Hepatitis C Virus Internal Ribosome Entry Site Activity Research Articles

Polypyrimidine-Tract-Binding Protein Isoforms Differentially Regulate the Hepatitis C Virus Internal Ribosome Entry Site.

Translation initiation of the hepatitis C virus (HCV) mRNA depends on an internal ribosome entry site (IRES) that encompasses most of the 5'UTR and includes nucleotides of the core coding region. This study shows that the polypyrimidine-tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the HCV 5'UTR, stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Our results show that PTB1 and PTB4, but not PTB2, stimulate HCV IRES activity in HuH-7 and HEK293T cells. In HuH-7 cells, PTB1 promotes HCV IRES-mediated initiation more strongly than PTB4. Mutations in PTB1, PTB4, RRM1/RRM2, or RRM3/RRM4, which disrupt the RRM's ability to bind RNA, abrogated the protein's capacity to stimulate HCV IRES activity in HuH-7 cells. In HEK293T cells, PTB1 and PTB4 stimulate HCV IRES activity to similar levels. In HEK293T cells, mutations in RRM1/RRM2 did not impact PTB1's ability to promote HCV IRES activity; and mutations in PTB1 RRM3/RRM4 domains reduced, but did not abolish, the protein's capacity to stimulate HCV IRES activity. In HEK293T cells, mutations in PTB4 RRM1/RRM2 abrogated the protein's ability to promote HCV IRES activity, and mutations in RRM3/RRM4 have no impact on PTB4 ability to enhance HCV IRES activity. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity in a cell type-specific manner. We conclude that PTB1 and PTB4, but not PTB2, act as IRES transacting factors of the HCV IRES.

Characterization of Hepatitis C Virus IRES Quasispecies - From the Individual to the Pool.

Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus from the genus Hepacivirus. The viral genomic +RNA is 9.6 kb long and contains highly structured 5′ and 3′ untranslated regions (UTRs) and codes for a single large polyprotein, which is co- and post-translationally processed by viral and cellular proteases into at least 11 different polypeptides. Most of the 5′ UTR and an initial part of the polyprotein gene are occupied by an internal ribosome entry site (IRES), which mediates cap-independent translation of the viral proteins and allows the virus to overcome cellular antiviral defense based on the overall reduction of the cap-dependent translation initiation. We reconsidered published results concerning a search for possible correlation between patient response to interferon-based antiviral therapy and accumulation of nucleotide changes within the HCV IRES. However, we were unable to identify any such correlation. Rather than searching for individual mutations, we suggest to focus on determination of individual and collective activities of the HCV IRESs found in patient specimens. We developed a combined, fast, and undemanding approach based on high-throughput cloning of the HCV IRES species to a bicistronic plasmid followed by determination of the HCV IRES activity by flow cytometry. This approach can be adjusted for measurement of the individual HCV IRES activity and for estimation of the aggregate ability of the whole HCV population present in the specimen to synthesize viral proteins. To detect nucleotide variations in the individual IRESs, we used denaturing gradient gel electrophoresis (DGGE) analysis that greatly improved identification and classification of HCV IRES variants in the sample. We suggest that determination of the collective activity of the majority of HCV IRES variants present in one patient specimen in a given time represents possible functional relations among variant sequences within the complex population of viral quasispecies better than bare information about their nucleotide sequences. A similar approach might be used for monitoring of sequence variations in quasispecies populations of other RNA viruses in all cases when changes in primary sequence represent changes in measurable and easily quantifiable phenotypes.

