scholarly journals Eukaryotic Initiation Factor 4G-Poly(A) Binding Protein Interaction Is Required for Poly(A) Tail-Mediated Stimulation of Picornavirus Internal Ribosome Entry Segment-Driven Translation but Not for X-Mediated Stimulation of Hepatitis C Virus Translation

2001 ◽  
Vol 21 (13) ◽  
pp. 4097-4109 ◽  
Author(s):  
Yanne M. Michel ◽  
Andrew M. Borman ◽  
Sylvie Paulous ◽  
Katherine M. Kean
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Initiation Factor ◽  
Translation System ◽  
Eukaryotic Initiation Factor ◽  
Internal Ribosome Entry ◽  
Internal Ribosome Entry Segment ◽  
Eukaryotic Initiation Factor 4G ◽  
Hcv Ires ◽  
Stimulation Of

ABSTRACT Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5′ m7GpppN cap and the 3′ poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRES-driven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3′ end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3′-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.

scholarly journals Translation initiation by the hepatitis C virus IRES requires eIF1A and ribosomal complex remodeling

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Zane A Jaafar ◽  
Akihiro Oguro ◽  
Yoshikazu Nakamura ◽  
Jeffrey S Kieft
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Translation Initiation ◽  
Rna Structure ◽  
Initiation Factor ◽  
Internal Ribosome Entry ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation ◽  
Hcv Ires ◽  
Type 3

Internal ribosome entry sites (IRESs) are important RNA-based translation initiation signals, critical for infection by many pathogenic viruses. The hepatitis C virus (HCV) IRES is the prototype for the type 3 IRESs and is also invaluable for exploring principles of eukaryotic translation initiation, in general. Current mechanistic models for the type 3 IRESs are useful but they also present paradoxes, including how they can function both with and without eukaryotic initiation factor (eIF) 2. We discovered that eIF1A is necessary for efficient activity where it stabilizes tRNA binding and inspects the codon-anticodon interaction, especially important in the IRES’ eIF2-independent mode. These data support a model in which the IRES binds preassembled translation preinitiation complexes and remodels them to generate eukaryotic initiation complexes with bacterial-like features. This model explains previous data, reconciles eIF2-dependent and -independent pathways, and illustrates how RNA structure-based control can respond to changing cellular conditions.

scholarly journals Differential Effects on the Hepatitis C Virus (HCV) Internal Ribosome Entry Site by Vitamin B12 and the HCV Core Protein

2004 ◽  
Vol 78 (21) ◽  
pp. 12075-12081 ◽  
Author(s):  
Dongsheng Li ◽  
William B. Lott ◽  
John Martyn ◽  
Gholamreza Haqshenas ◽  
Eric J. Gowans
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Core Protein ◽  
Vitamin B ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Hcv Core Protein ◽  
Hcv Ires

ABSTRACT To investigate the role of the hepatitis C virus internal ribosome entry site (HCV IRES) domain IV in translation initiation and regulation, two chimeric IRES elements were constructed to contain the reciprocal domain IV in the otherwise HCV and classical swine fever virus IRES elements. This permitted an examination of the role of domain IV in the control of HCV translation. A specific inhibitor of the HCV IRES, vitamin B12, was shown to inhibit translation directed by all IRES elements which contained domain IV from the HCV and the GB virus B IRES elements, whereas the HCV core protein could only suppress translation from the wild-type HCV IRES. Thus, the mechanisms of translation inhibition by vitamin B12 and the core protein differ, and they target different regions of the IRES.

scholarly journals The 5′ Untranslated Region of Protein Kinase Cδ Directs Translation by an Internal Ribosome Entry Segment That Is Most Active in Densely Growing Cells and during Apoptosis

2002 ◽  
Vol 22 (17) ◽  
pp. 6089-6099 ◽  
Author(s):  
Bronwyn C. Morrish ◽  
Martin G. Rumsby
Keyword(s):  
Protein Kinase ◽  
Untranslated Region ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Serum Starvation ◽  
Proliferating Cells ◽  
Internal Ribosome Entry ◽  
Internal Ribosome Entry Segment ◽  
Eukaryotic Initiation Factor 4G ◽  
Protein Kinase Cδ

