Translational efficiency of poliovirus mRNA: mapping inhibitory cis-acting elements within the 5'' noncoding region.

Poliovirus mRNA contains a long 5' noncoding region of about 750 nucleotides (the exact number varies among the three virus serotypes), which contains several AUG codons upstream of the major initiator AUG. Unlike most eucaryotic mRNAs, poliovirus does not contain a m7GpppX (where X is any nucleotide) cap structure at its 5' end and is translated by a cap-independent mechanism. To study the manner by which poliovirus mRNA is expressed, we examined the translational efficiencies of a series of deletion mutants within the 5' noncoding region of the mRNA. In this paper we report striking translation system-specific differences in the ability of the altered mRNAs to be translated. The results suggest the existence of an inhibitory cis-acting element(s) within the 5' noncoding region of poliovirus (between nucleotides 70 and 381) which restricts mRNA translation in reticulocyte lysate, wheat germ extract, and Xenopus oocytes, but not in HeLa cell extracts. In addition, we show that HeLa cell extracts contain a trans-acting factor(s) that overcomes this restriction.     

In vitro mutational analysis of cis-acting RNA translational elements within the poliovirus type 2 5'' untranslated region.

Initiation of translation on poliovirus RNA occurs by internal binding of ribosomes to a region within the 5' untranslated region (UTR) of the mRNA. This region has been previously roughly mapped between nucleotides 140 and 631 of the 5' UTR and termed the ribosome landing pad. To identify cis-acting elements in the 5' UTR of poliovirus type 2 (Lansing strain) RNA that confer cap-independent internal initiation, we determined the in vitro translational efficiencies of a series of deletion and point mutations within the 5' UTR of the mRNA. The results demonstrate that the 3' border of the core poliovirus ribosome landing pad is located between nucleotides 556 and 585, whereas a region extending between nucleotides 585 and 612 confers enhanced translation. We studied two cis-acting elements within this region of the 5' UTR: a pyrimidine stretch which is critical for translation and an AUG (number 7 from the 5' end) that is located approximately 20 nucleotides downstream from the pyrimidine stretch and augments translation. We also show that the stem-loop structure which contains this AUG is not required for translation.     

Selective destabilization of short-lived mRNAs with the granulocyte-macrophage colony-stimulating factor AU-rich 3'' noncoding region is mediated by a cotranslational mechanism.

The 3' noncoding region element (AUUUA)n specifically targets many short-lived mRNAs for degradation. Although the mechanism by which this sequence functions is not yet understood, a potential link between facilitated mRNA turnover and translation has been implied by the stabilization of cellular mRNAs in the presence of protein synthesis inhibitors. We therefore directly investigated the role of translation on mRNA stability. We demonstrate that mRNAs which are poorly translated through the introduction of stable secondary structure in the 5' noncoding region are not efficiently targeted for selective destabilization by the (AUUUA)n element. These results suggest that AUUUA-mediated degradation involves either a 5'-->3' exonuclease or is coupled to ongoing translation of the mRNA. To distinguish between these two possibilities, we inserted the poliovirus internal ribosome entry site, which promotes internal ribosome initiation, downstream of the 5' secondary structure. Translation directed by internal ribosome binding was found to fully restore targeted destabilization of AUUUA-containing mRNAs despite the presence of 5' secondary structure. This study therefore demonstrates that selective degradation mediated by the (AUUUA)n element is coupled to ribosome binding or ongoing translation of the mRNA and does not involve 5'-to-3' exonuclease activity.     

Linker scanning mutagenesis of the internal ribosome entry site of poliovirus RNA.

The initiation of cap-independent translation of poliovirus mRNA occurs as a result of ribosome entry at an internal site(s) within the 5' noncoding region. A series of linker scanning mutations was constructed to define the genetic determinants of RNA-protein interactions that lead to high-fidelity translation of this unusual viral mRNA. The mutations are located within two distinct stem-loop structures in the 5' noncoding region of poliovirus RNA that constitute a major portion of a putative internal ribosome entry site. On the basis of our data derived from genetic and biochemical assays, the stability of one of the stem-loop structures appears to be essential for translation initiation via internal binding of ribosomes. However, the second stem-loop structure may function in a manner that requires base pairing and proper spacing between specific nucleotide sequences. By employing RNA electrophoretic mobility shift assays, an RNA-protein interaction was detected for this latter stem-loop structure that does not occur in RNAs containing mutations which perturb the predicted hairpin structure. Analysis of in vivo-selected virus revertants, in combination with mobility shift assays, suggests that extensive genetic rearrangement can lead to restoration of 5' noncoding region functions, possibly by the repositioning of specific RNA sequence or structure motifs.     

Internal binding of eucaryotic ribosomes on poliovirus RNA: translation in HeLa cell extracts.

