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).     

Positioning of subdomain IIId and apical loop of domain II of the hepatitis C IRES on the human 40S ribosome

The 5′-untranslated region of the hepatitis C virus (HCV) RNA contains a highly structured motif called IRES (Internal Ribosome Entry Site) responsible for the cap-independent initiation of the viral RNA translation. At first, the IRES binds to the 40S subunit without any initiation factors so that the initiation AUG codon falls into the P site. Here using an original site-directed cross-linking strategy, we identified 40S subunit components neighboring subdomain IIId, which is critical for HCV IRES binding to the subunit, and apical loop of domain II, which was suggested to contact the 40S subunit from data on cryo-electron microscopy of ribosomal complexes containing the HCV IRES. HCV IRES derivatives that bear a photoactivatable group at nucleotide A275 or at G263 in subdomain IIId cross-link to ribosomal proteins S3a, S14 and S16, and HCV IRES derivatized at the C83 in the apex of domain II cross-link to proteins S14 and S16.     

Crystal structure of an RNA tertiary domain essential to HCV IRES-mediated translation initiation

The hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA drives internal initiation of viral protein synthesis during host cell infection. In the tertiary structure of the IRES RNA, two helical junctions create recognition sites for direct binding of the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). The 2.8 A resolution structure of the IIIabc four-way junction, which is critical for binding eIF3, reveals how junction nucleotides interact with an adjacent helix to position regions directly involved in eIF3 recognition. Two of the emergent helices stack to form a nearly continuous A-form duplex, while stacking of the other two helices is interrupted by the insertion of junction residues into the helix minor groove. This distorted stack probably serves as an important recognition surface for the translational machinery.     

eIF2A mediates translation of hepatitis C viral mRNA under stress conditions

Translation of most mRNAs is suppressed under stress conditions. Phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF2), which delivers initiator tRNA (Met-tRNA(i)) to the P site of the 40S ribosomal subunit, is responsible for such translational suppression. However, translation of hepatitis C viral (HCV) mRNA is refractory to the inhibitory effects of eIF2α phosphorylation, which prevents translation by disrupting formation of the eIF2-GTP-Met-tRNA(i) ternary complex. Here, we report that eIF2A, an alternative initiator tRNA-binding protein, has a key role in the translation of HCV mRNA during HCV infection, in turn promoting eIF2α phosphorylation by activating the eIF2α kinase PKR. Direct interaction of eIF2A with the IIId domain of the HCV internal ribosome entry site (IRES) is required for eIF2A-dependent translation. These data indicate that stress-independent translation of HCV mRNA occurs by recruitment of eIF2A to the HCV IRES via direct interaction with the IIId domain and subsequent loading of Met-tRNA(i) to the P site of the 40S ribosomal subunit.     

Antisense oligonucleotides targeted to the domain IIId of the hepatitis C virus IRES compete with 40S ribosomal subunit binding and prevent in vitro translation

Initiation of protein synthesis on the hepatitis C virus (HCV) mRNA involves a structured element corresponding to the 5′ untranslated region and constituting an internal ribosome entry site (IRES). The domain IIId of the HCV IRES, an imperfect RNA hairpin extending from nucleotides 253 to 279 of the viral mRNA, has been shown to be essential for translation and for the binding of the 40S ribosomal subunit. We investigated the properties of a series of antisense 2′-O-methyloligoribonucleotides targeted to various portions of the domain IIId. Several oligomers, 14–17 nt in length, selectively inhibited in vitro translation of a bicistronic RNA construct in rabbit reticulocyte lysate with IC₅₀s <10 nM. The effect was restricted to the second cistron (the Renilla luciferase) located downstream of the HCV IRES; no effect was observed on the expression of the first cistron (the firefly luciferase) which was translated in a cap-dependent manner. Moreover, antisense 2′-O-methyloligoribonucleotides specifically competed with the 40S ribosomal subunit for binding to the IRES RNA in a filter- retention assay. The antisense efficiency of the oligonucleotides was nicely correlated to their affinity for the IIId subdomain and to their ability to displace 40S ribosomal subunit, making this process a likely explanation for in vitro inhibition of HCV-IRES-dependent translation.     

Ribosomal protein S5 interacts with the internal ribosomal entry site of hepatitis C virus

Translational initiation of hepatitis C virus (HCV) genome RNA occurs via its highly structured 5' noncoding region called the internal ribosome entry site (IRES). Recent studies indicate that HCV IRES and 40 S ribosomal subunit form a stable binary complex that is believed to be important for the subsequent assembly of the 48 S initiation complex. Ribosomal protein (rp) S9 has been suggested as the prime candidate protein for binding of the HCV IRES to the 40 S subunit. RpS9 has a molecular mass of approximately 25 kDa in UV cross-linking experiments. In the present study, we examined the approximately 25-kDa proteins of the 40 S ribosome that form complexes with the HCV IRES upon UV cross-linking. Immunoprecipitation with specific antibodies against two 25-kDa 40 S proteins, rpS5 and rpS9, clearly identified rpS5 as the protein bound to the IRES. Thus, our results support rpS5 as the critical element in positioning the HCV RNA on the 40 S ribosomal subunit during translation initiation.     

