Our understanding of picornavirus RNA replication has improved over the past 10 years, due in large part to the discovery of cis-active RNA elements (CREs) within picornavirus RNA genomes. CREs function as templates for the conversion of VPg, the Viral Protein of the genome, into VPgpUpU OH . These so called CREs are different from the previously recognized cis-active RNA sequences and structures within the 5′ and 3′ NTRs of picornavirus genomes. Two adenosine residues in the loop of the CRE RNA structures allow the viral RNA-dependent RNA polymerase 3D Pol to add two uridine residues to the tyrosine residue of VPg. Because VPg and/or VPgpUpU OH prime the initiation of viral RNA replication, the asymmetric replication of viral RNA could not be explained without an understanding of the viral RNA template involved in the conversion of VPg into VPgpUpU OH primers. We review the growing body of knowledge regarding picornavirus CREs and discuss how CRE RNAs work coordinately with viral replication proteins and other cis-active RNAs in the 5′ and 3′ NTRs during RNA replication.

A 3 poly(A) tail is a common feature of picornavirus RNA genomes and the RNA genomes of many other positive-strand RNA viruses. We examined the manner in which the homopolymeric poly(A) and poly(U) portions of poliovirus (PV) positive-and negative-strand RNAs were used as reciprocal templates during RNA replication. Poly(A) sequences at the 3 end of viral positive-strand RNA were transcribed into VPg-linked poly(U) products at the 5 end of negative-strand RNA during PV RNA replication. Subsequently, VPg-linked poly(U) sequences at the 5 ends of negative-strand RNA templates were transcribed into poly(A) sequences at the 3 ends of positive-strand RNAs. The homopolymeric poly(A) and poly(U) portions of PV RNA products of replication were heterogeneous in length and frequently longer than the corresponding homopolymeric sequences of the respective viral RNA templates. The data support a model of PV RNA replication wherein reiterative transcription of homopolymeric templates ensures the synthesis of long 3 poly(A) tails on progeny RNA genomes.Many positive-strand RNA viruses (e.g., members of the Picornavirales, Nidovirales, Togaviridae, Caliciviridae, and Astroviridae) have long poly(A) sequences at the 3Ј termini of their RNA genomes (16,25), yet the mechanisms by which 3Ј poly(A) sequences are derived during viral replication are unclear. Picornavirus RNA genomes, including that of poliovirus (PV), have a covalently linked 5Ј-terminal protein called VPg (viral protein, genome linked), a 5Ј untranslated region (UTR), a single large open reading frame, a 3Ј UTR, and a poly(A) tail of variable length (ϳ20 to 150 adenosine residues) (1). RNA polymerases encoded by viruses in the order Picornavirales utilize viral proteins (and their nucleotidylylated intermediates) to prime the initiation of RNA replication at the 3Ј termini of viral RNA templates (28,29,31,34). In this mechanism, the viral protein VPg becomes covalently linked to the 5Ј ends of both positive-and negative-strand RNAs during viral RNA replication (30, 32). PV, which is commonly studied to elucidate mechanisms of picornavirus replication, is viable when the 3Ј UTR of the genome is deleted (12, 44); however, the 3Ј poly(A) tail is essential for RNA replication (33, 39). The length of the 3Ј poly(A) tail required for virus viability and for efficient negative-strand RNA synthesis has been examined in some detail (35,45). PV RNAs with 3Ј poly(A) tails less than 9 bases long support less than 1% of wild-type negative-strand RNA synthesis, whereas poly(A) tails Ն20 bases long support wild-type levels of negative-strand RNA synthesis (35).In this investigation, we programmed PV RNAs with defined 3Ј 84-, 51-, and 32-base poly(A) sequences [designated poly(A) (84) , poly(A) (51) , and poly(A) (32) , respectively] into cell-free reactions that faithfully reconstitute all of the metabolic steps of viral mRNA translation (11,22,23) and viral RNA replication (5, 7, 27). A significant advantage of this experimental system is the ability to study one cycle of seq...