ABSTRACT Protein kinase Cδ (PKCδ) is a member of the PKC family of phospholipid-dependent serine/threonine kinases and is involved in cell proliferation, apoptosis, and differentiation. Previous studies have suggested that different PKC isoforms might be translationally regulated. We report here that the 395-nt-long 5′ untranslated region (5′ UTR) of PKCδ is predicted to form very stable secondary structures with free energies (ΔG values) of around −170 kcal/mol. The 5′ UTR of PKCδ can significantly repress luciferase translation in rabbit reticulocyte lysate but does not repress luciferase translation in a number of transiently transfected cell lines. By using a bicistronic luciferase reporter, we show that the 5′ UTR of PKCδ contains a functional internal ribosome entry segment (IRES). The activity of the PKCδ IRES is greatest in densely growing cells and during apoptosis, when total protein synthesis and levels of full-length eukaryotic initiation factor 4G are reduced. However, the IRES activity of the 5′ UTR of PKCδ is not enhanced during serum starvation, another condition shown to inhibit cap-dependent translation, suggesting that its potency is dependent on specific cellular conditions. Accumulating data suggest that PKCδ has a function as proliferating cells reach high density and in early and later events of apoptosis. Our studies suggest a mechanism whereby PKCδ synthesis can be maintained under these conditions when cap-dependent translation is inhibited.

scholarly journals The Ribosome Binding Site of Hepatitis C Virus mRNA

2001 ◽  
Vol 75 (16) ◽  
pp. 7629-7636 ◽  
Author(s):  
J. Robin Lytle ◽  
Lily Wu ◽  
Hugh D. Robertson
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Binding Site ◽  
Ribosome Binding Site ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Promising Target ◽  
Hcv Ires

ABSTRACT Hepatitis C virus (HCV) infects an estimated 170 million people worldwide, the majority of whom develop a chronic infection which can lead to severe liver disease, and for which no generally effective treatment yet exists. A promising target for treatment is the internal ribosome entry site (IRES) of HCV, a highly conserved domain within a highly variable RNA. Never before have the ribosome binding sites of any IRES domains, cellular or viral, been directly characterized. Here, we reveal that the HCV IRES sequences most closely associated with 80S ribosomes during protein synthesis initiation are a series of discontinuous domains together comprising by far the largest ribosome binding site yet discovered.

scholarly journals Functional Analyses of RNA Structures Shared between the Internal Ribosome Entry Sites of Hepatitis C Virus and the Picornavirus Porcine Teschovirus 1 Talfan

2006 ◽  
Vol 80 (3) ◽  
pp. 1271-1279 ◽  
Author(s):  
Louisa S. Chard ◽  
Yoshihiro Kaku ◽  
Barbara Jones ◽  
Arabinda Nayak ◽  
Graham J. Belsham
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mutant Form ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Porcine Teschovirus ◽  
High Level ◽  
Hcv Ires

ABSTRACT 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.

The hepatitis C virus internal ribosome-entry site: a new target for antiviral research

2002 ◽  
Vol 30 (2) ◽  
pp. 140-145 ◽  
Author(s):  
J. Gallego ◽  
G. Varani
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Entry Site ◽  
Viral Protein Synthesis ◽  
Initiation Mechanism ◽  
Internal Ribosome Entry

The hepatitis C virus (HCV) is the main causative agent of non-A, non-B hepatitis in humans and a major cause of mortality and morbidity in the world. Currently there is no effective treatment available for the infection caused by this virus, whose replication depends on an unusual translation-initiation mechanism. The viral RNA contains an internal ribosome-entry site (IRES) that is recognized specifically by the small ribosomal subunit and by eukaryotic initiation factor 3, and these interactions allow cap (7-methylguanine nucleotide)-independent initiation of viral protein synthesis. In this article, we review the structure and mechanism of translation initiation of the HCV IRES, and its potential as a target for novel antivirals.