HCV IRES interacts with the 18S rRNA to activate the 40S ribosome for subsequent steps of translation initiation

Previous analyses of complexes of 40S ribosomal subunits with the hepatitis C virus (HCV) internal ribosome entry site (IRES) have revealed contacts made by the IRES with ribosomal proteins. Here, using chemical probing, we show that the HCV IRES also contacts the backbone and bases of the CCC triplet in the 18S ribosomal RNA (rRNA) expansion segment 7. These contacts presumably provide interplay between IRES domain II and the AUG codon close to ribosomal protein S5, which causes a rearrangement of 18S rRNA structure in the vicinity of the universally conserved nucleotide G1639. As a result, G1639 becomes exposed and the corresponding site of the 40S subunit implicated in transfer RNA discrimination can select . These data are the first demonstration at nucleotide resolution of direct IRES–rRNA interactions and how they induce conformational transition in the 40S subunit allowing the HCV IRES to function without AUG recognition initiation factors.     

The pathway of HCV IRES-mediated translation initiation

The HCV internal ribosome entry site (IRES) directly regulates the assembly of translation initiation complexes on viral mRNA by a sequential pathway that is distinct from canonical eukaryotic initiation. The HCV IRES can form a binary complex with an eIF-free 40S ribosomal subunit. Next, a 48S-like complex assembles at the AUG initiation codon upon association of eIF3 and ternary complex. 80S complex formation is rate limiting and follows the GTP-dependent association of the 60S subunit. Efficient assembly of the 48S-like and 80S complexes on the IRES mRNA is dependent upon maintenance of the highly conserved HCV IRES structure. This revised model of HCV IRES translation initiation provides a context to understand the function of different HCV IRES domains during translation initiation.     

Structure of HCV IRES domain II determined by NMR

Complex RNA structures regulate many biological processes, but are often too large for structure determination by NMR methods. The 5' untranslated region (5' UTR) of the hepatitis C viral (HCV) RNA genome contains an internal ribosome entry site (IRES) that binds to 40S ribosomal subunits with high affinity and specificity to control translation. Domain II of the HCV IRES forms a 25-kDa folded subdomain that may alter ribosome conformation. We report here the structure of domain II as determined using an NMR approach that combines short- and long-range structural data. Domain II adopts a distorted L-shape structure, and its overall shape in the free form is markedly similar to its 40S subunit-bound form; this suggests how domain II may modulate 40S subunit conformation. The results show how NMR can be used for structural analysis of large biological RNAs.     

Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site

The hepatitis C viral mRNA initiates translation using an internal ribosome entry site (IRES) located in the 5' noncoding region of the viral genome. At physiological magnesium ion concentrations, the HCV IRES forms a binary complex with the 40S ribosomal subunit, recruits initiation factor eIF3 and the ternary eIF2/GTP/Met-tRNA(i)Met complex, and joins 60S subunits to assemble translation-competent 80S ribosomes. Here we show that in the presence of 5 mM MgCl2, the HCV IRES can initiate translation by an alternative mechanism that does not require known initiation factors. Specifically, the HCV IRES was shown to initiate translation in a reconstituted system consisting only of purified 40S and 60S subunits, elongation factors, and aminoacylated tRNAs at high magnesium concentration. Analyses of assembled complexes supported a mechanism by which preformed 80S ribosomes can assemble directly on the HCV IRES at high cation concentrations. This mechanism is reminiscent of that employed by the divergent IRES elements in the Dicistroviridae, exemplified by the cricket paralysis virus, which mediates initiation of protein synthesis without initiator tRNA.     

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem–loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit''s decoding groove. This configuration depends on the sequence and structure of a different stem–loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.     

Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S ribosomal subunit

The molecular environment of the internal ribosome entry site (IRES element) of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group at nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the subunit between the head and the body aside the “beak” near the exit from the mRNA-binding channel.     

Domains on the hepatitis C virus internal ribosome entry site for 40s subunit binding

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA is known to interact with the 40S ribosomal subunit alone, in the absence of any additional initiation factors or Met-tRNAi. Previous work from this laboratory on the 80S and 48S ribosomal initiation complexes involving the HCV IRES showed that stem-loop III, the pseudoknot domain, and some coding sequence were protected from pancreatic RNase digestion. Stem-loop II is never protected by these complexes. Furthermore, there is no prior evidence reported showing extensive direct binding of stem-loop II to ribosomes or subunits. Using direct analysis of RNase-protected HCV IRES domains bound to 40S ribosomal subunits, we have determined that stem-loops II and III and the pseudoknot of the HCV IRES are involved in this initial binding step. The start AUG codon is only minimally protected. The HCV-40S subunit binary complex thus involves recognition and binding of stem-loop II, revealing its role in the first step of a multistep initiation process that may also involve rearrangement of the bound IRES RNA as it progresses.     