HCV NS5A co-operates with PKR in modulating HCV IRES-dependent translation

Translation initiation of the Hepatitis C virus (HCV) genome is driven by an internal ribosome entry site (IRES), located within the 5′ non-coding region. Several studies have suggested that different cellular non canonical proteins or viral proteins can regulate the HCV IRES activity. However, the role of the viral proteins on HCV translation remains controversial. In this report, we confirmed previous studies showing that NS5A down-regulates IRES activity in HepG2 but not in Huh7 cells suggesting that the NS5A effect on HCV IRES is cell-type dependent. Additionally, we provide strong evidence that activated PKR up-regulates the IRES activity while silencing of endogenous PKR had the opposite effect. Furthermore, we present data indicating that the NS5A-mediated inhibitory effect on IRES-dependent translation could be linked with the PKR inactivation. Finally, we show that NS5A from GBV-C but not from GBV-B down-regulates HCV IRES activity in the absence or the presence of PKR over expression. Notably, HCV and GBV-C but not GBV-B NS5A contains a previously identified PKR interacting protein domain.

La Protein Required for Internal Ribosome Entry Site–Directed Translation Is a Potential Therapeutic Target for Hepatitis C Virus Replication

Translation of the hepatitis C virus (HCV) is mediated by an internal ribosome entry site (IRES). Here, we analyzed the functional relevance of La protein for replication of HCV using an infectious HCV clone, JFH-1. A single-nucleotide mutation from A to U was introduced at the 338th nucleotide in the stem-loop domain IV structure of HCV IRES, which stabilized stem-loop IV and abolished translation and replication of JFH-1 almost completely. During JFH-1 replication, translation initiation factors required for HCV IRES activity, including La protein, polypyrimidine tract binding protein (PTB), PSMA7, and PCBP2, were significantly induced in Huh-7.5 cells. Interestingly, JFH-1 infection increased telomerase activity and induced the expression of human telomerase RNA (hTR) in Huh-7.5 cells. In 37 tissue specimens from patients with chronic hepatitis C, La protein significantly correlated with the representative essential telomerase components hTR, p23, and HSP90 (P<.001). Recombinant adenovirus that expressed short-hairpin RNA against La protein successfully suppressed the levels of La protein and core protein of JFH-1 to 30% of that in the control cells. HCV infection might be strongly related to telomerase activity in the liver through La protein induction. Inhibition of La protein substantially repressed JFH-1 replication; therefore, La protein is a potential therapeutic target for HCV.

HnRNP L is required for the translation mediated by HCV IRES

Translation of hepatitis C virus (HCV) RNA is initiated by internal loading of the ribosome into the HCV internal ribosome entry site (IRES). Previously, heterogeneous ribonucleoprotein L (hnRNP L) was shown to bind specifically to the 3′ border region of the HCV IRES and enhance HCV mRNA translation. Here, we provide evidence for the functional requirement of hnRNP L for the HCV IRES-mediated translation initiation using specific RNA aptamers. In vitro selection techniques were employed to isolate RNA aptamers against hnRNP L, which were shown to contain consensus sequences with repetitive ACAC/U. The hnRNP L-specific RNA aptamers efficiently inhibited the in vitro translation reactions mediated by the HCV IRES in rabbit reticulocyte lysates. RNA ligands with only (ACAU)5 or (AC)10 nucleotide sequences could also specifically bind to hnRNP L, and specifically and effectively impeded in vitro translation reactions controlled by the HCV IRES. Importantly, the hnRNP L-specific RNA aptamers inhibited the HCV IRES function in cells in a dose-dependent manner, and the aptamer-mediated inhibition of the HCV IRES was considerably relieved by the addition of hnRNP L-expressing vector. These results strongly demonstrate the functional requirement of cellular hnRNP L for the HCV IRES activity.

Hepatitis C virus internal ribosome entry site‐mediated translation is stimulated by cis‐acting RNA elements and trans‐acting viral factors

