A new cis-acting element for RNA replication within the 5' noncoding region of poliovirus type 1 RNA.

Mouse cells expressing the human poliovirus receptor (PVR-mouse cells) as well as human HeLa cells are susceptible to poliovirus type 1 Mahoney strains and produce a large amount of progeny virus at 37 degrees C. However, the virus yield is markedly reduced at 40 degrees C in PVR-mouse cells but not in HeLa cells. The reduction in virus yield at 40 degrees C appears to be due to a defective initiation process in positive-strand RNA synthesis (K. Shiroki, H. Kato, S. Koike, T. Odaka, and A. Nomoto, J. Virol. 67:3989-3996, 1993). To gain insight into the molecular mechanisms involved in this detective process, naturally occurring heat-resistant (Hr)-mutants which show normal growth ability in PVR-mouse cells even at 40 degrees C were isolated from a virus stock of the Mahoney strain and their mutation sites that affect the phenotype were identified. The key mutation was a change from adenine (A) to guanine (G) at nucleotide position (nt) 133 within the 5' noncoding region of the RNA. This mutation also gave an Hr phenotype to the viral plus-strand RNA synthesis in PVR-mouse cells. Mutant Mahoney strains with a single point mutation at nt 133 (A to G, C, or T or deletion) were investigated for their ability to grow in PVR-mouse cells at 40 degrees C. Only the mutant carrying G at nt 133 showed an Hr growth phenotype in PVR-mouse cells. These results suggest that a host cellular factor(s) interacts with an RNA segment around nt 133 of the plus-strand RNA or the corresponding region of the minus-strand RNA, contributing to efficiency of plus-strand RNA synthesis.     

Host cell proteins binding to domain IV of the 5' noncoding region of poliovirus RNA.

Translation of poliovirus RNA occurs by the binding of ribosomes to an internal segment of RNA sequence within the 5' untranslated region of the viral RNA. This region is predicted to consist of six domains (I to VI) that possess complex secondary and tertiary structures. Domain IV is a large region in which alterations in the sequence or structure markedly reduce translational efficiency. In this study, we employed RNA mobility shift assays to demonstrate that a protein(s) from uninfected HeLa cell extracts, as well as from neuroblastoma extracts, interacts with the domain IV structure. A mutation in domain IV caused reduced binding of HeLa cell proteins and reduced translation both in vitro and in vivo, suggesting that the binding of at least one of these proteins plays a role in the mechanism of viral translation. UV cross-linking indicated that a protein(s) with a size of approximately 40 kDa interacted directly with the RNA. Using streptavidin beads to capture biotinylated RNA bound to proteins, we were able to visualize a number of HeLa and neuroblastoma cell proteins that interact with domain IV. These proteins have molecular masses of approximately 39, approximately 40, and approximately 42 kDa.     

Attenuation stem-loop lesions in the 5' noncoding region of poliovirus RNA: neuronal cell-specific translation defects.

The nucleotide at position 480 in the 5' noncoding region of the viral RNA genome plays an important role in directing the attenuation phenotype of the Sabin vaccine strain of poliovirus type 1. In vitro translation studies have shown that the attenuated viral genomes of the Sabin strains direct levels of viral protein synthesis lower than those of their neurovirulent counterparts. We previously described the isolation of pseudorevertant polioviruses derived from transfections of HeLa cells with genome-length RNA harboring an eight-nucleotide lesion in a stem-loop structure (stem-loop V) that contains the attenuation determinant at position 480 (A. A. Haller and B. L. Semler, J. Virol. 66:5075-5086, 1992). This stem-loop structure is a major component of the poliovirus internal ribosome entry site required for initiation of viral protein synthesis. The eight-nucleotide lesion (X472) was lethal for virus growth and gave rise only to viruses which had partially reverted nucleotides within the original substituted sequences. In this study, we analyzed two of the poliovirus revertants (X472RI and X472R2) for cell-type-specific growth properties. The X472RI and X472R2 RNA templates directed protein synthesis to wild-type levels in in vitro translation reaction mixtures supplemented with crude cytoplasmic HeLa cell extracts. In contrast, the same X472 revertant RNAs displayed a decreased translation initiation efficiency when translated in a cell-free system supplemented with extracts from neuronal cells. This translation initiation defect of the X472R templates correlated with reduced yields of infectious virus particles in neuronal cells compared with those obtained from HeLa cells infected with the X472 poliovirus revertants. Our results underscore the important of RNA secondary structures within the poliovirus internal ribosome entry site in directing translation initiation and suggest that such structures interact with neuronal cell factors in a specific manner.     

Replication-competent picornaviruses with complete genomic RNA 3' noncoding region deletions.

The genomic RNA 3' noncoding region is believed to be a major cis-acting molecular genetic determinant for regulating picornavirus negative-strand RNA synthesis by promoting replication complex recognition. We report the replication of two picornavirus RNAs harboring complete deletions of the genomic RNA 3' noncoding regions. Our results suggest that while specific 3'-terminal RNA sequences and/or secondary structures may have evolved to promote or regulate negative-strand RNA synthesis, the basic mechanism of replication initiation is not strictly template specific and may rely primarily upon the proximity of newly translated viral replication proteins to the 3' terminus of template RNAs within tight membranous replication complexes.     