Proteins surrounding hairpin IIIe of the hepatitis C virus internal ribosome entry site on the human 40S ribosomal subunit

Binding of the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA to the eIF-free 40S ribosomal subunit is the first step of initiation of translation of the viral RNA. Hairpins IIId and IIIe comprising 253–302 nt of the IRES are known to be essential for binding to the 40S subunit. Here we have examined the molecular environment of the HCV IRES in its binary complex with the human 40S ribosomal subunit. For this purpose, two RNA derivatives were used that bore a photoactivatable perfluorophenyl azide cross-linker. In one derivative the cross-linker was at the nucleotide A296 in hairpin IIIe, and in the other at G87 in domain II. Site-specific introduction of the cross-linker was performed using alkylating derivatives of oligodeoxyribonucleotides complementary to the target RNA sequences. No cross-links with the rRNA were detected with either RNA derivative. The RNA with the photoactivatable group at A296 cross-linked to proteins identified as S5 and S16 (major) and p40 and S3a (minor), while no cross-links with proteins were detected with RNA modified at G87. The results obtained indicate that hairpin IIIe is located on the solvent side of the 40S subunit head on a site opposite the beak.     

Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S ribosomal subunit

The molecular environment of the internal ribosome entry site (IRES) element of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 was crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group in nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the outer subunit surface between the head and the body next to the "beak" near the entrance into the mRNA-binding channel. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.     

Binding of the IRES of hepatitis C virus RNA to the 40S ribosomal subunit: Role of p40

Ribosomal protein p40 is a structural component of the eukaryotic 40S ribosomal subunit, is partly homologous to prokaryotic ribosomal protein S2, and has a long eukaryote-specific C-terminal region. The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA was tested for the binding to 40S ribosomal subunits deficient in p40, saturated with recombinant p40, or pretreated with monoclonal antibody (MAB) 4F6 against p40. The apparent association constant of HCV IRES binding to 40S subunits was shown to directly depend on the p40 content in the subunits. MAB 4F6 prevented HCV IRES binding to 40S subunits and blocked translation of IRES-containing RNA in a cell-free translation system. The results implicate p40 in the binding of the HCV IRES to the ribosome and, therefore, in translation initiation on HCV RNA.     

Ribosomal proteins mediate the hepatitis C virus IRES-HeLa 40S interaction

Translation of the hepatitis C virus genomic RNA is mediated by an internal ribosome entry site (IRES). The 330-nt IRES RNA forms a binary complex with the small 40S ribosomal subunit as a first step in translation initiation. Here chemical probing and 4-thiouridine-mediated crosslinking are used to characterize the interaction of the HCV IRES with the HeLa 40S subunit. No IRES-18S rRNA contacts were detected, but several specific crosslinks to 40S ribosomal proteins were observed. The identity of the crosslinked proteins agrees well with available structural information and provides new insights into HCV IRES function. The protein-rich surface of the 40S subunit thus mediates the IRES-ribosome interaction.     

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.     

Conserved functional domains and a novel tertiary interaction near the pseudoknot drive translational activity of hepatitis C virus and hepatitis C virus-like internal ribosome entry sites

The translational activity of the hepatitis C virus (HCV) internal ribosome entry site (IRES) and other HCV-like IRES RNAs depends on structured RNA elements in domains II and III, which serve to recruit the ribosomal 40S subunit, eukaryotic initiation factor (eIF) 3 and the ternary eIF2/Met-tRNA_i^Met/GTP complex and subsequently domain II assists subunit joining. Porcine teschovirus-1 talfan (PTV-1) is a member of the Picornaviridae family, with a predicted HCV-like secondary structure, but only stem-loops IIId and IIIe in the 40S-binding domain display significant sequence conservation with the HCV IRES. Here, we use chemical probing to show that interaction sites with the 40S subunit and eIF3 are conserved between HCV and HCV-like IRESs. In addition, we reveal the functional role of a strictly conserved co-variation between a purine–purine mismatch near the pseudoknot (A–A/G) and the loop sequence of domain IIIe (GAU/CA). These nucleotides are involved in a tertiary interaction, which serves to stabilize the pseudoknot structure and correlates with translational efficiency in both the PTV-1 and HCV IRES. Our data demonstrate conservation of functional domains in HCV and HCV-like IRESs including a more complex structure surrounding the pseudoknot than previously assumed.