A small yeast RNA inhibits HCV IRES mediated translation and inhibits replication of poliovirus in vivo

AIM: To investigate the anti-virus infection activity of internal ribosome entry site (IRES) specific inhibitor RNA (IRNA). METHODS: IRNA eukaryotic vector pcRz-IRNA or mIRNA eukaryotic vector pcRz-mIRNA was transfected into human hepatocarcinoma cells (HHCC), then selected with neomycin G418 for 4 to 8 weeks, and then infected with polio virus vaccines line. The cytopethogenesis effect was investigated and the cell extract was collected. At last the polio virus titer of different cells was determined by plaque assay. RESULTS: Constructive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constructively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell line. Additionally, HHCC cells constitutively expressing IRNA became refractory to infection of polio virus. CONCLUSION: IRES specific IRNA can inhibit HCV IRES mediated translation and poliovirus replication.     

Inhibition of hepatitis C virus translation and subgenomic replication by siRNAs directed against highly conserved HCV sequence and cellular HCV cofactors

BACKGROUND/AIMS: Small interfering RNAs (siRNAs) are an efficient tool to specifically inhibit gene expression by RNA interference. Since hepatitis C virus (HCV) replicates in the cytoplasm of liver cells without integration into the host genome, RNA-directed antiviral strategies are likely to successfully block the HCV replication cycle. Additional benefit might arise from inhibition of cellular cofactors of HCV replication, such as proteasome alpha-subunit 7 (PSMA7) or Hu antigen R (HuR). METHODS: In this study, we investigated direct and cofactor-mediated inhibition of HCV by a panel of DNA-based retroviral vectors expressing siRNAs against highly conserved HCV sequences or the putative HCV cofactors PSMA7 and HuR. Effects were determined in HCV IRES-mediated translation assays and subgenomic HCV replicon cells. RESULTS: PSMA7- and HuR-directed siRNAs successfully inhibited expression of the endogenous genes, and PSMA7 and HuR silencing significantly diminished HCV replicon RNA and NS5B protein levels. HCV-directed siRNAs substantially inhibited HCV IRES-mediated translation and subgenomic HCV replication. Combinations of PSMA7- and HuR-directed siRNAs with HCV-directed siRNAs revealed additive HCV RNA inhibitory effects in monocistronic replicon cells. CONCLUSIONS: A dual approach of direct- and cofactor-mediated inhibition of HCV replication might avoid selection of mutants and thereby become a powerful strategy against HCV.     

Eukaryotic translation initiation factor 4GI and p97 promote cellular internal ribosome entry sequence-driven translation

Numerous cellular mRNAs encoding proteins critical during cell stress, apoptosis, and the cell cycle seem to be translated by means of internal ribosome entry sequences (IRES) when cap-dependent translation is compromised. The underlying molecular mechanisms are largely unknown. Using a HeLa-based cell-free translation system that mirrors the function of cellular IRESs in vitro, we recently demonstrated that translation from the c-myc IRES continues after proteolytic cleavage of eukaryotic translation initiation factor (eIF) 4G. To address the role of eIF4G in cellular IRES-driven translation directly, we immunodepleted eIF4GI from the HeLa cell translation extracts. After efficient depletion of eIF4GI (>90%), both cap-dependent and c-myc IRES-dependent translations are diminished to residual levels (<5%). In striking contrast to cap-dependent translation, c-myc IRES-dependent translation is fully restored by addition of the conserved middle fragment of eIF4GI, harboring the eIF3- and eIF4A-binding sites. p97, an eIF4G-related protein that has been described both as an inhibitor of translation and as a modulator of apoptosis, not only suffices to also rescue c-myc IRES-driven (but not cap-dependent) translation, but it even superinduces IRES-mediated translation 3-fold compared with nondepleted extracts. Interestingly, both p97 and the middle fragment of eIF4GI also rescue translation driven by proapoptotic (p97) and antiapoptotic [X-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis 1 (c-IAP1)] IRESs, reflecting a broader role of these polypeptides in cellular IRES-mediated translation and indicating their importance in apoptosis.     

Interferons alpha, beta, gamma each inhibit hepatitis C virus replication at the level of internal ribosome entry site-mediated translation.

