Hepatitis C virus-related internal ribosome entry sites are found in multiple genera of the family Picornaviridae (original) (raw)
Related papers
Journal of Virology, 2004
Typically, picornavirus IRES elements contain about 450 nucleotides (nt) and use most of the cellular translation initiation factors. However, it is now shown that just 280 nt of the porcine teschovirus type 1 Talfan (PTV-1) 5 untranslated region direct the efficient internal initiation of translation in vitro and within cells. In toeprinting assays, assembly of 48S preinitiation complexes from purified components on the PTV-1 IRES was achieved with just 40S ribosomal subunits plus eIF2 and Met-tRNA i Met . Indeed, a binary complex between 40S subunits and the PTV-1 IRES is formed. Thus, the PTV-1 IRES has properties that are entirely different from other picornavirus IRES elements but highly reminiscent of the hepatitis C virus (HCV) IRES. Comparison between the PTV-1 IRES and HCV IRES elements revealed islands of high sequence identity that occur in regions critical for the interactions of the HCV IRES with the 40S ribosomal subunit and eIF3. Thus, there is significant functional and structural similarity between the IRES elements from the picornavirus PTV-1 and HCV, a flavivirus.
Journal of Virology, 2006
The internal ribosome entry site (IRES) of porcine teschovirus 1 (PTV-1), a member of the Picornaviridae family, is quite distinct from other well-characterized picornavirus IRES elements, but it displays functional similarities to the IRES from hepatitis C virus (HCV), a member of the Flaviviridae family. In particular, a dominant negative mutant form of eIF4A does not inhibit the activity of the PTV-1 IRES. Furthermore, there is a high level (ca. 50%) of identity between the PTV-1 and HCV IRES sequences. A secondary-structure model of the whole PTV-1 IRES has been derived which includes a pseudoknot. Validation of specific features within the model has been achieved by mutagenesis and functional assays. The differences and similarities between the PTV-1 and HCV IRES elements should assist in defining the critical features of this type of IRES. Downloaded from 1272 CHARD ET AL. J. VIROL. on December 18, 2015 by guest http://jvi.asm.org/ Downloaded from FIG. 2. Predicted secondary structure of the PTV-1 IRES.
Journal of Virology, 2008
Avian encephalomyelitis virus (AEV) is a picornavirus that causes disease in poultry worldwide, and flocks must be vaccinated for protection. AEV is currently classified within the hepatovirus genus, since its proteins are most closely related to those of hepatitis A virus (HAV). We now provide evidence that the 494-nucleotidelong 5 untranslated region of the AEV genome contains an internal ribosome entry site (IRES) element that functions efficiently in vitro and in mammalian cells. Unlike the HAV IRES, the AEV IRES is relatively short and functions in the presence of cleaved eIF4G and it is also resistant to an inhibitor of eIF4A. These properties are reminiscent of the recently discovered class of IRES elements within certain other picornaviruses, such as porcine teschovirus 1 (PTV-1). Like the PTV-1 IRES, the AEV IRES shows significant similarity to the hepatitis C virus (HCV) IRES in sequence, function, and predicted secondary structure. Furthermore, mutational analysis of the predicted pseudoknot structure at the 3 end of the AEV IRES lends support to the secondary structure we present. AEV is therefore another example of a picornavirus harboring an HCV-like IRES element within its genome, and thus, its classification within the hepatovirus genus may need to be reassessed in light of these findings.
Journal of Virology, 2011
The RNA genome of Seneca Valley virus (SVV), a recently identified picornavirus, contains an internal ribosome entry site (IRES) element which has structural and functional similarity to that from classical swine fever virus (CSFV) and hepatitis C virus, members of the Flaviviridae. The SVV IRES has an absolute requirement for the presence of a short region of virus-coding sequence to allow it to function either in cells or in rabbit reticulocyte lysate. The IRES activity does not require the translation initiation factor eIF4A or intact eIF4G. The predicted secondary structure indicates that the SVV IRES is more closely related to the CSFV IRES, including the presence of a bipartite IIId domain. Mutagenesis of the SVV IRES, coupled to functional assays, support the core elements of the IRES structure model, but surprisingly, deletion of the conserved IIId 2 domain had no effect on IRES activity, including 40S and eIF3 binding. This is the first example of a picornavirus IRES that is most closely related to the CSFV IRES and suggests the possibility of multiple, independent recombination events between the genomes of the Picornaviridae and Flaviviridae to give rise to similar IRES elements.
The internal ribosome entry site (IRES) of hepatitis C virus visualized by electron microscopy
RNA, 2001
Translation of hepatitis C virus (HCV) RNA is initiated via the internal ribosome entry site (IRES), located within the 59 untranslated region. Although the secondary structure of this element has been predicted, little information on the tertiary structure is available. Here we report the first structural characterization of the HCV IRES using electron microscopy. In vitro transcribed RNA appeared as particles with characteristic morphology and gold labeling using a specific oligonucleotide confirmed them to be HCV IRES. Dimerization of the IRES by hybridization with tandem repeat oligonucleotides allowed the identification of domain III and an assignment of domains II and IV to distinct regions within the molecule. Using immunogold labeling, the pyrimidine tract binding protein (PTB) was shown to bind to domain III. Structure-function relationships based on the flexible hinge between domains II and III are suggested. Finally, the architecture of the HCV IRES was seen to be markedly different from that of a picornavirus, foot-and-mouth disease virus (FMDV).