Translation initiation of hepatitis C virus (HCV) occurs through an internal ribosome entry site (IRES) located at its 5'-end. As a positive-stranded RNA virus, HCV uses its genome as a common template for translation and replication, but the coordination between these two processes remains poorly characterized. Moreover, although genetic evidence of RNA-protein interactions for viral replication is accumulating because of subgenomic replicons and a recent culture system for HCV, such interactions are still contentious in the regulation of translation. To gain insight into such mechanisms, we addressed the involvement of cis and trans viral factors in HCV IRES activity by using a cell-based RNA reporter system. We found that the HCV 3' noncoding region (NCR) strongly stimulates IRES efficiency in cis, depending on the genotype and the cell line. Moreover, we confirmed the role of the core protein in viral gene expression as previously reported in vitro. Surprisingly, we observed a similar effect, i.e. a twofold increase under low amounts of NS5B RNA polymerase, followed by a decrease at higher concentrations. However, no contribution of NS5A to HCV IRES-mediated translation was noted and no cooperative effect could be detected between 3' NCR and viral proteins or between proteins. Collectively, these results suggest that HCV RNA translation is regulated, and that the switch from translation to replication might involve a sequential requirement for both cis and trans viral factors, because of their apparent lack of synergy, probably with the aid of host factors.

A Peptide Derived from RNA Recognition Motif 2 of Human La Protein Binds to Hepatitis C Virus Internal Ribosome Entry Site, Prevents Ribosomal Assembly, and Inhibits Internal Initiation of Translation

Human La protein is known to interact with hepatitis C virus (HCV) internal ribosome entry site (IRES) and stimulate translation. Previously, we demonstrated that mutations within HCV SL IV lead to reduced binding to La-RNA recognition motif 2 (RRM2) and drastically affect HCV IRES-mediated translation. Also, the binding of La protein to SL IV of HCV IRES was shown to impart conformational alterations within the RNA so as to facilitate the formation of functional initiation complex. Here, we report that a synthetic peptide, LaR2C, derived from the C terminus of La-RRM2 competes with the binding of cellular La protein to the HCV IRES and acts as a dominant negative inhibitor of internal initiation of translation of HCV RNA. The peptide binds to the HCV IRES and inhibits the functional initiation complex formation. An Huh7 cell line constitutively expressing a bicistronic RNA in which both cap-dependent and HCV IRES-mediated translation can be easily assayed has been developed. The addition of purified TAT-LaR2C recombinant polypeptide that allows direct delivery of the peptide into the cells showed reduced expression of HCV IRES activity in this cell line. The study reveals valuable insights into the role of La protein in ribosome assembly at the HCV IRES and also provides the basis for targeting ribosome-HCV IRES interaction to design potent antiviral therapy.

Hepatitis C Virus Core Protein Acts as a trans-Modulating Factor on Internal Translation Initiation of the Viral RNA

Translation initiation of hepatitis C virus (HCV) RNA occurs through an internal ribosome entry site (IRES) located at its 5' end. As a positive-stranded virus, HCV uses the genomic RNA template for translation and replication, but the transition between these two processes remains poorly understood. HCV core protein (HCV-C) has been proposed as a good candidate to modulate such a regulation. However, current data are still the subject of controversy in attributing any potential role in HCV translation to the HCV core protein. Here we demonstrate that HCV-C displays binding activities toward both HCV IRES and the 40 S ribosomal subunit by using centrifugation on sucrose gradients. To gain further insight into these interactions, we investigated the effect of exogenous addition of purified HCV-C on HCV IRES activity by using an in vitro reporter assay. We found that HCV IRES-mediated translation was specifically modulated by HCV-C provided in trans, in a dose-dependent manner, with up to a 5-fold stimulation of the IRES efficiency upon addition of low amounts of HCV-C, followed by a decrease at high doses. Interestingly, mutations within some domains of the IRES as well as the presence of an upstream reporter gene both lead to changes in the expected effects, consistent with the high dependence of HCV IRES function on its overall structure. Collectively, these results indicate that the HCV core protein is involved in a tight modulation of HCV translation initiation, depending on its concentration, and they suggest an important biological role of this protein in viral gene expression.

