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.     

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.     

A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry

Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotide-long 5' untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translation systems. Here, the mechanism of action of the RhPV IRES has been characterized by reconstitution of mammalian 48S initiation complexes on the IRES from purified components combined with the toeprint assay. There is an absolute requirement for the initiation factors eIF2 and eIF3 and the scanning factor eIF1 to form 48S complexes on the IRES. In addition, eIF1A, eIF4F (or the C-terminal fragment of eIF4G), and eIF4A strongly stimulated the assembly of this complex, whereas eIF4B had no effect. Although the eIF4-dependent pathway is dominant in the RhPV IRES-directed cell-free translation, omission of either eIF4G or eIF4A or both still allowed the assembly of 48S complexes from purified components with approximately 23% of maximum efficiency. Deletions of up to 100 nucleotides throughout the 5'-UTR sequence produced at most a marginal effect on the IRES activity, suggesting the absence of specific binding sites for initiation factors. Only deletion of the U-rich unstructured 380-nucleotide region proximal to the initiation codon resulted in a complete loss of the IRES activity. We suggest that the single-stranded nature of the RhPV IRES accounts for its strong but less selective potential to bind key mRNA recruiting components of the translation initiation apparatus from diverse origins.     

Effect of mutations downstream of the internal ribosome entry site on initiation of poliovirus protein synthesis.

Initiation of poliovirus translation is mediated by a large, structured segment of the 5' nontranslated region known as the internal ribosome entry site (IRES) and normally occurs 155 nucleotides (nt) downstream of the IRES at AUG743 (the AUG at nucleotide 743). Functional AUG codons introduced at nt 611 or 614 reduced initiation at AUG743 by 10 to 40% in vitro but had no effect on virus phenotype. To investigate the role of the nt 586-743 spacer in greater detail, four intervening termination codons were removed, and an additional AUG triplet at nt 683 was introduced by nucleotide substitution. Initiation at AUG743 was reduced by only 50 to 80%, depending on the number of upstream initiation codons. Initiation at AUG743 was also reduced following insertion of a stable hairpin at nt 630, but the reduction was modest in an ascites carcinoma cell extract. Initiation was more frequent at AUG743 than at AUG683 if mRNAs contained either an upstream initiation codon or the stable hairpin. These results suggested that not all initiation events at AUG743 can be accounted for by a scanning-dependent mechanism. Translation of bicistronic mRNAs in which the intercistronic spacer contained nt 630 to 742 of the poliovirus 5' nontranslated region indicated that these residues are not able to act as an entry point for ribosomes independently of the IRES. Insertion of increasingly longer sequences immediately downstream of the stable hairpin progressively reduced initiation at AUG743 without affecting initiation at AUG683. These results are discussed in terms of a model for initiation of poliovirus translation in which a complex RNA superstructure upstream of nt 586 promotes ribosome binding at an entry point determined by specific downstream cis-acting elements.     

A cell cycle-dependent internal ribosome entry site

The eukaryotic mRNA 5' cap structure facilitates translation. However, cap-dependent translation is impaired at mitosis, suggesting a cap-independent mechanism for mRNAs translated during mitosis. Translation of ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, peaks twice during the cell cycle, at the G1/S transition and at G2/M. Here, we describe a cap-independent internal ribosome entry site (IRES) in the ODC mRNA that functions exclusively at G2/M. This ensures elevated levels of polyamines, which are implicated in mitotic spindle formation and chromatin condensation. c-myc mRNA also contains an IRES that functions during mitosis. Thus, IRES-dependent translation is likely to be a general mechanism to synthesize short-lived proteins even at mitosis, when cap-dependent translation is interdicted.     