The structures of polio-, coxsackie-, and rhinovirus polymerases have revealed a conserved yet unusual protein conformation surrounding their buried N termini where a ␤-strand distortion results in a solventexposed hydrophobic amino acid at residue 5. In a previous study, we found that coxsackievirus polymerase activity increased or decreased depending on the size of the amino acid at residue 5 and proposed that this residue becomes buried during the catalytic cycle. In this work, we extend our studies to show that poliovirus polymerase activity is also dependent on the nature of residue 5 and further elucidate which aspects of polymerase function are affected. Poliovirus polymerases with mutations of tryptophan 5 retain wild-type elongation rates, RNA binding affinities, and elongation complex formation rates but form unstable elongation complexes. A large hydrophobic residue is required to maintain the polymerase in an elongation-competent conformation, and smaller hydrophobic residues at position 5 progressively decrease the stability of elongation complexes and their processivity on genome-length templates. Consistent with this, the mutations also reduced viral RNA production in a cell-free replication system. In vivo, viruses containing residue 5 mutants produce viable virus, and an aromatic phenylalanine was maintained with only a slightly decreased virus growth rate. However, nonaromatic amino acids resulted in slow-growing viruses that reverted to wild type. The structural basis for this polymerase phenotype is yet to be determined, and we speculate that amino acid residue 5 interacts directly with template RNA or is involved in a protein structural interaction that stabilizes the elongation complex.Members of the Picornaviridae family of small RNA viruses cause a wide range of diseases in humans, including liver disease, heart disease, aseptic meningitis, the common cold, and poliomyelitis. The picornaviruses include the most common human viruses, which are the rhinoviruses that spread through respiratory pathways, and the second most common viruses, which are enteroviruses that spread by fecal-oral transmission. These viruses have Ϸ7.5-kb positive-sense genomes containing a single large open reading frame encoding a Ϸ250-kDa polyprotein that is cleaved into about a dozen different proteins by viral proteases (20). Their genome life cycle is completely RNA based, with replication being driven by the viral 3D pol protein, an RNA-dependent RNA polymerase (RdRP). After viral RNA translation and polyprotein processing, 3D pol replicates the infecting positive-strand RNA template into a negative-strand intermediate that is subsequently used as a template for positive-strand synthesis. During these processes, 3D pol interacts with multiple templates, substrates, and other viral proteins; however, many aspects of these events remain obscure. The crystal structures of several picornaviral 3D pol enzymes have been solved, and these all conform to the "right hand" analogy commonly used to describe polymerases as ...

cis-acting RNA sequences and structures in the 5 and 3 nontranslated regions of poliovirus RNA interact with host translation machinery and viral replication proteins to coordinately regulate the sequential translation and replication of poliovirus RNA. The poliovirus internal ribosome entry site (IRES) in the 5 nontranslated region (NTR) has been implicated as a cis-active RNA required for both viral mRNA translation and viral RNA replication. To evaluate the role of the IRES in poliovirus RNA replication, we exploited the advantages of cell-free translation-replication reactions and preinitiation RNA replication complexes. Genetic complementation with helper mRNAs allowed us to create preinitiation RNA replication complexes containing RNA templates with defined deletions in the viral open reading frame and the IRES. A series of deletions revealed that no RNA elements of either the viral open reading frame or the IRES were required in cis for negative-strand RNA synthesis. The IRES was dispensable for both negative-and positive-strand RNA syntheses. Intriguingly, although small viral RNAs lacking the IRES replicated efficiently, the replication of genome length viral RNAs was stimulated by the presence of the IRES. These results suggest that RNA replication is not directly dependent on a template RNA first functioning as an mRNA. These results further suggest that poliovirus RNA replication is not absolutely dependent on any protein-RNA interactions involving the IRES. Poliovirus (PV), the prototypic member of the viral familyPicornaviridae, is a positive-polarity RNA virus 7,441 nucleotides (nt) in length (44). PV RNA is composed of a 5Ј nontranslated region (NTR), an open reading frame (ORF) encoding the viral proteins, a 3Ј NTR, and a 3Ј-terminal poly(A) tail. PV RNA is sequentially translated and replicated within the cytoplasm of an infected cell. The 5Ј NTR of PV RNA is composed of two functionally discrete RNA elements. The first 88 nt of the 5Ј NTR form a cloverleaf structure involved in RNA stability and RNA replication (31, 38). The 5Ј NTR also contains the internal ribosome entry site (IRES). The IRES is composed of nt 124 to ϳ630 [PV type 1(M) nucleotide numbering] (21) and is known to interact with canonical and noncanonical translation factors to direct ribosomes to an internal translation initiation site at nt 743 (15). The 5Ј-terminal cloverleaf and 3Ј NTR function coordinately to mediate viral negative-strand RNA synthesis (9, 24, 31), suggesting that viral RNA may assume a conformation involving direct interactions between ribonucleoprotein complexes containing the 5Ј and 3Ј NTRs for the initiation of RNA replication. It is possible that interactions of PV mRNA with the cellular translation machinery alter the conformation of viral RNA, bringing the 5Ј and 3Ј NTRs into a proximal orientation favorable for the subsequent formation of functional RNA replication complexes.Previous investigations suggested that the IRES possesses signals required for both viral mRNA translation and viral RNA repli...

Six different adjuvants, each in combination with inactivated polio vaccine (IPV) produced with attenuated Sabin strains (sIPV), were evaluated for their ability to enhance virus neutralizing antibody titers (VNTs) in the rat potency model. The increase of VNTs was on average 3-, 15-, 24-fold with adjuvants after one immunization (serotype 1, 2, and 3, respectively). Also after a boost immunization the VNTs of adjuvanted sIPV were on average another 7- 20- 27 times higher than after two inoculations of sIPV without adjuvant. The results indicate that it is feasible to increase the potency of inactivated polio vaccines by using adjuvants.