Hepatitis C virus (HCV) NS5A protein downregulates HCV IRES-dependent translation

Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located mainly within the 5' non-translated region of the viral genome. In this study, the effect of the HCV non-structural 5A (NS5A) protein on the HCV IRES-dependent translation was investigated by using a transient transfection system. Three different cell lines (HepG2, WRL-68 and BHK-21) were co-transfected with a plasmid vector containing a bicistronic transcript carrying the chloramphenicol acetyltransferase (CAT) and the firefly luciferase genes separated by the HCV IRES sequences, and an expression vector producing the NS5A protein. Here, it was shown that the HCV NS5A protein inhibited HCV IRES-dependent translation in a dose-dependent manner. In contrast, NS5A had no detectable effect on cap-dependent translation of the upstream gene (CAT) nor on translation from another viral IRES. Further analysis using deleted forms of the NS5A protein revealed that a region of about 120 aa located just upstream of the nuclear localization signal of the protein is critical for this suppression. Overall, these results suggest that HCV NS5A protein negatively modulates the HCV IRES activity in a specific manner.

Hepatitis C virus translation and cell cycle

The 5' nontranslated region (NTR) of hepatitis C virus (HCV) contains highly structured segments which form internal ribosome entry site (IRES). This cis-active RNA element direct the cap-independent initiation of translation of the viral polyprotein in association with trans-acting cellular proteins. Thus HCV IRES plays a key role in the replication of virus, especially in liver in which various cellular proteins changes in their expression levels under the condition of chronic hepatitis. To construct a system in which HCV IRES activity could be continuously monitored in vivo, 2 stably transformed cell lines from Huh-7 cells (human hepatocellular carcinoma cells) that express a reporter protein (firefly luciferase) under the translational control of the IRES were established. The activity of the HCV IRES varies in different phases of the cell cycle and HCV IRES activity is highest during the mitotic (G2/M) phases of the cell cycle and lower in the quiescent (G0) phases. The gene expression dynamics of host factors and kinetics of HCV IRES activity in cells at various points during the cell cycle were evaluated using a cDNA microarray. HCV TRES activity correlated with a gene cluster induced in S and G2/M phases. Interestingly, most initiation factors known to bind or interact with HCV IRES (PCBP2, PTB, eIF3, eIF2 gamma, eIF2 beta, La protein and RNLPL) were induced during S and G2/M phases. Of these factors, the expression of La protein, PTB and eIF3 (p116, 170) were predominantly repressed in quiescent (G0) and induced in S and G2/M phases. Suppression or overexpression of La protein and PTB in cells significantly changed HCV IRES activity. In the livers of patients with chronic hepatitis C, the expression of La protein was significantly increased and correlated with the amount of HCV-RNA. Collectively, these data indicated that HCV utilizes host factors induced during cell division but not during quiescence for replication. Of these, La protein is a potent regulator and enhances HCV replication in regenerating hepatocytes in patients with chronic hepatitis C. Biomedical Reviews 2004; 15: 37-46.

Involvement of proteasome alpha-subunit PSMA7 in hepatitis C virus internal ribosome entry site-mediated translation.

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 alpha-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 bicistronic Renilla-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.

Core protein-coding sequence, but not core protein, modulates the efficiency of cap-independent translation directed by the internal ribosome entry site of hepatitis C virus.

Among a myriad of putative functions assigned to the hepatitis C virus (HCV) core protein, several studies suggest that it may modulate internal ribosome entry site (IRES)-mediated initiation of translation. We compared the translational activity of dicistronic reporter transcripts containing the HCV IRES within the intercistronic space fused to downstream sequence encoding either 22 amino acids (aa) or 173 aa of the core protein. The inclusion of the nearly full-length core protein-coding sequence significantly suppressed translation in vitro and in transfected HepG2 cells. However, this suppression was not eliminated by frameshift mutations introduced into the core sequence, suggesting that it occurred at the RNA level and not as a result of core protein expression in cis. Similarly, the expression of core protein (aa 1 to 191) in trans from a recombinant baculovirus did not suppress IRES-directed translation from any of these transcripts in transfected Huh-7 cells. While core protein expression did decrease IRES activity in HepG2 cells (up to 79% suppression), the expression of beta-galactosidase from a control baculovirus also suppressed IRES activity (up to 56%), strongly suggesting that this suppression was nonspecific. Finally, the addition of purified recombinant core protein (aa 1 to 179) to in vitro translation reactions at concentrations up to a 10-fold molar excess over the RNA transcripts resulted in no significant reduction in IRES activity. Consistent with these results, a gel retention assay indicated no difference in the affinities of the recombinant HCV core protein and a recombinant Venezuelan equine encephalitis virus capsid protein for HCV IRES-containing RNA transcripts. We conclude that while the inclusion of core protein-coding sequence downstream of the IRES may reduce the efficiency of cap-independent translation on HCV RNA, the core protein itself has no biologically relevant activity in modulating HCV IRES activity.

