BiP internal ribosomal entry site activity is controlled by heat-induced interaction of NSAP1

TheBiP protein, a stress response protein, plays an important role in the proper folding and assembly of nascent protein and in the scavenging of misfolded proteins in the endoplasmic reticulum lumen. Translation of BiP is directed by an internal ribosomal entry site (IRES) in the 5' nontranslated region of the BiP mRNA. BiP IRES activity increases when cells are heat stressed. Here we report that NSAP1 specifically enhances the IRES activity of BiP mRNA by interacting with the IRES element. Overexpression of NSAP1 in 293T cells increased the IRES activity of BiP mRNA, whereas knockdown of NSAP1 by small interfering RNA (siRNA) reduced the IRES activity of BiP mRNA. The amount of NSAP1 bound to the BiP IRES increased under heat stress conditions, and the IRES activity of BiP mRNA was increased. Moreover, the increase in BiP IRES activity with heat treatment was not observed in cells lacking NSAP1 after siRNA treatment. BiP mRNAs were redistributed from the heavy polysome to the light polysome in NSAP1 knockdown cells. Together, these data indicate that NSAP1 modulates IRES-dependent translation of BiP mRNA through an RNA-protein interaction under heat stress conditions.     

Heterogeneous Nuclear Ribonucleoprotein L Interacts with the 3′ Border of the Internal Ribosomal Entry Site of Hepatitis C Virus

Translation initiation of hepatitis C virus (HCV) RNA occurs by internal entry of a ribosome into the 5′ nontranslated region in a cap-independent manner. The HCV RNA sequence from about nucleotide 40 up to the N terminus of the coding sequence of the core protein is required for efficient internal initiation of translation, though the precise border of the HCV internal ribosomal entry site (IRES) has yet to be determined. Several cellular proteins have been proposed to direct HCV IRES-dependent translation by binding to the HCV IRES. Here we report on a novel cellular protein that specifically interacts with the 3′ border of the HCV IRES in the core-coding sequence. This protein with an apparent molecular mass of 68 kDa turned out to be heterogeneous nuclear ribonucleoprotein L (hnRNP L). The binding of hnRNP L to the HCV IRES correlates with the translational efficiencies of corresponding mRNAs. This finding suggests that hnRNP L may play an important role in the translation of HCV mRNA through the IRES element.     

Translation-competent 48S complex formation on HCV IRES requires the RNA-binding protein NSAP1

Translation of many cellular and viral mRNAs is directed by internal ribosomal entry sites (IRESs). Several proteins that enhance IRES activity through interactions with IRES elements have been discovered. However, the molecular basis for the IRES-activating function of the IRES-binding proteins remains unknown. Here, we report that NS1-associated protein 1 (NSAP1), which augments several cellular and viral IRES activities, enhances hepatitis C viral (HCV) IRES function by facilitating the formation of translation-competent 48S ribosome-mRNA complex. NSAP1, which is associated with the solvent side of the 40S ribosomal subunit, enhances 80S complex formation through correct positioning of HCV mRNA on the 40S ribosomal subunit. NSAP1 seems to accomplish this positioning function by directly binding to both a specific site in the mRNA downstream of the initiation codon and a 40S ribosomal protein (or proteins).     

Interaction of hepatitis C virus core protein with viral sense RNA and suppression of its translation