Nucleic Acids Research, 2009
The translational activity of the hepatitis C virus (HCV) internal ribosome entry site (IRES) and other HCV-like IRES RNAs depends on structured RNA elements in domains II and III, which serve to recruit the ribosomal 40S subunit, eukaryotic initiation factor (eIF) 3 and the ternary eIF2/Met-tRNA i Met /GTP complex and subsequently domain II assists subunit joining. Porcine teschovirus-1 talfan (PTV-1) is a member of the Picornaviridae family, with a predicted HCV-like secondary structure, but only stem-loops IIId and IIIe in the 40S-binding domain display significant sequence conservation with the HCV IRES. Here, we use chemical probing to show that interaction sites with the 40S subunit and eIF3 are conserved between HCV and HCV-like IRESs. In addition, we reveal the functional role of a strictly conserved co-variation between a purine-purine mismatch near the pseudoknot (A-A/G) and the loop sequence of domain IIIe (GAU/CA). These nucleotides are involved in a tertiary interaction, which serves to stabilize the pseudoknot structure and correlates with translational efficiency in both the PTV-1 and HCV IRES. Our data demonstrate conservation of functional domains in HCV and HCV-like IRESs including a more complex structure surrounding the pseudoknot than previously assumed.
Journal of Virology, 2002
The teschoviruses constitute a recently defined picornavirus genus. Most of the genome sequence of the porcine teschovirus-1 (PTV) Talfan and several other strains is known. We now demonstrate that initiation of protein synthesis occurs at nucleotide (nt) 412 on the PTV Talfan RNA and that nt 1 to 405 contains an internal ribosome entry site (IRES) that functions efficiently in vitro and within mammalian cells. In comparison with other picornavirus IRES elements, the PTV IRES is relatively short and lacks a significant polypyrimidine tract near the 3 end. Expression of an enterovirus 2A protease, which induces cleavage of eIF4G within the translation initiation complex eIF4F, has little effect on the PTV IRES activity within BHK cells. The PTV IRES has a unique set of properties and represents a new class of picornavirus IRES element.
Viral Internal Ribosome Entry Site Structures Segregate into Two Distinct Morphologies
Journal of Virology, 2003
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 back...
RNA, 2001
Some studies suggest that the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires downstream 59 viral polyprotein-coding sequence for efficient initiation of translation, but the role of this RNA sequence in internal ribosome entry remains unresolved. We confirmed that the inclusion of viral sequence downstream of the AUG initiator codon increased IRES-dependent translation of a reporter RNA encoding secretory alkaline phosphatase, but found that efficient translation of chloramphenicol acetyl transferase (CAT) required no viral sequence downstream of the initiator codon. However, deletion of an adenosine-rich domain near the 59 end of the CAT sequence, or the insertion of a small stable hairpin structure (DG 5-18 kcal/mol) between the HCV IRES and CAT sequences (hpCAT) substantially reduced IRES-mediated translation. Although translation could be restored to both mutants by the inclusion of 14 nt of the polyprotein-coding sequence downstream of the AUG codon, a mutational analysis of the inserted protein-coding sequence demonstrated no requirement for either a specific nucleotide or amino acid-coding sequence to restore efficient IRES-mediated translation to hpCAT. Similar results were obtained with the structurally and phylogenetically related IRES elements of classical swine fever virus and GB virus B. We conclude that there is no absolute requirement for viral protein-coding sequence with this class of IRES elements, but that there is a requirement for an absence of stable RNA structure immediately downstream of the AUG initiator codon. Stable RNA structure immediately downstream of the initiator codon inhibits internal initiation of translation but, in the case of hpCAT, did not reduce the capacity of the RNA to bind to purified 40S ribosome subunits. Thus, stable RNA structure within the 59 proximal protein-coding sequence does not alter the capacity of the IRES to form initial contacts with the 40S subunit, but appears instead to prevent the formation of subsequent interactions between the 40S subunit and viral RNA in the vicinity of the initiator codon that are essential for efficient internal ribosome entry.
The Hepatitis C Virus Internal Ribosome Entry Site Adopts an Ion-dependent Tertiary Fold
Journal of Molecular Biology, 1999
Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) located in the 5' untranslated region of the genomic RNA that drives cap-independent initiation of translation of the viral message. The approximate secondary structure and minimum functional length of the HCV IRES are known, and extensive mutagenesis has established that nearly all secondary structural domains are critical for activity. However, the presence of an IRES RNA tertiary fold and its functional relevance have not been established. Using chemical and enzymatic probes of the HCV IRES RNA in solution, we show that the IRES adopts a unique three-dimensional structure at physiological salt concentrations in the absence of additional cofactors or the translation apparatus. Folding of the IRES involves cooperative uptake of magnesium and is driven primarily by charge neutralization. This tertiary structure contains at least two independently folded regions which closely correspond to putative binding sites for the 40 S ribosomal subunit and initiation factor 3 (eIF3). Point mutations that inhibit IRES folding also inhibit its function, suggesting that the IRES tertiary structure is essential for translation initiation activity. Chemical and enzymatic probing data and small-angle X-ray scattering (SAXS) experiments in solution show that upon folding, the IRES forms an extended structure in which functionally important loops are exposed. These results suggest that the 40 S ribosomal subunit and eIF3 bind an HCV IRES that is prefolded to spatially organize recognition domains.