Interferon (IFN)-alpha is the standard therapy for the treatment of chronic hepatitis C, but the mechanisms underlying its antiviral action are not well understood. In this report, we demonstrated that IFN-alpha, -beta and -gamma inhibit replication of the hepatitis C virus (HCV) in a cell culture model at concentrations between 10 and 100 IU/ml. We demonstrated that the antiviral actions each of each these IFNs are targeted to the highly conserved 5' untranslated region of the HCV genome, and that they directly inhibit translation from a chimeric clone between full-length HCV genome and green fluorescent protein (GFP). This effect is not limited to HCV internal ribosome entry site (IRES), since these IFNs also inhibit translation of the encephalomyocardititis virus (EMCV) chimeric mRNA in which GFP is expressed by IRES-dependent mechanisms (pCITE-GFP). These IFNs had minimal effects on the expression of mRNAs from clones in which translation is not IRES dependent. We conclude that IFN-alpha, -beta and -gamma inhibit replication of sub-genomic HCV RNA in a cell culture model by directly inhibiting two internal translation initiation sites of HCV- and EMCV-IRES sequences present in the dicistronic HCV sub-genomic RNA. Results of this in vitro study suggest that selective inhibition of IRES-mediated translation of viral polyprotein is a general mechanism by which IFNs inhibits HCV replication.     

Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3)

Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli, revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.     

Internal initiation in Saccharomyces cerevisiae mediated by an initiator tRNA/eIF2-independent internal ribosome entry site element

Internal initiation of translation can be mediated by specific internal ribosome entry site (IRES) elements that are located in certain mammalian and viral mRNA molecules. Thus far, these mammalian cellular and viral IRES elements have not been shown to function in the yeast Saccharomyces cerevisiae. We report here that a recently discovered IRES located in the genome of cricket paralysis virus can direct the efficient translation of a second URA3 cistron in dicistronic mRNAs in S. cerevisiae, thereby conferring uracil-independent growth. Curiously, the IRES functions poorly in wild-type yeast but functions efficiently either in the presence of constitutive expression of the eIF2 kinase GCN2 or in cells that have two initiator tRNA(met) genes disrupted. Both of these conditions have been shown to lower the amounts of ternary eIF2-GTP/initiator tRNA(met) complexes. Furthermore, tRNA(met)-independent initiation was also observed in translation-competent extracts prepared from S. cerevisiae in the presence of edeine, a compound that has been shown to interfere with start codon recognition by ribosomal subunits carrying ternary complexes. Therefore, the cricket paralysis virus IRES is likely to recruit ribosomes by internal initiation in S. cerevisiae in the absence of eIF2 and initiator tRNA(met), by the same mechanism of factor-independent ribosome recruitment used in mammalian cells. These findings will allow the use of yeast genetics to determine the mechanism of internal ribosome entry.     

Design of highly specific cytotoxins by using trans-splicing ribozymes

We have designed ribozymes based on a self-splicing group I intron that can trans-splice exon sequences into a chosen RNA target to create a functional chimeric mRNA and provide a highly specific trigger for gene expression. We have targeted ribozymes against the coat protein mRNA of a widespread plant pathogen, cucumber mosaic virus. The ribozymes were designed to trans-splice the coding sequence of the diphtheria toxin A chain in frame with the viral initiation codon of the target sequence. Diphtheria toxin A chain catalyzes the ADP ribosylation of elongation factor 2 and can cause the cessation of protein translation. In a Saccharomyces cerevisiae model system, ribozyme expression was shown to specifically inhibit the growth of cells expressing the virus mRNA. A point mutation at the target splice site alleviated this ribozyme-mediated toxicity. Increasing the extent of base pairing between the ribozyme and target dramatically increased specific expression of the cytotoxin and reduced illegitimate toxicity in vivo. Trans-splicing ribozymes may provide a new class of agents for engineering virus resistance and therapeutic cytotoxins.     

Translation of hepatitis C virus RNA

The 341-nucleotide 5' non-translated region is the most conserved part of the hepatitis C virus (HCV) genome. It contains a highly structured internal ribosomal entry site (IRES) that mediates cap-independent initiation of translation of the viral polyprotein by a mechanism that is unprecedented in eukaryotes. The first step in translation initiation is assembly of eukaryotic initiation factor (eIF) 3, eIF2, GTP, initiator tRNA and a 40S ribosomal subunit into a 43S preinitiation complex. The HCV IRES recruits this complex and directs its precise attachment at the initiation codon to form a 48S complex in a process that does not involve eIFs 4A, 4B or 4F. The IRES contains sites that bind independently with the eIF3 and 40S subunit components of 43S complexes, and structural determinants that ensure the correct spatial orientation of these binding sites so that the 48S complex assembles precisely at the initiation codon.     