To clarify the binding properties of hepatitis C virus (HCV) core protein and its viral RNA for the encapsidation, morphogenesis, and replication of HCV, the specific interaction of HCV core protein with its genomic RNA synthesized in vitro was examined in an in vivo system. The positive-sense RNA from the 5' end to nucleotide (nt) 2327, which covers the 5' untranslated region (5'UTR) and a part of the coding region of HCV structural proteins, interacted with HCV core protein, while no interaction was observed in the same region of negative-sense RNA and in other regions of viral and antiviral sense RNAs. The internal ribosome entry site (IRES) exists around the 5'UTR of HCV; therefore, the interaction of the core protein with this region of HCV RNA suggests that there is some effect on its cap-independent translation. Cells expressing HCV core protein were transfected with reporter RNAs consisting of nt 1 to 709 of HCV RNA (the 5'UTR of HCV and about two-thirds of the core protein coding regions) followed by a firefly luciferase gene (HCV07Luc RNA). The translation of HCV07Luc RNA was suppressed in cells expressing the core protein, whereas no significant suppression was observed in the case of a reporter RNA possessing the IRES of encephalomyocarditis virus followed by a firefly luciferase. This suppression by the core protein occurred in a dose-dependent manner. The expression of the E1 envelope protein of HCV or beta-galactosidase did not suppress the translation of both HCV and EMCV reporter RNAs. We then examined the regions that are important for suppression of translation by the core protein and found that the region from nt 1 to 344 was enough to exert this suppression. These results suggest that the HCV core protein interacts with viral genomic RNA at a specific region to form nucleocapsids and regulates the expression of HCV by interacting with the 5'UTR.     

Inhibition of hepatitis C virus IRES-mediated translation by small RNAs analogous to stem-loop structures of the 5'-untranslated region

Translation of the hepatitis C virus (HCV) RNA is mediated by the interaction of ribosomes and cellular proteins with an internal ribosome entry site (IRES) located within the 5′-untranslated region (5′-UTR). We have investigated whether small RNA molecules corresponding to the different stem–loop (SL) domains of the HCV IRES, when introduced in trans, can bind to the cellular proteins and antagonize their binding to the viral IRES, thereby inhibiting HCV IRES-mediated translation. We have found that a RNA molecule corresponding to SL III could efficiently inhibit HCV IRES-mediated translation in a dose-dependent manner without affecting cap-dependent translation. The SL III RNA was found to bind to most of the cellular proteins which interacted with the HCV 5′-UTR. A smaller RNA corresponding to SL e+f of domain III also strongly and selectively inhibited HCV IRES-mediated translation. This RNA molecule interacted with the ribosomal S5 protein and prevented the recruitment of the 40S ribosomal subunit. This study reveals valuable insights into the role of the SL structures of the HCV IRES in mediating ribosome entry. Finally, these results provide a basis for developing anti-HCV therapy using small RNA molecules mimicking the SL structures of the 5′-UTR to specifically block viral RNA translation.     

Poly(C)-binding protein 2 interacts with sequences required for viral replication in the hepatitis C virus (HCV) 5' untranslated region and directs HCV RNA replication through circularizing the viral genome

Sequences in the 5' untranslated region (5'UTR) of hepatitis C virus (HCV) RNA is important for modulating both translation and RNA replication. The translation of the HCV genome depends on an internal ribosome entry site (IRES) located within the 341-nucleotide 5'UTR, while RNA replication requires a smaller region. A question arises whether the replication and translation functions require different regions of the 5'UTR and different sets of RNA-binding proteins. Here, we showed that the 5'-most 157 nucleotides of HCV RNA is the minimum 5'UTR for RNA replication, and it partially overlaps with the IRES. Stem-loops 1 and 2 of the 5'UTR are essential for RNA replication, whereas stem-loop 1 is not required for translation. We also found that poly(C)-binding protein 2 (PCBP2) bound to the replication region of the 5'UTR and associated with detergent-resistant membrane fractions, which are the sites of the HCV replication complex. The knockdown of PCBP2 by short hairpin RNA decreased the amounts of HCV RNA and nonstructural proteins. Antibody-mediated blocking of PCBP2 reduced HCV RNA replication in vitro, indicating that PCBP2 is directly involved in HCV RNA replication. Furthermore, PCBP2 knockdown reduced IRES-dependent translation preferentially from a dual reporter plasmid, suggesting that PCBP2 also regulated IRES activity. These findings indicate that PCBP2 participates in both HCV RNA replication and translation. Moreover, PCBP2 interacts with HCV 5'- and 3'UTR RNA fragments to form an RNA-protein complex and induces the circularization of HCV RNA, as revealed by electron microscopy. This study thus demonstrates the mechanism of the participation of PCBP2 in HCV translation and replication and provides physical evidence for HCV RNA circularization through 5'- and 3'UTR interaction.     

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.     

