scholarly journals A Small Yeast RNA Blocks Hepatitis C Virus Internal Ribosome Entry Site (HCV IRES)-Mediated Translation and Inhibits Replication of a Chimeric Poliovirus under Translational Control of the HCV IRES Element

1998 ◽  
Vol 72 (7) ◽  
pp. 5638-5647 ◽  
Author(s):  
Saumitra Das ◽  
Michael Ott ◽  
Akemi Yamane ◽  
Weimin Tsai ◽  
Matthias Gromeier ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Internal Ribosome Entry Site ◽  
Hcv Infection ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Ires Element ◽  
Hcv Ires

ABSTRACT Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis and cirrhosis of the liver and has been linked to development of hepatocellular carcinoma. We previously identified a small yeast RNA (IRNA) capable of specifically inhibiting poliovirus (PV) internal ribosome entry site (IRES)-mediated translation. Here we report that IRNA specifically inhibits HCV IRES-mediated translation both in vivo and in vitro. A number of human hepatoma (Huh-7) cell lines expressing IRNA were prepared and characterized. Constitutive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constitutively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell lines. Additionally, Huh-7 cells constitutively expressing IRNA became refractory to infection by a PV-HCV chimera in which the PV IRES is replaced by the HCV IRES. In contrast, replication of a PV-encephalomyocarditis virus (EMCV) chimera containing the EMCV IRES element was not affected significantly in the IRNA-producing cell line. Finally, the binding of the La autoantigen to the HCV IRES element was specifically and efficiently competed by IRNA. These results provide a basis for development of novel drugs effective against HCV infection.

scholarly journals Involvement of Proteasome α-Subunit PSMA7 in Hepatitis C Virus Internal Ribosome Entry Site-Mediated Translation

2001 ◽  
Vol 21 (24) ◽  
pp. 8357-8364 ◽  
Author(s):  
Martin Krüger ◽  
Carmela Beger ◽  
Peter J. Welch ◽  
Jack R. Barber ◽  
Michael P. Manns ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Inhibitory Effect ◽  
Encephalomyocarditis Virus ◽  
Luciferase Reporter ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Α Subunit ◽  
Hcv Ires

ABSTRACT Ribozymes are small catalytic RNA molecules that can be engineered to enzymatically cleave RNA transcripts in a sequence-specific fashion and thereby inhibit expression and function of the corresponding gene product. With their simple structures and site-specific cleavage activity, they have been exploited as potential therapeutic agents in a variety of human disorders, including hepatitis C virus (HCV) infection. We have designed a hairpin ribozyme (Rz3′X) targeting the HCV minus-strand replication intermediate at position 40 within the 3′X tail. Surprisingly, Rz3′X was found to induce ganciclovir (GCV)-resistant colonies in a bicistronic cellular reporter system with HCV internal ribosome entry site (IRES)-dependent translation of herpes simplex virus thymidine kinase (TK). Rz3′X-transduced GCV-resistant HeLa reporter cells showed substantially reduced IRES-mediated HCV core protein translation compared with control vector-transduced cells. Since these reporter systems do not contain the HCV 3′X tail sequences, the results indicate that Rz3′X probably exerted an inhibitory effect on HCV IRES activity fortuitously through another gene target. A novel technique of ribozyme cleavage-based target gene identification (cleavage-specific amplification of cDNA ends) (M. Krüger, C. Beger, P. J. Welch, J. R. Barber, and F. Wong-Staal, Nucleic Acids Res. 29:e94, 2001) revealed that human 20S proteasome α-subunit PSMA7 mRNA was a target RNA recognized and cleaved by Rz3′X. We then showed that additional ribozymes directed against PSMA7 RNA inhibited HCV IRES activity in two assay systems: GCV resistance in the HeLa IRES TK reporter cell system and a transient transfection assay performed with a bicistronicRenilla-HCV IRES-firefly luciferase reporter in Huh7 cells. In contrast, ribozymes were inactive against IRES of encephalomyocarditis virus and human rhinovirus. Additionally, proteasome inhibitor MG132 exerted a dose-dependent inhibitory effect on HCV IRES-mediated translation but not on cap-dependent translation. These data suggest a principal role for PSMA7 in regulating HCV IRES activity, a function essential for HCV replication.