Interaction of translation initiation factor eIF4B with the poliovirus internal ribosome entry site

Poliovirus translation is initiated at the internal ribosome entry site (IRES). Most likely involving the action of standard initiation factors, this highly structured cis element in the 5" noncoding region of the viral RNA guides the ribosome to an internal silent AUG. The actual start codon for viral protein synthesis further downstream is then reached by ribosomal scanning. In this study we show that two of the secondary structure elements of the poliovirus IRES, domain V and, to a minor extent, domain VI, are the determinants for binding of the eukaryotic initiation factor eIF4B. Several mutations in domain V which are known to greatly affect poliovirus growth also seriously impair the binding of eIF4B. The interaction of eIF4B with the IRES is not dependent on the presence of the polypyrimidine tract-binding protein, which also binds to the poliovirus IRES. In contrast to its weak interaction with cellular mRNAs, eIF4B remains tightly associated with the poliovirus IRES during the formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. These results indicate that the interaction of eIF4B with the 3" region of the poliovirus IRES may be directly involved in translation initiation.     

Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis.

The 5' untranslated region of poliovirus RNA has been reported to possess two functional elements: (i) the 5' proximal 88 nucleotides form a cloverleaf structure implicated in positive-strand RNA synthesis during viral replication, and (ii) nucleotides 134 to at least 556 function as a highly structured internal ribosome entry segment (IRES) during cap-independent, internal initiation of translation. We show here that the IRES itself is bifunctional and contains sequences necessary for viral RNA synthesis per se. For this purpose, we used a dicistronic poliovirus RNA in which the translation of the viral non-structural (replication) proteins is uncoupled from the poliovirus IRES. In this system, RNA synthesis is readily detectable in transfected cells, even when the poliovirus IRES is inactivated by point mutation. However, deletion of the major part of the poliovirus IRES renders viral-specific RNA synthesis undetectable. Using the same system, we show that a three nucleotide deletion at position 500 in the 5' untranslated region drastically affects both translation efficiency and RNA synthesis. Furthermore, disruption of the secondary structure of the IRES around nucleotide 343 has minimal effects on IRES function, but dramatically reduces viral RNA replication. Taken together, these results provide direct evidence that sequences essential for viral RNA synthesis are located in the 3' region of the poliovirus IRES.     

Mutation of a single conserved nucleotide between the cloverleaf and internal ribosome entry site attenuates poliovirus neurovirulence

The chemical synthesis of poliovirus (PV) cDNA combined with the cell-free synthesis of infectious particles yielded virus whose mouse neurovirulence was highly attenuated (J. Cello, A. V. Paul, and E. Wimmer, Science 297:1016-1018, 2002). Compared to the wild-type PV1 (Mahoney) [PV1(M)] sequence, the synthetic virus genome harbored 27 nucleotide (nt) changes deliberately introduced as genetic markers. Of the 27 nucleotide substitutions, the UA-to-GG exchanges at nucleotides 102/103, mapping to a region between the cloverleaf and the internal ribosome entry site (IRES) in the 5'-nontranslated region, were found to be involved in the observed attenuation phenotype in mice. The UA/GG mutation at nt 102/103 in the synthetic PV1(M) [sPV1(M)] background conferred also a ts phenotype of replication to the virus in human neuroblastoma cells. Conversely, the exchange of GG to wild-type (wt) UA at 102/103 in an sPV1(M) background restored wt neurovirulence in CD155 transgenic (tg) mice and suppressed the ts phenotype in SK-N-MC cells. All poliovirus variants replicated well in HeLa cells at the two temperatures, regardless of the sequence at the 102/103 locus. Analyses of variants isolated from sPV(M)-infected CD155 tg mice revealed that the G(102)G(103)-to-G(102)A(103) reversion alone reestablished the neurovirulent phenotype. This suggests that a single mutation is responsible for the observed change of the neurovirulence phenotype. sPV1(M) RNA is translated in cell extracts of SK-N-MC cells with significantly lower efficiency than PV1(M) RNA or sPV1(M) RNA with a G(102)-to-A(102) reversion. These studies suggest a function for the conserved nucleotide (A(103)) located between the cloverleaf and the IRES which is important for replication of PV in the central nervous system of CD155 tg mice and in human cells of neuronal origin.     