Cytoplasmic expression of mRNAs containing the internal ribosome entry site and 3' noncoding region of hepatitis C virus: effects of the 3' leader on mRNA translation and mRNA stability

Translation initiation in many eukaryotic mRNAs is modulated by an interaction between the cap binding protein complex, bound to the 5' end of the mRNA, and the polyadenosine binding protein, bound to the 3'-terminal polyadenosine sequences. A few cellular and viral mRNAs, such as the hepatitis C virus (HCV) mRNA genome, lack 3'-terminal polyadenosine sequences. For such mRNAs, the question of whether their 3'-end sequences also regulate the initiation phase of protein synthesis via an interaction with their 5' ends has received intense scrutiny. For HCV mRNA, various experimental designs have led to conflicting interpretations, that the 3' end of the RNA can modulate translation initiation either in a positive or in a negative fashion. To examine the possibility of end-to-end communication in HCV in detail, mRNAs containing the HCV internal ribosome entry site linked to a luciferase coding region, followed by different 3' noncoding regions, were expressed in the cytoplasm of cultured cells by T7 RNA polymerase. The intracellular translation efficiencies, steady-state levels, stabilities, and 3'-end sequences of these chimeric RNAs were examined. It was found that the HCV 3' noncoding region modulates neither the translation nor the stability of the mRNAs. Thus, there is no detectable end-to-end communication in cytoplasmically expressed chimeric mRNAs containing the HCV noncoding regions. However, it remains an open question whether end-to-end communication occurs in full-length HCV mRNAs in the infected liver.     

IGF2BP1 enhances HCV IRES-mediated translation initiation via the 3′UTR

The positive-strand RNA genome of the Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) in the 5′untranslated region (5′UTR) and structured sequence elements within the 3′UTR, but no poly(A) tail. Employing a limited set of initiation factors, the HCV IRES coordinates the 5′cap-independent assembly of the 43S pre-initiation complex at an internal initiation codon located in the IRES sequence. We have established a Huh7 cell-derived in vitro translation system that shows a 3′UTR-dependent enhancement of 43S pre-initiation complex formation at the HCV IRES. Through the use of tobramycin (Tob)-aptamer affinity chromatography, we identified the Insulin-like growth factor-II mRNA-binding protein 1 (IGF2BP1) as a factor that interacts with both, the HCV 5′UTR and 3′UTR. We report that IGF2BP1 specifically enhances translation at the HCV IRES, but it does not affect 5′cap-dependent translation. RNA interference against IGF2BP1 in HCV replicon RNA-containing Huh7 cells reduces HCV IRES-mediated translation, whereas replication remains unaffected. Interestingly, we found that endogenous IGF2BP1 specifically co-immunoprecipitates with HCV replicon RNA, the ribosomal 40S subunit, and eIF3. Furthermore eIF3 comigrates with IGF2BP1 in 80S ribosomal complexes when a reporter mRNA bearing both the HCV 5′UTR and HCV 3′UTR is translated. Our data suggest that IGF2BP1, by binding to the HCV 5′UTR and/or HCV 3′UTR, recruits eIF3 and enhances HCV IRES-mediated translation.     

RNA-binding protein hnRNP D modulates internal ribosome entry site-dependent translation of hepatitis C virus RNA

Hepatitis C virus (HCV) is one of the major causative agents of virus-related hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. Translation of the HCV polyprotein is mediated by an internal ribosomal entry site (IRES) in the 5′ nontranslated region of the genome. Here, we report that a cellular protein, hnRNP D, interacts with the 5′ border of HCV IRES (stem-loop II) and promotes translation of HCV mRNA. Overexpression of hnRNP D in mammalian cells enhances HCV IRES-dependent translation, whereas knockdown of hnRNP D with small interfering RNAs (siRNAs) inhibits translation. In addition, sequestration of hnRNP D with an interacting DNA oligomer inhibits the translation of HCV mRNA in an in vitro system. Ribosome profiling experiments reveal that HCV RNA is redistributed from heavy to light polysome fractions upon suppression of the hnRNP D level using specific siRNA. These results collectively suggest that hnRNP D plays an important role in the translation of HCV mRNA through interactions with the IRES. Moreover, knockdown of hnRNP D with siRNA significantly hampers infection by HCV. A potential role of hnRNP D in HCV proliferation is discussed.     