Translation of hepatitis C virus RNA

The 341-nucleotide 5' non-translated region is the most conserved part of the hepatitis C virus (HCV) genome. It contains a highly structured internal ribosomal entry site (IRES) that mediates cap-independent initiation of translation of the viral polyprotein by a mechanism that is unprecedented in eukaryotes. The first step in translation initiation is assembly of eukaryotic initiation factor (eIF) 3, eIF2, GTP, initiator tRNA and a 40S ribosomal subunit into a 43S preinitiation complex. The HCV IRES recruits this complex and directs its precise attachment at the initiation codon to form a 48S complex in a process that does not involve eIFs 4A, 4B or 4F. The IRES contains sites that bind independently with the eIF3 and 40S subunit components of 43S complexes, and structural determinants that ensure the correct spatial orientation of these binding sites so that the 48S complex assembles precisely at the initiation codon.     

Coordinated assembly of human translation initiation complexes by the hepatitis C virus internal ribosome entry site RNA

Protein synthesis in all cells begins with recruitment of the small ribosomal subunit to the initiation codon in a messenger RNA. In some eukaryotic viruses, RNA upstream of the coding region forms an internal ribosome entry site (IRES) that directly binds to the 40S ribosomal subunit and enables translation initiation in the absence of many canonical translation initiation factors. The hepatitis C virus (HCV) IRES RNA requires just two initiation factors, eukaryotic initiation factor (eIF) 2 and eIF3, to form preinitiation 48S ribosomal complexes that subsequently assemble into translation-competent ribosomes. Using an RNA-based affinity purification approach, we show here that HCV IRES RNA facilitates eIF2 function through its interactions with eIF3 and the 40S ribosomal subunit. Although the wild-type IRES assembles normally into 48S and 80S ribosomal complexes in human cell extract, mutant IRES RNAs become trapped at the 48S assembly stage. Trapped 48S complexes formed by IRES mutants with reduced eIF3 binding affinity nonetheless contain eIF3, consistent with inherent eIF3-40S subunit affinity. Intriguingly, however, one of these IRES mutants prevents stable association of both eIF3 and eIF2, preventing initiator tRNA deposition and explaining the block in 80S assembly. In contrast, an IRES mutant unable to induce a conformational change in the 40S subunit, as observed previously by single-particle cryoelectron microscopy, blocks 80S formation at a later stage in assembly. These data suggest that the IRES RNA coordinates interactions of eIF3 and eIF2 on the ribosome required to position the initiator tRNA on the mRNA in the ribosomal peptidyl-tRNA site (P site).     

Inhibitor RNA blocks the protein translation mediated by hepatitis C virus internal ribosome entry site in vivo

AIM:To investigate the inhibitory effect of hepatitis C virus internal ribosome entry site (HCV IRES) specific inhibitor RNA(IRNA) on gene expression mediated by HCV IRES in vivo.METHODS:By using G418 screening system, hepatoma cells constitutively expressing IRNA or mutant IRNA (mIRNA) were established and characterized, and HCV replicons containing the 5′ untranslated region (5′UTR) were constructed by using the same method. Cotransfection of pCMVNCRluc containing HCV 5′UTR-Iuc fusion genes and eukaryotic vector of IRNA into human hepatic carcinoma cells (HepG2) was performed and the eukaryotic expression plasmid of IRNA was transfected transiently into HCV replicons, pCMVNCRluc or pCDNA-luc was cotransfected with pSV40-β Gal into IRNA expressing hepatoma cells by using lipofectamine 2000 in vitro. Then the reporting gene expression level was examined at 48h after transfection by using a luminometer and the expressing level of HCV C antigen was analysed with a confocal microscope.RESULTS: Transient expression of IRES specific IRNA could significantly inhibit the expression of reporter gene and viral antigen mediated by HCV IRES by 50% to 90% in vivo, but mIRNA lost its inhibitory activity completely. The luciferase gene expression mediated by HCV IRES was blocked in the HHCC constitutively expressing IRNA. At 48h after transfection, the expression level of reportor gene descreased by 20%, but cap-dependent luciferase gene expression was not affected. IRNA could inhibit the HCV replicon expression 24h after transfection and the highest inhibitory activity was 80% by 72h, and the inhibitory activity was not increased until 7d after transfection.CONCLUSION:IRNA can inhibit HCV IRES mediated gene expression in vivo.     