Translation initiation on poliovirus mRNA in poliovirus-infected cells has been shown to occur by internal binding of ribosomes to the 5' noncoding region (J. Pelletier and N. Sonenberg, Nature [London] 334:320-325, 1988). Here we show that internal ribosome binding can occur in HeLa cell extracts in vitro. Internal binding to the 5' noncoding region of poliovirus mRNA in a bicistronic context was independent of the upstream open reading frame and did not require poliovirus proteins.     

Poliovirus translation: a paradigm for a novel initiation mechanism

All eukaryotic cellular mRNAs, and most viral mRNAs, are blocked at their 5' ends with a cap structure (m7GpppX, where X is any nucleotide). Poliovirus, along with a small number of other animal and plant viral mRNAs, does not contain a 5' cap structure. Since the cap structure functions to facilitate ribosome binding to mRNA, translation of polio-virus must proceed by a cap-independent mechanism. Consistent with this, recent studies have shown that ribosomes can bind to an internal region within the long 5' noncoding sequence of poliovirus RNA. Possible mechanisms for cap-independent translation are discussed. Cap-independent translation of poliovirus RNA is of major importance to the mechanism of shut-off of host protein synthesis after infection. Moreover, it is likely to play a role in determining poliovirus neurovirulence and attenuation.     

Minimum internal ribosome entry site required for poliovirus infectivity.

Translation initiation by internal ribosome binding is a recently discovered mechanism of eukaryotic viral and cellular protein synthesis in which ribosome subunits interact with the mRNAs at internal sites in the 5' untranslated RNA sequences and not with the 5' methylguanosine cap structure present at the extreme 5' ends of mRNA molecules. Uncapped poliovirus mRNAs harbor internal ribosome entry sites (IRES) in their long and highly structured 5' noncoding regions. Such IRES sequences are required for viral protein synthesis. In this study, a novel poliovirus was isolated whose genomic RNA contains two gross deletions removing approximately 100 nucleotides from the predicted IRES sequences within the 5' noncoding region. The deletions originated from previously in vivo-selected viral revertants displaying non-temperature-sensitive phenotypes. Each revertant had a different predicted stem-loop structure within the 5' noncoding region of their genomic RNAs deleted. The mutant poliovirus (Se1-5NC-delta DG) described in this study contains both stem-loop deletions in a single RNA genome, thereby creating a minimum IRES. Se1-5NC-delta DG exhibited slow growth and a pinpoint plaque phenotype following infection of HeLa cells, delayed onset of protein synthesis in vivo, and defective initiation during in vitro translation of the mutated poliovirus mRNAs. Interestingly, the peak levels of viral RNA synthesis in cells infected with Se1-5NC-delta DG occurred at slightly later times in infection than those achieved by wild-type poliovirus, but these mutant virus RNAs accumulated in the host cells during the late phases of virus infection. UV cross-linking assays with the 5' noncoding regions of wild-type and mutated RNAs were carried out in cytoplasmic extracts from HeLa cells and neuronal cells and in reticulocyte lysates to identify the cellular factors that interact with the putative IRES elements. The cellular proteins that were cross-linked to the minimum IRES may represent factors playing an essential role in internal translation initiation of poliovirus mRNAs.     

cis-acting translational effects of the 5'' noncoding region of c-myc mRNA.

We have previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte lysate (A. Darveau, J. Pelletier, and N. Sonenberg, Proc. Natl. Acad. Sci. USA 82:2315-2319, 1985). We wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. Here we report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, we report that the cis-inhibitory effect is also exhibited in microinjected Xenopus oocytes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.     

Translation of poliovirus RNA: role of an essential cis-acting oligopyrimidine element within the 5'' nontranslated region and involvement of a cellular 57-kilodalton protein.

Translation of poliovirus RNA is initiated by cap-independent internal entry of ribosomes into the 5' nontranslated region. This process is dependent on elements within the 5' nontranslated region (the internal ribosomal entry site) and may involve novel translation factors. Systematic mutation of a conserved oligopyrimidine tract has revealed a cis-acting element that is essential for translation in vitro. The function of this element is related to its position relative to other cis-acting domains. This element is part of a more complex structure that interacts with several cellular factors, but changes in protein binding after mutation of this element were not detected in a UV cross-linking assay. A 57-kDa protein from the ribosomal salt wash fraction of HeLa cells was identified that binds upstream of the oligopyrimidine tract. Translation of poliovirus mRNA in vitro was strongly and specifically inhibited by competition with the p57-binding domain (nucleotides 260 to 488) of the 5' nontranslated region of encephalomyocarditis virus, indicating a probable role for p57 in poliovirus translation. p57 is likely to be identical to the ribosome-associated factor that binds to and is necessary for the function of the internal ribosomal entry site of encephalomyocarditis virus RNA.     

Mutational analysis of upstream AUG codons of poliovirus RNA.