The hepatitis C virus 3'-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase

Enhancement of eukaryotic messenger RNA (mRNA) translation initiation by the 3′ poly(A) tail is mediated through interaction of poly(A)-binding protein with eukaryotic initiation factor (eIF) 4G, bridging the 5′ terminal cap structure. In contrast to cellular mRNA, translation of the uncapped, non-polyadenylated hepatitis C virus (HCV) genome occurs independently of eIF4G and a role for 3′-untranslated sequences in modifying HCV gene expression is controversial. Utilizing cell-based and in vitro translation assays, we show that the HCV 3′-untranslated region (UTR) or a 3′ poly(A) tract of sufficient length interchangeably stimulate translation dependent upon the HCV internal ribosomal entry site (IRES). However, in contrast to cap-dependent translation, the rate of initiation at the HCV IRES was unaffected by 3′-untranslated sequences. Analysis of post-initiation events revealed that the 3′ poly(A) tract and HCV 3′-UTR improve translation efficiency by enabling termination and possibly ribosome recycling for successive rounds of translation.     

The sequence element of the internal ribosome entry site and a 25-kilodalton cellular protein contribute to efficient internal initiation of translation of hepatitis C virus RNA.

Translation of hepatitis C virus (HCV) RNA is initiated by internal entry of ribosomes into the 5' noncoding region (NCR). This process depends on genomic elements within the 5' NCR called the internal ribosome entry site (IRES) and may involve host factors. The alpha-branch structure (nucleotides 47 to 67) of the HCV IRES is considered a cis-acting element critical for translation initiation because it is indispensable for translation in vitro (S. Fukushi, K. Katayama, C. Kurihara, N. Ishiyama, F. B. Hoshino, T. Ando, and A. Oya, Biochem. Biophys. Res. Commun. 199:425-432, 1994). In order to further characterize the function of the alpha-branch, we determined whether sequence exchange within the alpha-branch had any effect on translation initiation. An in vitro translation study revealed that the stem sequences of this region played an important role in efficient IRES function. In addition to several HeLa cell proteins, which had a binding affinity for the 5' NCR, a novel 25-kDa protein that specifically interacted with the HCV IRES was discovered. The binding affinity of the 25-kDa protein for the 5' NCR was correlated with the efficiency of translation initiation of HCV RNA, indicating a critical role for the 25-kDa protein in HCV translation.     

Hepatitis C virus 3′UTR regulates viral translation through direct interactions with the host translation machinery

The 3′ untranslated region (3′UTR) of hepatitis C virus (HCV) messenger RNA stimulates viral translation by an undetermined mechanism. We identified a high affinity interaction, conserved among different HCV genotypes, between the HCV 3′UTR and the host ribosome. The 3′UTR interacts with 40S ribosomal subunit proteins residing primarily in a localized region on the 40S solvent-accessible surface near the messenger RNA entry and exit sites. This region partially overlaps with the site where the HCV internal ribosome entry site was found to bind, with the internal ribosome entry site-40S subunit interaction being dominant. Despite its ability to bind to 40S subunits independently, the HCV 3′UTR only stimulates translation in cis, without affecting the first round translation rate. These observations support a model in which the HCV 3′UTR retains ribosome complexes during translation termination to facilitate efficient initiation of subsequent rounds of translation.     

Polypyrimidine tract-binding protein inhibits translation of bip mRNA

Translation initiation of human Bip mRNA is directed by an internal ribosomal entry site (IRES) located in the 5' non-translated region. No trans-acting factor possibly involved in this process has as of yet been identified. For the encephalomyocarditis virus and other picornaviruses, polypyrimidine tract-binding protein (PTB) has been found to enhance the translation through IRES elements, probably by interaction with the IRES structure. Here, we report that PTB specifically binds to the central region (nt 50-117) of the Bip 5' non-translated region. Addition of purified PTB to rabbit reticulocyte lysate and overexpression of PTB in Cos-7 cells selectively inhibited Bip IRES-dependent translation. On the other hand, depletion of endogenous PTB or addition of an RNA interacting with PTB enhanced the translational initiation directed by Bip IRES. These suggest that PTB can either enhance or inhibit IRES-dependent translation depending on mRNAs.     

Interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation.

Initiation of translation of the human hepatitis C virus (HCV) RNA genome occurs by internal ribosome entry into the 5' noncoding region (5'NCR) in a cap-independent manner. The internal ribosome entry site of the HCV 5'NCR has been previously defined to encompass almost the entire 5'NCR. Here we report the interaction of polypyrimidine tract-binding protein (PTB) at three distinct regions within the 5'NCR by UV cross-linking assays. All three regions contain a consensus polypyrimidine tract motif. The evidence for the interaction of recombinant PTB at multiple sites within the 5'NCR is based on the use of 5'NCR mutants as competitors and by direct UV cross-linking of the mutant RNAs. Furthermore, the PTB isomers from HeLa nuclear extracts interact with the HCV 5'NCR, as shown by immunoprecipitation of a UV cross-linked complex with anti-PTB serum. Immunodepletion of PTB from translation lysates suggested the functional requirement for PTB during translation initiation of the HCV RNA. Addition of purified PTB to immunodepleted lysates did not restore translation mediated by the HCV 5'NCR, indicating the requirement of PTB-associated factors that were removed during immunodepletion.     

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.     

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.     

Long-range RNA-RNA interaction between the 5' nontranslated region and the core-coding sequences of hepatitis C virus modulates the IRES-dependent translation

Hepatitis C virus (HCV) is a positive-sense RNA virus approximately 9600 bases long. An internal ribosomal entry site (IRES) spans the 5' nontranslated region, which is the most conserved and highly structured region of the HCV genome. In this study, we demonstrate that nucleotides 428-442 of the HCV core-coding sequence anneal to nucleotides 24-38 of the 5'NTR, and that this RNA-RNA interaction modulates IRES-dependent translation in rabbit reticulocyte lysate and in HepG2 cells. The inclusion of the core-coding sequence (nucleotides 428-442) significantly suppressed the translational efficiency directed by HCV IRES in dicistronic reporter systems, and this suppression was relieved by site-directed mutations that blocked the long-range interaction between nucleotides 24-38 and 428-442. These findings suggest that the long-range interaction between the HCV 5'NTR and the core-coding nucleotide sequence down-regulate cap-independent translation via HCV IRES. The modulation of protein synthesis by long-range RNA-RNA interaction may play a role in the regulation of viral gene expression.     

The 3′-Untranslated Region of Hepatitis C Virus RNA Enhances Translation from an Internal Ribosomal Entry Site

Translation of most eukaryotic mRNAs and many viral RNAs is enhanced by their poly(A) tails. Hepatitis C virus (HCV) contains a positive-stranded RNA genome which does not have a poly(A) tail but has a stretch of 98 nucleotides (X region) at the 3′-untranslated region (UTR), which assumes a highly conserved stem-loop structure. This X region binds a polypyrimidine tract-binding protein (PTB), which also binds to the internal ribosome entry site (IRES) in HCV 5′-UTR. These RNA-protein interactions may regulate its translation. We generated a set of HCV RNAs differing only in their 3′-UTRs and compared their translation efficiencies. HCV RNA containing the X region was translated three- to fivefold more than the corresponding RNAs without this region. Mutations that abolished PTB binding in the X region reduced, but did not completely abolish, enhancement in translation. The X region also enhanced translation from another unrelated IRES (from encephalomyocarditis virus RNA), but did not affect the 5′-end-dependent translation of globin mRNA in either monocistronic or bicistronic RNAs. It did not appear to affect RNA stability. The free X region added in trans, however, did not enhance translation, indicating that the translational enhancement by the X region occurs only in cis. These results demonstrate that the highly conserved 3′ end of HCV RNA provides a novel mechanism for enhancement of HCV translation and may offer a target for antiviral agents.