A ferritin-responsive internal ribosome entry site regulates folate metabolism

Cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme levels are elevated by the expression of the heavy chain ferritin (H ferritin) cDNA in cultured cells without corresponding changes in mRNA levels, resulting in enhanced folate-dependent de novo thymidylate biosynthesis and impaired homocysteine remethylation. In this study, the mechanism whereby H ferritin regulates cSHMT expression was determined. cSHMT translation is shown to be regulated by an H ferritin-responsive internal ribosome entry site (IRES) located within the cSHMT mRNA 5'-untranslated region (5'-UTR). The cSHMT 5'-UTR exhibited IRES activity during in vitro translation of bicistronic mRNA templates, and in MCF-7 and HeLa cells transfected with bicistronic mRNAs. IRES activity was depressed in H ferritin-deficient mouse embryonic fibroblasts and elevated in cells expressing the H ferritin cDNA. H ferritin was shown to interact with the mRNA-binding protein CUGBP1, a protein known to interact with the alpha and beta subunits of eukaryotic initiation factor eIF2. Small interference RNA-mediated depletion of CUGBP1 decreased IRES activity from bicistronic templates that included the cSHMT 3'-UTR in the bicistronic construct. The identification of this H ferritin-responsive IRES represents a mechanism that accounts for previous observations that H ferritin regulates folate metabolism.     

Conserved structural motifs located in distal loops of aphthovirus internal ribosome entry site domain 3 are required for internal initiation of translation.

A comparison of picornavirus internal ribosome entry site (IRES) secondary structures revealed the existence of conserved motifs located on loops. We have carried out a mutational analysis to test their requirement for IRES-driven translation. The GUAA sequence, located in the aphthovirus 3A loop, did not tolerate substitutions that disrupt the GNRA motif. Interestingly, this motif was found at similar positions in all picornavirus IRESs, suggesting that it may form part of a tertiary-structure element. The RAAA tetranucleotide located in the 3B loop was conserved only in cardiovirus and aphthovirus. A mutational analysis of the RAAA motif revealed that activities of 3B loop mutants correlated with both the presence of a sequence close to CAAA at the new 3B loop and the absence of reorganization of the 3B and 3C stem-loops. In support of this conclusion, insertion of a large number of nucleotides close to the 3B loop, which was predicted to reorganize the 3B-3C stem-loop structure, led to defective IRES elements. We conclude that the aphthovirus IRES loops located at the most distal part of domain 3, which carries GNRA and RAAA motifs, are essential for IRES function.     

Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus

The cricket paralysis virus (CrPV), a member of the CrPV-like virus family, contains a single positive-stranded RNA genome that encodes two non-overlapping open reading frames separated by a short intergenic region (IGR). The CrPV IGR contains an internal ribosomal entry site (IRES) that directs the expression of structural proteins. Unlike previously described IRESs, the IGR IRES initiates translation by recruiting 80S ribosomes in the absence of initiator Met-tRNA(i) or any canonical initiation factors, from a GCU alanine codon located in the A-site of the ribosome. Here, we have shown that a variety of mutations, designed to disrupt individually three pseudoknot (PK) structures and alter highly conserved nucleotides among the CrPV-like viruses, inhibit IGR IRES-mediated translation. By separating the steps of translational initiation into ribosomal recruitment, ribosomal positioning and ribosomal translocation, we found that the mutated IRES elements could be grouped into two classes. One class, represented by mutations in PKII and PKIII, bound 40S subunits with significantly reduced affinity, suggesting that PKIII and PKII are involved in the initial recruitment of the ribosome. A second class of mutations, exemplified by alterations in PKI, did not affect 40S binding but altered the positioning of the ribosome on the IRES, indicating that PKI is involved in the correct positioning of IRES-associated ribosomes. These results suggest that the IGR IRES has distinct pseudoknot-like structures that make multiple contacts with the ribosome resulting in initiation factor-independent recruitment and correct positioning of the ribosome on the mRNA.     