Two functional complexes formed by KH domain containing proteins with the 5' noncoding region of poliovirus RNA.

The 5' noncoding region of the poliovirus genome contains RNA structures important for replication and translation. Here we show that two closely related cellular poly(rC) binding proteins (PCBP1 and PCBP2) bind to the terminal cloverleaf structure and facilitate the interaction of the viral protein 3CD (the uncleaved precursor of the protease-polymerase). In addition, these cellular proteins bind to stem-loop IV of the internal ribosomal entry site. The proteins are cytoplasmic and largely associated with ribosomes; they appear to dimerize in solution and to form heterodimers when binding to stem-loop IV. Initiation of viral translation in Xenopus oocytes is strongly inhibited by co-injection of specific antibodies directed against PCBP1 or PCBP2, indicating that the poly(rC) binding proteins may facilitate this process. Furthermore, PCPB-depleted HeLa extracts translate poliovirus RNA inefficiently and the activity is partially restored by addition of recombinant PCBP proteins.     

Substitutions in the protease (3Cpro) gene of poliovirus can suppress a mutation in the 5' noncoding region.

The poliovirus mutant 5NC-11 has a 4-base insertion at position 70 within the 5' untranslated region and is deficient in RNA synthesis. Revertants from 5NC-11 were isolated, showing a partial recovery of wild-type levels of RNA synthesis. The 5' noncoding region of those revertants contained the mutation intact; mix-and-match experiments with the cDNA from these revertants revealed that a restricted region within the 3C gene was the site of the suppressing mutations in the revertants. The suppressors were point mutations, confirmed by introducing them into the 3C gene by site-directed mutagenesis. Although complementation studies indicated that the suppressors were cis active, we believe that protein changes rather than RNA sequence alterations are responsible for the suppression because RNA changes that did not alter protein sequence had no effect, whereas various protein alterations were suppressive. The results therefore imply that protein 3C interacts with the 5' end of the RNA and may play a role in RNA replication.     

5' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis

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.     

Determinants in the 5'' noncoding region of poliovirus Sabin 1 RNA that influence the attenuation phenotype.

A number of recombinants between the virulent Mahoney and attenuated Sabin strains of type 1 poliovirus were constructed by using infectious cDNA clones of the two strains. To identify a strong neurovirulence determinant(s) residing in the genome region upstream of nucleotide position 1122, these recombinant viruses were subjected to biological tests, including monkey neurovirulence tests. The results of the monkey neurovirulence tests suggested the important contribution of an adenine residue (Mahoney type) at position 480 to the expression of the neurovirulence phenotype of type 1 poliovirus. This nucleotide, however, had only a minor effect, if any, on viral temperature sensitivity. Monkey neurovirulence tests on the recombinant virus whose genome had a guanine residue (Sabin type) at position 480 and variants generated from this recombinant virus in the central nervous system of monkeys strongly suggested that only one nucleotide change, from adenine to guanine, was not sufficient for full expression of the attenuation phenotype encoded by this genome region. These results suggest that the expression of the attenuation phenotype depends on the highly ordered structure formed in the 5' noncoding sequence and that the formation of such a structure is possibly influenced by the nucleotide at position 480. Furthermore, in vitro biological tests performed on viruses recovered from the central nervous system of monkeys injected with a temperature-sensitive recombinant virus showing the small-plaque and d phenotypes revealed that most of the recovered viruses had even higher temperature sensitivities and that all of the recovered viruses that had acquired the large-plaque phenotype had lost the d phenotype to some extent. These results indicate that there may be an unknown selection pressure(s) in the central nervous system and that common determinants might be involved in the expression of the small-plaque and d phenotypes.     

Poliovirus CRE-dependent VPg uridylylation is required for positive-strand RNA synthesis but not for negative-strand RNA synthesis

The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpU(OH), the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpU(OH), however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpU(OH) synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpU(OH) were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpU(OH) in the asymmetric replication of poliovirus RNA.     

Genetic variation in vivo and proposed functional domains of the 5'' noncoding region of poliovirus RNA.

Poliovirus has a single-stranded RNA genome of about 7,440 nucleotides (nt) with an unusually long 750-nt noncoding region in the 5' end (5'NCR). Several regulatory functions have been assigned to the 5'NCR. We sequenced the 5'NCRs of 33 wild-type 3 poliovirus strains to study the range and distribution of naturally occurring sequence variations. In this regard, the 5'NCR can be divided into a conserved part (nt 1 to 650) and a hypervariable part (nt 651 to 750). In the conserved part, altogether 234 unevenly distributed nucleotide positions (36%) showed variation. When these positions were plotted against the predicted secondary-structure models, it was found that the existence of most of the proposed stem-loop structures was supported by extensive structure-conserving substitutions in the stems. Regions with conserved sequences, as well as mutational hot spots, were observed. The hypervariable part of the 5'NCR varied up to 56% between the strains studied. The A + U percentage was significantly higher than in the conserved part. The number of AUG codons varied between 5 and 15 in the conserved part of the 5'NCR, while none was found in the hypervariable part. These results provide information that can be used in site-directed mutagenesis and other approaches targeted to reveal the functional domains of the 5'NCR.     