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 Cell Type-Specific Enhancement of Hepatitis C Virus Internal Ribosome Entry Site-Directed Translation due to 5′ Nontranslated Region Substitutions Selected during Passage of Virus in Lymphoblastoid Cells

2000 ◽  
Vol 74 (15) ◽  
pp. 7024-7031 ◽  
Author(s):  
Hervé Lerat ◽  
Yoko K. Shimizu ◽  
Stanley M. Lemon
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Internal Ribosome Entry Site ◽  
Nucleotide Substitutions ◽  
Entry Site ◽  
Lymphoblastoid Cells ◽  
Internal Ribosome Entry ◽  
Translational Activity ◽  
Nontranslated Region

ABSTRACT Low-level replication of hepatitis C virus (HCV) in cultured lymphoblastoid cells inoculated with H77 serum inoculum led to the appearance of new virus variants containing identical substitutions at three sites within the viral 5′ nontranslated RNA (5′NTR): G107→A, C204→A, and G243→A (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325–3329, 1996). These results suggest that virus with this 5′NTR sequence may have a greater capacity for replication in such cells, possibly due to more efficient cap-independent translation, since these nucleotide substitutions reside within the viral internal ribosome entry site (IRES). To test this hypothesis, we examined the translation of dicistronic RNAs containing upstream and downstream reporter sequences (Renilla and firefly luciferases, respectively) separated by IRES sequences containing different combinations of these substitutions. The activity of the IRES was assessed by determining the relative firefly and Renillaluciferase activities expressed in transfected cells. Compared with the IRES present in the dominant H77 quasispecies, an IRES containing all three nucleotide substitutions had significantly greater translational activity in three of five human lymphoblastoid cell lines (Raji, Bjab, and Molt4 but not Jurkat or HPBMa10-2 cells). In contrast, these substitutions did not enhance IRES activity in cell lines derived from monocytes or granulocytes (HL-60, KG-1, or THP-1) or hepatocytes (Huh-7) or in cell-free translation assays carried out with rabbit reticulocyte lysates. Each of the three substitutions was required for maximally increased translational activity in the lymphoblastoid cells. The 2- to 2.5-fold increase in translation observed with the modified IRES sequence may facilitate the replication of HCV, possibly accounting for differences in quasispecies variants recovered from liver tissue and peripheral blood mononuclear cells of the same patient.

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.

scholarly journals Tissue-Specific Replicating Capacity of a Chimeric Poliovirus That Carries the Internal Ribosome Entry Site of Hepatitis C Virus in a New Mouse Model Transgenic for the Human Poliovirus Receptor

2003 ◽  
Vol 77 (19) ◽  
pp. 10479-10487 ◽  
Author(s):  
Akiko Yanagiya ◽  
Seii Ohka ◽  
Noriyasu Hashida ◽  
Masahito Okamura ◽  
Choji Taya ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mouse Model ◽  
Internal Ribosome Entry Site ◽  
Mammalian Cells ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Tissue Specific ◽  
Hcv Ires ◽  
The Brain

ABSTRACT Nucleotides (nt) 108 to 742 of an infectious cDNA clone of poliovirus (PV) Mahoney strain, including the corresponding region of the internal ribosome entry site (IRES), was replaced by nt 28 to 710 of hepatitis C virus (HCV) cDNA corresponding to the whole HCV IRES. A chimeric PV (2A-369) was generated by transfecting mammalian cells with an RNA transcribed in vitro from the cDNA. To examine replicating capacity of virus 2A-369 in the brain and liver of a mouse model for poliomyelitis, a new mouse model (MPVRTg25-61) that is transgenic for human PV receptor (hPVR; CD155) was generated in order to obtain a higher expression level of hPVR in the liver than those of hPVRTg mouse lines generated by us so far. The transgene used was constructed by combining a putative regulatory region of the mouse PVR homolog and the whole structural region of the hPVR gene. Virus 2A-369 replicated well in the liver of MPVRTg25-61 but not in the brain, whereas control Mahoney virus replicated well both in the liver and in the brain. The data suggest that the HCV IRES works more efficiently in the liver than in the brain and that PV IRES works well both in the liver and in the brain. The results support the notion that tissue-specific activity of IRES may be reflected in tissue tropism of a virus whose specific translation initiation is driven by IRES, that is, an IRES-dependent virus tropism.

scholarly journals Hepatitis C virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast

