A mucosal adjuvant for the inactivated poliovirus vaccine.

The eradication of poliovirus from the majority of the world has been achieved through the use of two vaccines: the inactivated poliovirus vaccine (IPV) and the live-attenuated oral poliovirus vaccine (OPV). Both vaccines are effective at preventing paralytic poliomyelitis, however, they also have significant differences. Most importantly for this work is the risk of revertant virus from OPV, the greater cost of IPV, and the low mucosal immunity induced by IPV. We and others have previously described the use of an alphavirus-based adjuvant that can induce a mucosal immune response to a co-administered antigen even when delivered at a non-mucosal site. In this report, we describe the use of an alphavirus-based adjuvant (GVI3000) with IPV. The IPV-GVI3000 vaccine significantly increased systemic IgG, mucosal IgG and mucosal IgA antibody responses to all three poliovirus serotypes in mice even when administered intramuscularly. Furthermore, GVI3000 significantly increased the potency of IPV in rat potency tests as measured by poliovirus neutralizing antibodies in serum. Thus, an IPV-GVI3000 vaccine would reduce the dose of IPV needed and provide significantly improved mucosal immunity. This vaccine could be an effective tool to use in the poliovirus eradication campaign without risking the re-introduction of revertant poliovirus derived from OPV.

Antigen sparing with adjuvanted inactivated polio vaccine based on Sabin strains.

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 titres (VNTs) in the rat potency model. The increase of VNTs was on average 3-, 15-, 24-fold with adjuvants after one immunization (serotypes 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.

Poliovirus polymerase residue 5 plays a critical role in elongation complex stability.

The structures of polio-, coxsackie-, and rhinovirus polymerases have revealed a conserved yet unusual protein conformation surrounding their buried N termini where a beta-strand distortion results in a solvent-exposed 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.

Poly(A) at the 3' end of positive-strand RNA and VPg-linked poly(U) at the 5' end of negative-strand RNA are reciprocal templates during replication of poliovirus RNA.

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.

Conversion of VPg into VPgpUpUOH before and during poliovirus negative-strand RNA synthesis.

There are two protein primers involved in picornavirus RNA replication, VPg, the viral protein of the genome, and VPgpUpU(OH). A cis-acting replication element (CRE) within the open reading frame of poliovirus (PV) RNA allows the viral RNA-dependent RNA polymerase 3D(Pol) to catalyze the conversion of VPg into VPgpUpU(OH). In this study, we used preinitiation RNA replication complexes (PIRCs) to determine when CRE-dependent VPg uridylylation occurs relative to the sequential synthesis of negative- and positive-strand RNA. Guanidine HCl (2 mM), a reversible inhibitor of PV 2C(ATPase), prevented CRE-dependent VPgpUpU(OH) synthesis and the initiation of negative-strand RNA synthesis. VPgpUpU(OH) and nascent negative-strand RNA molecules were synthesized coincident in time following the removal of guanidine, consistent with PV RNA functioning simultaneously as a template for CRE-dependent VPgpUpU(OH) synthesis and negative-strand RNA synthesis. The amounts of [(32)P]UMP incorporated into VPgpUpU(OH) and negative-strand RNA products indicated that 100 to 400 VPgpUpU(OH) molecules were made coincident in time with each negative-strand RNA. 3'-dCTP inhibited the elongation of nascent negative-strand RNAs without affecting CRE-dependent VPg uridylylation. A 3' nontranslated region mutation which inhibited negative-strand RNA synthesis did not inhibit CRE-dependent VPg uridylylation. Together, the data implicate 2C(ATPase) in the mechanisms whereby PV RNA functions as a template for reiterative CRE-dependent VPg uridylylation before and during negative-strand RNA synthesis.

Cis-active RNA elements (CREs) and picornavirus RNA replication.

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.

Poliovirus cis-acting replication element-dependent VPg Uridylylation lowers the Km of the initiating nucleoside triphosphate for viral RNA replication.

Initiation of RNA synthesis by RNA-dependent RNA polymerases occurs when a phosphodiester bond is formed between the first two nucleotides in the 5' terminus of product RNA. The concentration of initiating nucleoside triphosphates (NTPi) required for RNA synthesis is typically greater than the concentration of NTPs required for elongation. VPg, a small viral protein, is covalently attached to the 5' end of picornavirus negative- and positive-strand RNAs. A cis-acting replication element (CRE) within picornavirus RNAs serves as a template for the uridylylation of VPg, resulting in the synthesis of VPgpUpU(OH). Mutations within the CRE RNA structure prevent VPg uridylylation. While the tyrosine hydroxyl of VPg can prime negative-strand RNA synthesis in a CRE- and VPgpUpU(OH)-independent manner, CRE-dependent VPgpUpU(OH) synthesis is absolutely required for positive-strand RNA synthesis. As reported herein, low concentrations of UTP did not support negative-strand RNA synthesis when CRE-disrupting mutations prevented VPg uridylylation, whereas correspondingly low concentrations of CTP or GTP had no negative effects on the magnitude of CRE-independent negative-strand RNA synthesis. The experimental data indicate that CRE-dependent VPg uridylylation lowers the K(m) of UTP required for viral RNA replication and that CRE-dependent VPgpUpU(OH) synthesis was required for efficient negative-strand RNA synthesis, especially when UTP concentrations were limiting. By lowering the concentration of UTP needed for the initiation of RNA replication, CRE-dependent VPg uridylylation provides a mechanism for a more robust initiation of RNA replication.

Replication of poliovirus RNA with complete internal ribosome entry site deletions.

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.