Hepatitis C virus internal ribosome entry site (IRES) stem loop IIId contains a phylogenetically conserved GGG triplet essential for translation and IRES folding.

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 and Pestivirus 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 T(1)-probing studies, demonstrating the importance of correct RNA folding to IRES function.

Demonstration of Functional Requirement of Polypyrimidine Tract-binding Protein by SELEX RNA during Hepatitis C Virus Internal Ribosome Entry Site-mediated Translation Initiation

Polypyrimidine tract-binding protein (PTB) has been previously shown to physically interact with the hepatitis C virus (HCV) RNA genome at its 5'- and 3'-noncoding regions. Using high affinity SELEX RNA molecules, we present evidence for the functional requirement of PTB during HCV internal ribosome entry site (IRES)-controlled translation initiation. This study was carried out in rabbit reticulocyte translation lysates in which the HCV IRES-driven reporter RNA was introduced along with the PTB-specific SELEX RNA molecules. The SELEX RNAs specifically inhibited the HCV IRES function in the context of mono- and dicistronic mRNAs. The cap-dependent translation of a reporter (chloramphenicol acetyltransferase) RNA or naturally capped brome mosaic virus RNA, however, was not affected by the presence of SELEX during in vitro translation assays. The SELEX-mediated inhibition of the HCV IRES is shown to be relieved by the addition of recombinant human PTB in an add-back experiment. The in vivo requirement of PTB was further confirmed by cotransfection of Huh7 cells with reporter RNA and PTB-specific SELEX RNA. The HCV IRES activity was inhibited by the SELEX RNA in these cells, but not by an unrelated control RNA. Together, these results demonstrate the functional requirement of cellular PTB in HCV translation and further support the feasible use of SELEX RNA strategy in demonstrating the functional relevance of cellular protein(s) in complex biological processes.

Human La Antigen Is Required for the Hepatitis C Virus Internal Ribosome Entry Site-mediated Translation

The 5'-noncoding region (5'-NCR) of the hepatitis C virus (HCV) RNA genome serves as an internal ribosome entry site (IRES) and mediates translation initiation in a cap-independent manner. Previously, we reported the interaction between La antigen and the HCV IRES, which appeared to occur in the context of initiator AUG. It was further shown that HCV IRES-mediated translation was stimulated in the presence of human La antigen. In this study, we have defined the cis- and trans-acting elements responsible for La-5'-NCR interactions and established the dependence of the HCV IRES efficiency on cellular La antigen. During the La-IRES interaction, initiator AUG but not the neighboring codons was found to be the direct target of La binding. The C terminus effector domain-dependent modulation of La binding to the HCV IRES is demonstrated by deletion and substitution mutagenesis of the protein. An RNA systematic evolution of ligands by exponential enrichment (SELEX), generated against La protein that selectively binds La in HeLa lysates and competes for the protein binding to the 5'-NCR, was used to demonstrate the requirement of La for the HCV IRES function in the context of mono- and dicistronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translation lysates blocked the HCV and poliovirus IRES-mediated translation in vitro. The functional requirement of La protein for the HCV IRES activity was further established in a liver-derived cell line and in an add-back experiment in which the inhibited IRES was rescued by recombinant human La. These results strongly argue for the novel role of La protein during selection of the initiator AUG and its participation during internal initiation of translation of the HCV RNA genome.