A peptide from autoantigen La blocks poliovirus and hepatitis C virus cap-independent translation and reveals a single tyrosine critical for La RNA binding and translation stimulation

La, a 52-kDa autoantigen in patients with systemic lupus erythematosus, was one of the first cellular proteins identified to interact with viral internal ribosome entry site (IRES) elements and stimulate poliovirus (PV) and hepatitis C virus (HCV) IRES-mediated translation. Previous results from our laboratory have shown that a small, yeast RNA (IRNA) could selectively inhibit PV and HCV IRES-mediated translation by sequestering the La protein. Here we have identified an 18-amino-acid-long sequence from the N-terminal "La motif" which is required for efficient interaction of La with IRNA and viral 5' untranslated region (5'-UTR) elements. A synthetic peptide (called LAP, for La peptide) corresponding to this sequence (amino acids 11 to 28) of La was found to efficiently inhibit viral IRES-mediated translation in vitro. The LAP efficiently enters Huh-7 cells and preferentially inhibits HCV IRES-mediated translation programmed by a bicistronic RNA in vivo. The LAP does not bind RNA directly but appears to block La binding to IRNA and PV 5'-UTR. Competition UV cross-link and translation rescue experiments suggested that LAP inhibits IRES-mediated translation by interacting with proteins rather than RNA. Mutagenesis of LAP demonstrates that single amino acid changes in a highly conserved sequence within LAP are sufficient to eliminate the translation-inhibitory activity of LAP. When one of these mutations (Y23Q) is introduced into full-length La, the mutant protein is severely defective in interacting with the PV IRES element and consequently unable to stimulate IRES-mediated translation. However, the La protein with a mutation of the next tyrosine moiety (Y24Q) could still interact with PV 5'-UTR and stimulate viral IRES-mediated translation significantly. These results underscore the importance of the La N-terminal amino acids in RNA binding and viral RNA translation. The possible role of the LAP sequence in La-RNA binding and stimulation of viral IRES-mediated translation is discussed.     

Targeting internal ribosome entry site (IRES)-mediated translation to block hepatitis C and other RNA viruses

A number of RNA-containing viruses such as hepatitis C (HCV) and poliovirus (PV) that infect human beings and cause serious diseases use a common mechanism for synthesis of viral proteins, termed internal ribosome entry site (IRES)-mediated translation. This mode of translation initiation involves entry of 40S ribosome internally to the 5' untranslated region (UTR) of viral RNA. Cap-dependent translation of cellular mRNAs, on the other hand, requires recognition of mRNA 5' cap by the translation machinery. In this review, we discuss two inhibitors that specifically inhibit viral IRES-mediated translation without interfering with cellular cap-dependent translation. We present evidence, which suggest that one of these inhibitors, a small RNA (called IRNA) originally isolated from the yeast Saccharomyces cerevisiae, inhibits viral IRES-mediated translation by sequestering both noncanonical transacting factors and canonical initiation factors required for IRES-mediated translation. The other inhibitor, a small peptide from the lupus autoantigen La (called LAP), appears to block binding of cellular transacting factors to viral IRES elements. These results suggest that it might be possible to target viral IRES-mediated translation for future development of therapeutic agents effective against a number of RNA viruses including HCV that exclusively use cap-independent translation for synthesis of viral proteins.     

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.     

Promotion of Viral IRES-Mediated Translation Initiation under Mild Hypothermia

Internal ribosome entry site (IRES)-mediated translation is an essential replication step for certain viruses. As IRES-mediated translation is regulated differently from cap-dependent translation under various cellular conditions, we sought to investigate whether temperature influences efficiency of viral IRES-mediated translation initiation by using bicistronic reporter constructs containing an IRES element of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), human rhinovirus (HRV) or poliovirus (PV). Under mild hypothermic conditions (30 and 35°C), we observed increases in the efficiency of translation initiation by HCV and HRV IRES elements compared to translation initiation at 37°C. The promotion of HRV IRES activity was observed as early as 2 hours after exposure to mild hypothermia. We also confirmed the promotion of translation initiation by HRV IRES under mild hypothermia in multiple cell lines. The expression levels and locations of polypyrimidine tract-binding protein (PTB) and upstream of N-Ras (unr), the IRES trans-acting factors (ITAFs) of HCV and HRV IRES elements, were not modulated by the temperature shift from 37°C to 30°C. Taken together, this study demonstrates that efficiency of translation initiation by some viral IRES elements is temperature dependent.     