Long-range RNA–RNA interactions between distant regions of the hepatitis C virus internal ribosome entry site element

2002 ◽  
Vol 83 (5) ◽  
pp. 1113-1121 ◽  
Author(s):  
Esther Lafuente ◽  
Ricardo Ramos ◽  
Encarnación Martínez-Salas
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Long Range ◽  
Internal Ribosome Entry Site ◽  
Translation Efficiency ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Hcv Ires ◽  
Rna Complexes

Efficient internal initiation of translation from the hepatitis C virus (HCV) internal ribosome entry site (IRES) requires sequences of domain II, but the precise role of these sequences is still unknown. In this study, the formation of RNA–RNA complexes in the HCV IRES was evaluated. Using transcripts that contain the sequences of the structural HCV IRES domains II, IIIabcd, IIIabc, IV and IIIef-IV, specific long-range interactions between domains II and IV, as well as domains II and IIIabcd, have been found. These interactions were readily detected in a gel mobility-shift assay and required the presence of magnesium ions. A high concentration of nonspecific competitors, an 80 nt fragment of 18S rRNA or poly(I:C), did not interfere with the formation of RNA complexes. Interestingly, an RNA oligonucleotide bearing the sequence of stem–loop IIId interacted with domain II but not with domain IV or IIIef-IV, strongly suggesting that the interaction between domains II and IIIabcd was mediated by the IIId hairpin. Interaction between domains IIIabcd and IV was barely detected, consistent with the result that the apical part of domain III folds independently of the rest of the IRES. Moreover, the addition of stem–loop IIIef sequences to domain IV significantly reduced its ability to interact, which is in agreement with the formation of a compact RNA structure of domain IV with IIIef. The interactions observed in the absence of proteins between domains II and IV as well as stem–loop IIId and domain II may be transient, having a regulatory role in the translation efficiency of the HCV IRES.

scholarly journals Cap-dependent and hepatitis C virus internal ribosome entry site-mediated translation are modulated by phosphorylation of eIF2α under oxidative stress

2006 ◽  
Vol 87 (11) ◽  
pp. 3251-3262 ◽  
Author(s):  
Paul R. MacCallum ◽  
Samantha C. Jack ◽  
Philip A. Egan ◽  
Benjamin T. McDermott ◽  
Richard M. Elliott ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Hepg2 Cells ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Huh7 Cells ◽  
Hcv Ires

Chronic hepatitis C is often associated with oxidative stress. Hepatitis C virus (HCV) utilizes an internal ribosome entry site (IRES) element for translation, in contrast to cap-dependent translation of the majority of cellular proteins. To understand how virus translation is modulated under oxidative stress, HCV IRES-mediated translation was compared with cap-dependent translation using a bicistronic reporter construct and hydrogen peroxide (H2O2) as a stress inducer. In H2O2-sensitive HeLa cells, H2O2 repressed translation in a time- and dose-dependent manner, concomitant with the kinetics of eIF2α phosphorylation. A phosphomimetic of eIF2α, which mimics the structure of the phosphorylated eIF2α, was sufficient to repress translation in the absence of H2O2. In H2O2-resistant HepG2 cells, H2O2 activated both HCV IRES-mediated and cap-dependent translation, associated with an increased level of phospho-eIF2α. It was postulated that H2O2 might stimulate translation in HepG2 cells via an eIF2α-independent mechanism, whereas the simultaneous phosphorylation of eIF2α repressed part of the translational activities. Indeed, the translational repression was released in the presence of a non-phosphorylatable mutant, eIF2α-SA, resulting in further enhancement of both translational activities after exposure to H2O2. In HuH7 cells, which exhibited an intermediate level of sensitivity towards H2O2, both HCV IRES-mediated and cap-dependent translational activities were upregulated after treatment with various doses of H2O2, but the highest level of induction was achieved with a low level of H2O2, which may represent the physiological level of H2O2. At this level, the HCV IRES-mediated translation was preferentially upregulated compared with cap-dependent translation.

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