect on negative-strand synthesis but completely inhibited positive-strand synthesis. Negative-strand RNA synthesized in these reactions immunoprecipitated with anti-VPg antibody and demonstrated that it was covalently linked to VPg. This indicated that VPg was used to initiate negative-strand RNA synthesis, although the cre(2C)-dependent synthesis of VPgpUpU was inhibited. Based on these results, we concluded that the cre(2C)-dependent synthesis of VPgpUpU was required for positive-but not negative-strand RNA synthesis. These findings suggest a replication model in which negative-strand synthesis initiates with VPg uridylylated in the 3 poly(A) tail in virion RNA and positive-strand synthesis initiates with VPgpUpU synthesized on the cre(2C) hairpin. The pool of excess VPgpUpU synthesized on the cre(2C) hairpin should support high levels of positive-strand synthesis and thereby promote the asymmetric replication of poliovirus RNA.Poliovirus is a prototypic positive-strand RNA virus with a single-stranded genome that contains a 3Ј-terminal poly(A) tail and a 5Ј-terminal covalently linked protein, VPg (1,15,27). Once released into the cytoplasm of infected cells, poliovirion RNA serves as an mRNA for translation and then as a template for negative-strand synthesis. Multiple rounds of positivestrand synthesis on each molecule of negative-strand RNA result in the synthesis of excess genomic RNA. Overall, the ratio of positive-to negative-strand synthesis is greater than 30:1 (10, 24), which is essential for the production of high yields of virion RNA and progeny virus in infected cells.cis-active replication sequences that are present at the 3Ј and 5Ј ends of poliovirion RNA play an important role in regulating poliovirus RNA replication. The 3Ј nontranslated region (NTR) and the associated poly(A) tail are important cis-active determinants that are required for efficient negative-strand RNA synthesis (14,21,22,28,30,36). The 5Ј-terminal cloverleaf structure in poliovirus RNA is a multifunctional element that is required in cis for negative-strand synthesis, RNA stability, and VPg uridylylation in membrane replication complexes (8,14,17,23,34). A new cis-acting replication element (cre) was recently identified in the genomes of several picornaviruses (9,11,16,18,20). The cre was originally identified in the P1 capsid coding region of HRV14 [cre(VP1)] (19). Mutational analysis of the cr...

The precise relationship between the length of the 3' poly(A) tail and the replication and infectivity of poliovirus RNA was examined in this study. With both poly(A)(11) and poly(A)(12) RNAs, negative-strand synthesis was 1-3% of the level observed with poly(A)(80) RNA. In contrast, increasing the length of the poly(A) tail from (A)(12) to (A)(13) resulted in about a ten-fold increase in negative-strand synthesis. This increase continued with each successive increase in poly(A) tail length. With poly(A)(20) RNA, RNA synthesis approached the level observed with poly(A)(80) RNA. A similar relationship was observed between poly(A) tail length and the infectivity of the viral RNA. A replication model is described which suggests that viral RNA replication is dependent on a poly(A) tail that is long enough to bind poly(A) binding protein and to act as a template for VPg uridylylation and negative-strand initiation.