Structure of the hepatitis C virus IRES bound to the human 80S ribosome: remodeling of the HCV IRES

Initiation of translation of the hepatitis C virus (HCV) polyprotein is driven by an internal ribosome entry site (IRES) RNA that bypasses much of the eukaryotic translation initiation machinery. Here, single-particle electron cryomicroscopy has been used to study the mechanism of HCV IRES-mediated initiation. A HeLa in vitro translation system was used to assemble human IRES-80S ribosome complexes under near physiological conditions; these were stalled before elongation. Domain 2 of the HCV IRES is bound to the tRNA exit site, touching the L1 stalk of the 60S subunit, suggesting a mechanism for the removal of the HCV IRES in the progression to elongation. Domain 3 of the HCV IRES positions the initiation codon in the ribosomal mRNA binding cleft by binding helix 28 at the head of the 40S subunit. The comparison with the previously published binary 40S-HCV IRES complex reveals structural rearrangements in the two pseudoknot structures of the HCV IRES in translation initiation.     

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.     

Novel fluorescence-based screen to identify small synthetic internal ribosome entry site elements

We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.     

Structure and function of a small RNA that selectively inhibits internal ribosome entry site-mediated translation.

A 60 nt long RNA termed IRNA, isolated from the yeast Saccharomyces cerevesiae, was previously shown to selectively block internal ribosome entry site (IRES)-mediated translation without interfering with cap-dependent translation of cellular mRNAs both in vivo and in vitro. IRNA specifically bound cellular proteins believed to be important for IRES-mediated translation. We demonstrate here that a complementary copy of IRNA (cIRNA) is also active in blocking IRES-mediated translation and that it binds many of the same cellular proteins that IRNA does. We have probed the secondary structure of both IRNA and cIRNA using single-strand- and double-strand-specific nucleases as well as using oligonucleotide hybridization followed by RNase H digestion. Both IRNA and cIRNA share secondary structural homology, although distinct differences do exist between the two structures. Mutational analysis of IRNA shows that sequences that form both the main stem and one loop are critical for its translation inhibitory activity. Maintenance of the established secondary structure appears to be required for both IRNA's ability to bind cellular trans -acting proteins believed to be required for IRES-mediated translation and its ability to block IRES-mediated translation.     

脑心肌炎病毒(EMCV)的内部核糖体进入位点活性的研究

脑心肌炎病毒(Encephalomyocarditis virus ,EMCV)的内部核糖体进入位点(internal ribosomal entry site ,IRES)已被广泛运用于蛋白的表达中,如应用于Clontech 公司推出的pIRES2 质粒对增强型绿色荧光蛋白(EGFP)的表达。但是由于对EMCV的IRES了解还不够深入而在利用EMCV的IRES进行基因表达过程中经常会遇到问题,很有必要对其进行更深入的研究。以红绿色荧光蛋白基因作为报告基因,通过分子克隆、荧光显微镜、荧光分光光度计、Western blot等分子细胞生物学手段,对EMCV的IRES末端序列与其活性的关系进行了深入研究。研究发现EMCV的IRES末端序列对其IRES的活性影响极大,而人们常用的报告基因EGFP本身也可能存在IRES。     

Oligonucleotide-based strategies to inhibit human hepatitis C virus

Hepatitis C virus (HCV) infection represents a worldwide problem, and current antiviral regimens are not satisfactory. The need to develop novel, specific, anti-HCV antiviral drugs is clear. Antisense oligonucleotides (AS-ON), ribozymes, and more recently, small interfering RNAs (siRNAs) have been widely used to control gene expression, and several clinical trials are in progress. The potential to use AS-ON as tools to control HCV infection, either by promoting an RNase H mediated cleavage of viral genomic RNA or by interfering with the assembly of a translation initiation complex on the internal ribosome entry site (IRES) is reviewed. Extensive knowledge of IRES structure and conservation among HCV genotypes have rendered the HCV IRES (and, in particular, its IIId loop) particularly attractive for antisense approaches. Encouraging data have been obtained with IRES-targeted RNase H-competent and incompetent ON analogs. We demonstrate here that very short steric blocking ONs can inhibit the formation of translation preinitiation complexes on the IRES and block IRES-mediated translation in a cell-free translation assay and in a transfected hepatoma cell line.     

The HCV IRES pseudoknot positions the initiation codon on the 40S ribosomal subunit

The hepatitis C virus (HCV) genomic RNA contains an internal ribosome entry site (IRES) in its 5′ untranslated region, the structure of which is essential for viral protein translation. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation and, using 35 mutants, conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S ribosomal subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine–Dalgarno sequence and the initiation codon in bacterial mRNAs. Finally, we validated the HCV IRES pseudoknot as a potential drug target using antisense 2′-OMe oligonucleotides.     