A cell-permeable hairpin peptide inhibits hepatitis C viral nonstructural protein 5A-mediated translation and virus production

NS5A is a key regulator of the hepatitis C virus (HCV) life cycle including RNA replication, assembly, and translation. We and others have shown that NS5A augments HCV internal ribosomal entry site (IRES)-mediated translation. Furthermore, Quercetin treatment and heat shock protein (HSP) 70 knockdown inhibit the NS5A-driven augmentation of IRES-mediated translation and infectious virus production. We have also coimmunoprecipitated HSP70 with NS5A and demonstrated cellular colocalization, leading to the hypothesis that the NS5A/HSP70 complex formation is important for IRES-mediated translation. Here, we have identified the NS5A region responsible for complex formation through in vitro deletion analyses. Deletion of NS5A domains II and III failed to reduce HSP70 binding, whereas domain I deletion eliminated complex formation. NS5A domain I alone also bound HSP70. Deletion mapping of domain I identified the C-terminal 34 amino acids (C34) as the interaction site. Furthermore, addition of C34 to domains II and III restored complex formation. C34 expression significantly reduced intracellular viral protein levels, in contrast to same-size control peptides from other NS5A domains. C34 also competitively inhibited NS5A-augmented IRES-mediated translation, whereas controls did not. Triple-alanine scan mutagenesis determined that an exposed beta-sheet hairpin in C34 was primarily responsible for NS5A-augmented IRES-mediated translation. Moreover, treatment with a 10-amino acid peptide derivative of C34 suppressed NS5A-augmented IRES-mediated translation and significantly inhibited intracellular viral protein synthesis, with no associated cytotoxicity. Conclusion: These results support the hypothesis that the NS5A/HSP70 complex augments viral IRES-mediated translation, identify a sequence-specific hairpin element in NS5A responsible for complex formation, and demonstrate the functional significance of C34 hairpin-mediated NS5A/HSP70 interaction. Identification of this element may allow for further interrogation of NS5A-mediated IRES activity, sequence-specific HSP recognition, and rational drug design. (HEPATOLOGY 2012;55:1662-1672).     

Vitamin B12 and hepatitis C: molecular biology and human pathology

Cobalamins are stored in high concentrations in the human liver and thus are available to participate in the regulation of hepatotropic virus functions. We show that cyanocobalamin (vitamin B12) inhibited the HCV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly affecting the cap-dependent mechanism. Vitamin B12 failed to inhibit translation by IRES elements from encephalomyocarditis virus (EMCV) or classical swine fever virus (CSFV). We also demonstrate a relationship between the total cobalamin concentration in human sera and HCV viral load (a measure of viral replication in the host). The mean viral load was two orders of magnitude greater when the serum cobalamin concentration was above 200 pM (P < 0.003), suggesting that the total cobalamin concentration in an HCV-infected liver is biologically significant in HCV replication.     

Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes

Ribozymes are catalytic RNA molecules that can be designed to cleave specific RNA sequences. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection, we designed and synthesized hammerhead ribozymes targeting 15 conserved sites in the 5' untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5' UTR/luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with several ribozymes resulted in significant down-regulation of HCV 5' UTR/luciferase reporter expression (range 40% to 80% inhibition, P <.05). Moreover, several ribozymes showed significant inhibition (>90%, P <.001) of chimeric HCV-PV replication. We further show that the inhibitory activity of ribozymes targeting site 195 of HCV RNA exhibits a sequence-specific dose response, requires an active catalytic ribozyme core, and is dependent on the presence of the HCV 5' UTR. Treatment with synthetic stabilized anti-HCV ribozymes has the potential to aid patients who are infected with HCV by reducing the viral burden through specific targeting and cleavage of the viral genome.     

Hammerhead ribozymes with cleavage site specificity for NUH and NCH display significant anti-hepatitis C viral effect in vitro and in recombinant HepG2 and CCL13 cells

BACKGROUND/AIMS: Four different ribozymes (Rz) targeting the hepatitis C virus (HCV) 5'-non-coding region (NCR) at nucleotide (nt) positions GUA 165 (Rz1), GUC 270 (Rz2), GUA 330 (Rz3) and GCA 348 (Rz1293) were compared for in vitro cleavage using a 455 nt HCV RNA substrate. The GUA 330 (Rz3) and GCA 348 (Rz1293) ribozymes, both targeting the HCV loop IV region, were found to be the most efficient, and were further analyzed in an in vitro translation system. METHODS: For this purpose RNA transcribed from a construct encoding a HCV-5'-NCR-luciferase fusion protein was used. Cleavage-inactive (Rz1426), mismatch (Rz1293m) or unrelated ribozymes (Rz1437) were synthesized as controls for Rz-1293. HCV specificity was analysed by competition experiments using sense and mismatch oligodeoxynucleotides HCVrzCI and HCVrzMM, respectively. RESULTS: A chemically modified nuclease-resistant variant of the GCA 348 cleaving ribozyme was selected for cell culture experiments using recombinant HepG2 or CCL13 cell lines stably transfected with a HCV-5'-NCR-luciferase target construct. CONCLUSIONS: This ribozyme (Rz1293) showed an inhibitory activity of translation of more than 70% thus verifying that the GCA 348 cleavage site in the HCV loop IV is an accessible target site in vivo and may be suitable for the development of novel optimized hammerhead structures.     