2007 ◽  
Vol 88 (7) ◽  
pp. 1992-2002 ◽  
Author(s):  
Tomas Masek ◽  
Vaclav Vopalensky ◽  
Ondrej Horvath ◽  
Lucie Vortelova ◽  
Zuzana Feketova ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Yeast Cells ◽  
Initiation Codon ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Broad Dynamic Range ◽  
Hcv Ires

Hepatitis C virus (HCV) is an important pathogen causing both acute and chronic infections in humans. The HCV polyprotein is synthesized by cap-independent translation initiation after ribosome binding to the highly structured internal ribosome entry site (IRES). The HCV IRES has been shown to have a low requirement for translation initiation factors and the ability to bind directly to the 40S ribosomal subunit. A novel yeast bicistronic reporter system, suitable for sensitive and accurate analysis of IRES activity, has been developed. It employs signal amplification based on the Gal4p transcription factor-mediated activation of a variety of secondary reporter genes. The system has a broad dynamic range and, depending on the nature of the particular secondary reporter, can be used both for precise measurements of IRES activity and for selection and screening for novel IRES variants and IRES trans-acting factors. By using this novel bicistronic system, it was shown that the HCV IRES is functional in yeast cells. Mutational analysis of the IRES loop IV and the adjacent region revealed that, in yeast, as in mammalian cells, translation initiates preferentially at the authentic 342AUG codon and that disruption of the HCV IRES loop IV abrogates its function, whilst minor positional changes or substitutions of the initiation codon within loop IV are largely tolerated. These findings bring more general insights to translation initiation, but also open the door for utilization of yeast and its sophisticated genetics for searching for new antiviral drugs and HCV IRES trans-acting proteins.

scholarly journals The Hepatitis C Virus RNA 3′-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site

2006 ◽  
Vol 80 (23) ◽  
pp. 11579-11588 ◽  
Author(s):  
Yutong Song ◽  
Peter Friebe ◽  
Eleni Tzima ◽  
Christiane Jünemann ◽  
Ralf Bartenschlager ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Lines ◽  
Internal Ribosome Entry Site ◽  
Hepatoma Cell ◽  
Rna Replication ◽  
Viral Rna ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Positive Strand Rna

ABSTRACT The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5′- and 3′-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5′-UTR, and subsequent viral RNA replication requires sequences in the 3′-UTR and in the 5′-UTR. Addressing previous conflicting reports on a possible function of the 3′-UTR for RNA translation in this study, we found that reporter construct design is an important parameter in experiments testing 3′-UTR function. A translation enhancer function of the HCV 3′-UTR was detected only after transfection of monocistronic reporter RNAs or complete RNA genomes having a 3′-UTR with a precise 3′ terminus. The 3′-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. The variable region, the poly(U · C) tract, and the most 3′ terminal stem-loop 1 of the highly conserved 3′ X region contribute significantly to translation enhancement, whereas stem-loops 2 and 3 of the 3′ X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minus-strand synthesis in the HCV 3′-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3′-5′ end interaction and a switch between translation and RNA replication.

scholarly journals Hepatitis C Virus Internal Ribosome Entry Site (IRES) Stem Loop IIId Contains a Phylogenetically Conserved GGG Triplet Essential for Translation and IRES Folding

2000 ◽  
Vol 74 (22) ◽  
pp. 10430-10437 ◽  
Author(s):  
Ronald Jubin ◽  
Nicole E. Vantuno ◽  
Jeffrey S. Kieft ◽  
Michael G. Murray ◽  
Jennifer A. Doudna ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry ◽  
Apical Loop ◽  
Hcv Ires

ABSTRACT The hepatitis C virus (HCV) internal ribosome entry site (IRES) is a highly structured RNA element that directs cap-independent translation of the viral polyprotein. Morpholino antisense oligonucleotides directed towards stem loop IIId drastically reduced HCV IRES activity. Mutagenesis studies of this region showed that the GGG triplet (nucleotides 266 through 268) of the hexanucleotide apical loop of stem loop IIId is essential for IRES activity both in vitro and in vivo. Sequence comparison showed that apical loop nucleotides (UUGGGU) were absolutely conserved across HCV genotypes and the GGG triplet was strongly conserved among related Flavivirus andPestivirus nontranslated regions. Chimeric IRES elements with IIId derived from GB virus B (GBV-B) in the context of the HCV IRES possess translational activity. Mutations within the IIId stem loop that abolish IRES activity also affect the RNA structure in RNase T1-probing studies, demonstrating the importance of correct RNA folding to IRES function.

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