Translational efficiency of BVDV IRES and EMCV IRES for T7 RNA polymerase driven cytoplasmic expression in mammalian cell lines

Mammalian T7 polymerase-based cytoplasmic expression systems are common tool for molecular studies. The majority of these systems include the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). To carry out a cap-independent translation process, this type of IRES might require the expression of an extensive array of host factors, what is a disadvantage. Other IRESes might be less dependent on the host cell factors, but their biology is characterized to a lesser degree. Here, we compare the translational efficiencies of bovine viral diarrhea virus (BVDV) IRES with that of ECMV. Both IRESes were tested in reporter vectors containing the T7 promoter, an IRES of choice and the coding sequence of the enhanced green fluorescent protein (EGFP). To provide for the expression of T7 RNA polymerase, the corresponding gene was isolated from Escherichia coli and inserted into pCDNA3.1-Hygro(+). After co-transfection of the T7 RNA polymerase encoding vector with either of the two IRES-containing reporter vectors into T7 baby hamster kidney (T7-BHK), human embryonic kidney (HEK) 293T, chinese hamster ovary (CHO) and HeLa cells, the translational efficiency of the reporter construct was studied by fluorescence microscopy and flow cytometry. In T7-BHK, HEK 293T and HeLa cells the translational efficiency of BVDV IRES was two to three times higher than that of EMCV IRES. In CHO cells, BVDV IRES and EMCV IRES were equally efficient. An analysis of the secondary structure of respective mRNAs showed that their ΔG values were–544.00 and–469.40 kcal/mol for EMCV IRES and BVDV IRES harboring molecules, respectively. As EMCV IRES-containing mRNA is more stable, it is evident that other, still unidentified factors should be held responsible for the enhanced translational efficiency of BDVD IRES. Taken together, our results indicate the potential of BVDV IRES as a replacement for EMCV IRES, which is now commonly used for T7 polymerase driven cytoplasmic expression of genes of interest or virus cDNA rescue experiments.     

Increased inhibitory ability of conjugated RNA aptamers against the HCV IRES

Hepatitis C virus (HCV) translation begins within the internal ribosome entry site (IRES). We have previously isolated two RNA aptamers, 2-02 and 3-07, which specifically bind to domain II and domain III-IV of the HCV IRES, respectively, and inhibit IRES-dependent translation. To improve the function of these aptamers, we constructed two conjugated molecules of 2-02 and 3-07. These bound to the target RNA more efficiently than the two parental aptamers. Furthermore, they inhibited IRES-dependent translation about 10 times as efficiently as the 3-07 aptamer. This result indicates that combining aptamers for different target recognition sites potentiates the inhibition activity by enhancing the domain-binding efficiency.     

An integrated transcriptomic and meta-analysis of hepatoma cells reveals factors that influence susceptibility to HCV infection

Hepatitis C virus (HCV) is a global problem. To better understand HCV infection researchers employ in vitro HCV cell-culture (HCVcc) systems that use Huh-7 derived hepatoma cells that are particularly permissive to HCV infection. A variety of hyper-permissive cells have been subcloned for this purpose. In addition, subclones of Huh-7 which have evolved resistance to HCV are available. However, the mechanisms of susceptibility or resistance to infection among these cells have not been fully determined. In order to elucidate mechanisms by which hepatoma cells are susceptible or resistant to HCV infection we performed genome-wide expression analyses of six Huh-7 derived cell cultures that have different levels of permissiveness to infection. A great number of genes, representing a wide spectrum of functions are differentially expressed between cells. To focus our investigation, we identify host proteins from HCV replicase complexes, perform gene expression analysis of three HCV infected cells and conduct a detailed analysis of differentially expressed host factors by integrating a variety of data sources. Our results demonstrate that changes relating to susceptibility to HCV infection in hepatoma cells are linked to the innate immune response, secreted signal peptides and host factors that have a role in virus entry and replication. This work identifies both known and novel host factors that may influence HCV infection. Our findings build upon current knowledge of the complex interplay between HCV and the host cell, which could aid development of new antiviral strategies.     

A conserved RNA structure within the HCV IRES eIF3-binding site

The hepatitis C virus (HCV) internal ribosome entry site (IRES) is recognized specifically by the small ribosomal subunit and eukaryotic initiation factor 3 (eIF3) before viral translation initiation. Using extensive mutagenesis and structure probing analysis, we show that the eIF3-binding domain of the HCV IRES contains an internal loop structure (loop IIIb) and an adjacent mismatched helix that are important for IRES-dependent initiation of translation. NMR studies reveal a unique three-dimensional structure for this internal loop that is conserved between viral isolates of varying primary sequence in this region. These data indicate that internal loop IIIb may be an attractive target for structure-based design of new antiviral agents.