Hepatitis C viral protein translation: mechanisms and implications in developing antivirals

Hepatitis C viral protein translation occurs in a cap‐independent manner through the use of an internal ribosomal entry site (IRES) present within the viral 5′‐untranslated region. The IRES is composed of highly conserved structural domains that directly recruit the 40S ribosomal subunit to the viral genomic RNA. This frees the virus from relying on a large number of translation initiation factors that are required for cap‐dependent translation, conferring a selective advantage to the virus especially in times when the availability of such factors is low. Although the mechanism of translation initiation on the Hepatitis C virus (HCV) IRES is well established, modulation of the HCV IRES activity by both cellular and viral factors is not well understood. As the IRES is essential in the HCV life cycle and as such remains well conserved in an otherwise highly heterogenic virus, the process of HCV protein translation represents an attractive target in the development of novel antivirals. This review will focus on the mechanisms of HCV protein translation and how this process is postulated to be modulated by cis‐acting viral factors, as well as trans‐acting viral and cellular factors. Numerous therapeutic approaches investigated in targeting HCV protein translation for the development of novel antivirals will also be discussed.     

Selection of cyclic peptide aptamers to HCV IRES RNA using mRNA display

The hepatitis C virus (HCV) is a positive strand RNA flavivirus that is a major causative agent of serious liver disease, making new treatment modalities an urgent priority. Because HCV translation initiation occurs by a mechanism that is fundamentally distinct from that of host mRNAs, it is an attractive target for drug discovery. The translation of HCV mRNA is initiated from an internal ribosomal entry site (IRES), independent of cap and poly(A) recognition and bypassing eIF4F complex formation. We used mRNA display selection technology combined with a simple and robust cyclization procedure to screen a peptide library of >10(13) different sequences and isolate cyclic peptides that bind with high affinity and specificity to HCV IRES RNA. The best peptide binds the IRES with subnanomolar affinity, and a specificity of at least 100-fold relative to binding to several other RNAs of similar length. The peptide specifically inhibits HCV IRES-initiated translation in vitro with no detectable effect on normal cap-dependent translation initiation. An 8-aa cyclic peptide retains most of the activity of the full-length 27-aa bicyclic peptide. These peptides may be useful tools for the study of HCV translation and may have potential for further development as an anti-HCV drug.     

Proteolysis of human eukaryotic translation initiation factor eIF4GII, but not eIF4GI, coincides with the shutoff of host protein synthesis after poliovirus infection

Eukaryotic initiation factor (eIF) 4GI is a component of the cap-binding protein complex eIF4F, which is required for cap-dependent translation. Infection of cells by poliovirus results in a precipitous decline of host cell protein synthesis, which is preceded by the cleavage of eIF4GI. Cleavage of eIF4GI results in the inhibition of cap-dependent translation. Poliovirus translation is not affected by eIF4GI cleavage, however, because poliovirus mRNA is translated by a cap-independent mechanism. Cleavage of eIF4GI alone cannot explain the shutoff of host protein synthesis, because after infection in the presence of inhibitors of virus replication, eIF4GI is cleaved, yet host protein synthesis is only partially inhibited. Here we show that eIF4GII, a recently discovered functional homolog of eIF4GI, is more resistant to poliovirus-mediated cleavage than eIF4GI, and that its proteolysis is concomitant with the shutoff of host cell protein synthesis. Moreover, infection with poliovirus in the presence of inhibitors of virus replication resulted in efficient cleavage of eIF4GI, but only partial proteolysis of eIF4GII. Thus, cleavage of both eIF4GI and eIF4GII appears to be required for the shutoff of host protein synthesis after poliovirus infection. These results explain several earlier reports documenting the lack of correlation between eIF4GI cleavage and inhibition of cellular mRNA translation after poliovirus infection.