Unr is required in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus

Translation of picornavirus RNAs is mediated by internal ribosomal entry site (IRES) elements and requires both standard eukaryotic translation initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). Unr, a cytoplasmic RNA-binding protein that contains five cold-shock domains and is encoded by the gene upstream of N-ras, stimulates translation directed by the human rhinovirus (HRV) IRES in vitro. To examine the role of Unr in translation of picornavirus RNAs in vivo, we derived murine embryonic stem (ES) cells in which either one (-/+) or both (-/-) copies of the unr gene were disrupted by homologous recombination. The activity of picornaviral IRES elements was analyzed in unr(+/+), unr(+/-), and unr(-/-) cell lines. Translation directed by the HRV IRES was severely impaired in unr(-/-) cells, as was that directed by the poliovirus IRES, revealing a requirement for Unr not previously observed in vitro. Transient expression of Unr in unr(-/-) cells efficiently restored the HRV and poliovirus IRES activities. In contrast, the IRES elements of encephalomyocarditis virus and foot-and-mouth-disease virus are not Unr dependent. Thus, Unr is a specific regulator of HRV and poliovirus translation in vivo and may represent a cell-specific determinant limiting replication of these viruses.     

细胞内部核糖体进入位点研究进展

细胞内部核糖体进入位点(IRES)mRNA5’端非编码区的一段特殊的序列,它允许核糖体不从mRNA的5’到3’端阅读而直接在此序列处结合mRNA并起始翻译。本综述了IRES的发现、IRES的识别及细胞IRES的特征、作用机理、生物学意义及其生物学应用等方面的研究进展。     

Connexin43 mRNA contains a functional internal ribosome entry site

A reporter gene construct was used to study the regulation of connexin43 (Cx43) expression, the major gap junction protein found in heart and uterus, in transfected cell lines. The construct had the firefly luciferase gene under the control of the Cx43 promoter. Inclusion of the 5'-untranslated region (UTR) of the mRNA in the construct increased luciferase expression by 70%. A bicistronic vector assay demonstrated that the Cx43 5'-UTR contains a strong internal ribosome entry site (IRES). Deletion analysis localized the IRES element to the upstream portion of the 5'-UTR.     

High-titer bicistronic retroviral vectors employing foot-and-mouth disease virus internal ribosome entry site.

Bicistronic retroviral vectors were constructed containing the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES) followed by the coding region of beta-galactosidase (beta-gal) or therapeutic genes, with the selectable neomycin phosphotransferase gene under the control of the viral long terminal repeat (LTR) promoter. LNFX, a vector with a multiple cloning site 3' to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine deaminase (ADA) and beta-gal. In transduced primary rat vascular smooth muscle cells the cytokines were expressed at high levels, similar to those obtained from vectors employing the viral LTR promoter. LNFZ, a vector encoding beta-gal, had a 10-fold increase in titer over that of LNPoZ, a comparable vector containing the poliovirus (Po) internal ribosome entry site. Primary canine vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar activities of beta-gal and neomycin phosphotransferase (NPT). Overall, these vectors had titers between 10(6) and 2 x 10(7) c.f.u./ml, indicating that foot-and-mouth disease virus IRES provides high-titer bicistronic vectors with high-level two gene expression.     

Aryl-substituted aminobenzimidazoles targeting the hepatitis C virus internal ribosome entry site

We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands were established that were consistent with the crystal structure of the translation inhibitor complex.     

Viral internal ribosome entry site structures segregate into two distinct morphologies

An increasing number of viruses have been shown to initiate protein synthesis by a cap-independent mechanism involving internal ribosome entry sites (IRESs). Predictions of the folding patterns of these RNA motifs have been based primarily on sequence and biochemical analyses. Biophysical confirmation of the models has been achieved only for the IRES of hepatitis C virus (HCV), which adopts an open structure consisting of two major stems. We have conducted an extensive comparison of flavivirus and picornavirus IRES elements by negative stain transmission electron microscopy. All of the flavivirus IRESs we examined (those of GB virus-B, GB virus-C, and classical swine fever virus) fold to give a structure similar to that of the HCV IRES, as does an IRES recently found on mRNA encoded by human herpesvirus 8. The larger picornavirus IRESs (those of foot-and-mouth disease virus, rhinovirus, encephalomyocarditis virus, and hepatitis A virus) are morphologically similar, comprising a backbone with two protruding stems, and distinct from the flavivirus IRESs.