A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element 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 form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.

Type 1 diabetes acceleration in nonobese diabetic (NOD) mice through coxsackievirus B4 (CVB4) infection requires a preexisting critical mass of autoreactive T cells in pancreatic islets

The 5 cloverleaf in poliovirus RNA has a direct role in regulating the stability, translation, and replication of viral RNA. In this study, we investigated the role of stem a in the 5 cloverleaf in regulating the stability and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Our results showed that disrupting the duplex structure of stem a destabilized viral RNA and inhibited efficient negative-strand synthesis. Surprisingly, the duplex structure of stem a was not required for positive-strand synthesis. In contrast, altering the primary sequence at the 5-terminal end of stem a had little or no effect on negative-strand synthesis but dramatically reduced positive-strand initiation and the formation of infectious virus. The inhibition of positivestrand synthesis observed in these reactions was most likely a consequence of nucleotide alterations in the conserved sequence at the 3 ends of negative-strand RNA templates. Previous studies suggested that VPgpUpU synthesized on the cre(2C) hairpin was required for positive-strand synthesis. Therefore, these results are consistent with a model in which preformed VPgpUpU serves as the primer for positive-strand initiation on the 3AAUUUUGUC5 sequence at the 3 ends of negative-strand templates. Our results suggest that this sequence is the primary cis-acting element that is required for efficient VPgpUpU-primed positive-strand initiation.Poliovirus (PV) is a member of the Picornaviridae family of single-stranded positive-sense RNA viruses. The viral genome contains a 3Ј-terminal poly(A) tail and a large open reading frame that is flanked by nontranslated regions (NTR) at the 5Ј and 3Ј termini. A small viral protein, VPg (3B), is covalently linked to the 5Ј-terminal ends of all newly synthesized viral RNAs (1,30,43). Poliovirus RNA is first translated in the cytoplasm of the host cell to synthesize the viral structural and replication proteins and is then copied by the viral polymerase (3D pol ) to form full-length negative-strand RNA. The negative-strand RNA then serves as a template for the synthesis of positive-strand progeny RNA. The replication of viral RNA is highly asymmetric, with the ratio of positive-strand to negativestrand RNA synthesis ranging from 3 to 70 in various studies (21,40,49).The 3Ј NTR and poly(A) tail, the internal cre(2C) hairpin, and the 5Ј cloverleaf are cis-acting elements that are required to replicate the viral genome. The 3Ј NTR and poly(A) tail are both required for the efficient initiation of negative-strand RNA synthesis (26,35,36,46,50). The cre hairpin is a highly conserved structure that is located in various regions in the genomes of different picornaviruses (20,22,31,33,34,42,52) and is required for the synthesis of VPgpUpU (19, 41, 51) and positive-strand RNA (37, 38). The 5Ј-terminal cloverleaf is another highly conserved structure that is implicated in various aspects of viral RNA replication. The 5Ј cloverleaf is organized into stem a, as well as stem-loops b, c, and d (Fig. 1) (2). Stem-loops b and d bin...

Full-length and subgenomic poliovirus RNAs were transcribed in vitro and transfected into HeLa cells to study viral RNA replication in vivo. RNAs with deletion mutations were analyzed for the ability to replicate in either the absence or the presence of helper RNA by using a cotransfection procedure and Northern (RNA) blot analysis. An advantage of this approach was that viral RNA replication and genetic complementation could be characterized without first isolating conditional-lethal mutants. A subgenomic RNA with a large in-frame deletion in the capsid coding region (Pl) replicated more efficiently than full-length viral RNA transcripts. In cotransfection experiments, both the full-length and subgenomic RNAs replicated at slightly reduced levels and appeared to interfere with each other's replication. In contrast, a subgenomic RNA with a similarly sized out-of-frame deletion in Pl did not replicate in transfected cells, either alone or in the presence of helper RNA. Similar results were observed with an RNA transcript containing a large in-frame deletion spanning the P1, P2, and P3 coding regions. A mutant RNA with an in-frame deletion in the P1-2A coding sequence was self-replicating but at a significantly reduced level. The replication of this RNA was fully complemented after cotransfection with a helper RNA that provided 2A in trans. A P1-2A-2B in-frame deletion, however, totally