The 5' untranslated region of poliovirus type 2 Lansing RNA consists of 744 nucleotides containing seven AUG codons which are followed by in-frame termination codons, thus forming short open reading frames (ORFs). To determine the biological significance of these small ORFs, all of the upstream AUG codons were mutated to UUG. The point mutations were introduced into an infectious poliovirus cDNA clone, and RNA transcribed in vitro from the altered cDNA was transfected into HeLa cells to recover the virus. Mutation of AUG 7 resulted in a virus (called R2-5NC-14) with a small-plaque phenotype, whereas mutation of the other six AUG codons produced virus with a wild-type plaque morphology. To determine whether the small-plaque phenotype of R2-5NC-14 was due to altered translational efficiency of the viral mRNA, we constructed chimeric mRNAs containing the 5' noncoding region of poliovirus mRNA fused to the chloramphenicol acetyltransferase (CAT) coding sequence. mRNA containing a mutated AUG 7 codon showed decreased translational efficiency in vitro. The results indicate that the upstream ORFs of poliovirus RNA are not essential for viral replication and do not act as barriers to the translation of poliovirus mRNA. AUG 7 and flanking sequences may play a positive acting role in poliovirus RNA translation.     

Feedback inhibition of poly(A)-binding protein mRNA translation. A possible mechanism of translation arrest by stalled 40 S ribosomal subunits

An adenine-rich cis element at the 5'-untranslated region (UTR) of Pabp1 mRNA is able to inhibit translation of its own mRNA. Similar inhibition of translation of a reporter beta-galactosidase mRNA is observed when the adenine-rich auto regulatory sequence (ARS) is placed within the 5'-UTR of this mRNA. For this translational control the distance of the ARS from the 5' cap is not important. However, it determines the number of 40 S ribosomal subunits bound to the translationally arrested mRNA. Inhibition of mRNA translation by this regulatory sequence occurs at the step of joining of the 60 S ribosomal subunit to the pre-initiation complex. Translational arrest of the ARS containing mRNA in a rabbit reticulocyte lysate cell-free system in the presence of exogenous Pabp1 protects the 5'-flanking region of the ARS from nuclease digestion. This protection depends on the binding of the 40 S ribosomal subunit to the mRNA. The size and the sequence of the nucleotide-protected fragment depends on the location of the ARS within the 5'-UTR. When the ARS is located at a distance of about 78 nucleotides from the 5' cap, a 40-nucleotide long region adjacent to the ARS is protected. On the other hand, when the ARS is moved further away from the 5' cap to a distance of approximately 267 nucleotides, a 100-nucleotide-long region adjacent to the ARS is protected from nuclease digestion. Nuclease protection is attributed to the presence of one or more stalled 40 S ribosomal subunits near the Pabp1-bound ARS.     

mRNAs containing the unstructured 5' leader sequence of alfalfa mosaic virus RNA 4 translate inefficiently in lysates from poliovirus-infected HeLa cells.

Poliovirus infection is accompanied by translational control that precludes translation of 5'-capped mRNAs and facilitates translation of the uncapped poliovirus RNA by an internal initiation mechanism. Previous reports have suggested that the capped alfalfa mosaic virus coat protein mRNA (AIMV CP RNA), which contains an unstructured 5' leader sequence, is unusual in being functionally active in extracts prepared from poliovirus-infected HeLa cells (PI-extracts). To identify the cis-acting nucleotide elements permitting selective AIMV CP expression, we tested capped mRNAs containing structured or unstructured 5' leader sequences in addition to an mRNA containing the poliovirus internal ribosome entry site (IRES). Translations were performed with PI-extracts and extracts prepared from mock-infected HeLa cells (MI-extracts). A number of control criteria demonstrated that the HeLa cells were infected by poliovirus and that the extracts were translationally active. The data strongly indicate that translation of RNAs lacking an internal ribosome entry site, including AIMV CP RNA, was severely compromised in PI-extracts, and we find no evidence that the unstructured AIMV CP RNA 5' leader sequence acts in cis to bypass the poliovirus translational control. Nevertheless, cotranslation assays in the MI-extracts demonstrate that mRNAs containing the unstructured AIMV CP RNA 5' untranslated region have a competitive advantage over those containing the rabbit alpha-globin 5' leader. Previous reports of AIMV CP RNA translation in PI-extracts likely describe inefficient expression that can be explained by residual cap-dependent initiation events, where AIMV CP RNA translation is competitive because of a diminished quantitative requirement for initiation factors.     

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.     

Poliovirus 5'-terminal cloverleaf RNA is required in cis for VPg uridylylation and the initiation of negative-strand RNA synthesis

Chimeric poliovirus RNAs, possessing the 5' nontranslated region (NTR) of hepatitis C virus in place of the 5' NTR of poliovirus, were used to examine the role of the poliovirus 5' NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3' NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5'-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5'-terminal cloverleaf and 3' NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA.     

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.     

Cell-dependent role for the poliovirus 3' noncoding region in positive-strand RNA synthesis

We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3' noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3' noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3' noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3' noncoding region is involved in positive-strand synthesis during genome replication.