Biochemical and genetic evidence for a pseudoknot structure at the 3' terminus of the poliovirus RNA genome and its role in viral RNA amplification.

The sequences in the plus-stranded poliovirus RNA genome that dictate the specific amplification of viral RNA in infected cells remain unknown. We have analyzed the structure of the 3' noncoding region of the viral genome by thermodynamic-based structure calculation and by chemical and enzymatic probing of in vitro-synthesized RNAs and provide evidence for the existence of an RNA pseudoknot structure in this region. To explore the functional significance of this structure, revertants of a mutant bearing a lesion in the proposed pseudoknot and exhibiting a temperature-sensitive defect in viral RNA synthesis were isolated and mapped. The results of this genetic analysis established a correlation between the structure of the 3' terminus of the viral RNA and its function in vivo in RNA amplification. Furthermore, phylogenetic analysis indicated that a similar structure could be formed in coxsackievirus B1, a related enterovirus, which further supports a role for the pseudoknot structure in viral RNA amplification in infected cells.     

trans complementation of cap-independent translation directed by poliovirus 5' noncoding region deletion mutants: evidence for RNA-RNA interactions.

Poliovirus (PV) RNA is translated by a cap-independent mechanism involving the internal entry of ribosomes onto the 5' noncoding region (NCR). Using the vaccinia virus-T7 RNA polymerase transient expression system, we showed previously that deletion of certain individual predicted secondary structures within the PV 5' NCR rendered the element defective in directing internal initiation when assayed alone. However, these defective 5' NCRs were functional when coexpressed within cells with full-length PV cDNA (N. Percy, G. J. Belsham, J. K. Brangwyn, M. Sullivan, D. M. Stone, and J. W. Almond, J. Virol. 66:1695-1701, 1992). We have extended the study to demonstrate that when these predicted secondary structures are deleted in combination, the enhanced activity in the presence of the full-length PV cDNA is still observed. Indeed, a poliovirus 5' NCR devoid of all predicted secondary structures is capable of initiating protein synthesis under these conditions. Surprisingly, we also found that this enhancement of activity requires neither any PV protein nor the inhibition of cap-dependent translation. The results indicate that the defective PV 5' NCR elements can be complemented in trans by functional 5' NCRs in a highly sequence specific manner.     

A region of the 5' noncoding region of foot-and-mouth disease virus RNA directs efficient internal initiation of protein synthesis within cells: involvement with the role of L protease in translational control.

Plasmids encoding bicistronic mRNAs have been constructed and used to identify a region from the 5' noncoding region of foot-and-mouth disease virus (FMDV) which directs efficient internal initiation of protein synthesis within cells. The loss of about 30 nucleotides (nt) from the 5' terminus or about 50 nt from the 3' terminus of the 435-nt region completely abolished the activity of this region. The expression of the FMDV L protease severely inhibited the expression of other genes unless they were preceded by this element. The regulation of protein synthesis mediated by FMDV is discussed.     

3'-Terminal sequence in poliovirus negative-strand templates is the primary cis-acting element required for VPgpUpU-primed positive-strand initiation

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 positive-strand 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 3'AAUUUUGUC5' 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.     

HCV RNA-dependent RNA polymerase replicates in vitro the 3' terminal region of the minus-strand viral RNA more efficiently than the 3' terminal region of the plus RNA.

The NS5B protein, or RNA-dependent RNA polymerase of the hepatitis virus type C, catalyzes the replication of the viral genomic RNA. Little is known about the recognition domains of the viral genome by the NS5B. To better understand the initiation of RNA synthesis on HCV genomic RNA, we used in vitro transcribed RNAs as templates for in vitro RNA synthesis catalyzed by the HCV NS5B. These RNA templates contained different regions of the 3' end of either the plus or the minus RNA strands. Large differences were obtained depending on the template. A few products shorter than the template were synthesized by using the 3' UTR of the (+) strand RNA. In contrast the 341 nucleotides at the 3' end of the HCV minus-strand RNA were efficiently copied by the purified HCV NS5B in vitro. At least three elements were found to be involved in the high efficiency of the RNA synthesis directed by the HCV NS5B with templates derived from the 3' end of the minus-strand RNA: (a) the presence of a C residue as the 3' terminal nucleotide; (b) one or two G residues at positions +2 and +3; (c) other sequences and/or structures inside the following 42-nucleotide stretch. These results indicate that the 3' end of the minus-strand RNA of HCV possesses some sequences and structure elements well recognized by the purified NS5B.