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.     

Cell type-specific enhancement of hepatitis C virus internal ribosome entry site-directed translation due to 5' nontranslated region substitutions selected during passage of virus in lymphoblastoid cells

Low-level replication of hepatitis C virus (HCV) in cultured lymphoblastoid cells inoculated with H77 serum inoculum led to the appearance of new virus variants containing identical substitutions at three sites within the viral 5' nontranslated RNA (5'NTR): G(107)-->A, C(204)-->A, and G(243)-->A (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325-3329, 1996). These results suggest that virus with this 5'NTR sequence may have a greater capacity for replication in such cells, possibly due to more efficient cap-independent translation, since these nucleotide substitutions reside within the viral internal ribosome entry site (IRES). To test this hypothesis, we examined the translation of dicistronic RNAs containing upstream and downstream reporter sequences (Renilla and firefly luciferases, respectively) separated by IRES sequences containing different combinations of these substitutions. The activity of the IRES was assessed by determining the relative firefly and Renilla luciferase activities expressed in transfected cells. Compared with the IRES present in the dominant H77 quasispecies, an IRES containing all three nucleotide substitutions had significantly greater translational activity in three of five human lymphoblastoid cell lines (Raji, Bjab, and Molt4 but not Jurkat or HPBMa10-2 cells). In contrast, these substitutions did not enhance IRES activity in cell lines derived from monocytes or granulocytes (HL-60, KG-1, or THP-1) or hepatocytes (Huh-7) or in cell-free translation assays carried out with rabbit reticulocyte lysates. Each of the three substitutions was required for maximally increased translational activity in the lymphoblastoid cells. The 2- to 2.5-fold increase in translation observed with the modified IRES sequence may facilitate the replication of HCV, possibly accounting for differences in quasispecies variants recovered from liver tissue and peripheral blood mononuclear cells of the same patient.     

3' nontranslated RNA signals required for replication of hepatitis C virus RNA

We describe a mutational analysis of the 3' nontranslated RNA (3'NTR) signals required for replication of subgenomic hepatitis C virus (HCV) RNAs. A series of deletion mutants was constructed within the background of an HCV-N replicon that induces the expression of secreted alkaline phosphatase in order to examine the requirements for each of the three domains comprising the 3'NTR, namely, the highly conserved 3' terminal 98-nucleotide (nt) segment (3'X), an upstream poly(U)-poly(UC) [poly(U/UC)] tract, and the variable region (VR) located at the 5' end of the 3'NTR. Each of these domains was found to contribute to efficient replication of the viral RNA in transiently transfected hepatoma cells. Replication was not detected when any of the three putative stem-loop structures within the 3'X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. Replacement of a minimum of 50 to 62 nt of poly(U/UC) sequence was required for detectable RNA replication when the native sequence was restored in a stepwise fashion from its 3' end. Lengthier poly(U/UC) sequences, and possibly pure homopolymeric poly(U) tracts, were associated with more efficient RNA amplification. Finally, while multiple deletion mutations were tolerated within VR, each led to a partial loss of replication capacity. The impaired replication capacity of the deletion mutants could not be explained by reduced translational activity or by decreased stability of the RNA, suggesting that each of these mutations may impair recognition of the RNA by the viral replicase during an early step in negative-strand RNA synthesis. The results indicate that the 3'-most 150 nt of the HCV-N genome [the 3'X region and the 3' 52 nt of the poly(U/UC) tract] contain RNA signals that are essential for replication, while the remainder of the 3'NTR plays a facilitating role in replication but is not absolutely required.     

A common RNA structural motif involved in the internal initiation of translation of cellular mRNAs.

The 5'-non-translated regions (5'NTR) of human immunoglobulin heavy chain binding protein (BiP), Antennapedia (Antp) ofDrosophilaand human fibroblast growth factor 2 (FGF-2) mRNAs are reported to mediate translation initiation by an internal ribosome binding mechanism. In this study, we investigate predicted features of the higher order structures folded in these 5'NTR sequences. Statistical analyses of RNA folding detected a 92 nt unusual folding region (UFR) from 129 to 220, close to the initiator AUG in the BiP mRNA. Details of the structural analyses show that the UFR forms a Y-type stem-loop structure with an additional stem-loop in the 3'-end resembling the common structure core found in the internal ribosome entry site (IRES) elements of picornavirus. The Y-type structural motif is also conserved among a number of divergent BiP mRNAs. We also find two RNA elements in the 5'-leader sequence of human FGF-2. The first RNA element (96 nt) is 2 nt upstream of the first CUG start codon located in the reported IRES element of human FGF-2. The second (107 nt) is immediately upstream of the authentic initiator AUG of the main open reading frame. Intriguingly, the folded RNA structural motif in the two RNA elements is conserved in other members of FGF family and shares the same structural features as that found in the 5'NTR of divergent BiP mRNAs. We suggest that the common RNA structural motif conserved in the diverse BiP and FGF-2 mRNAs has a general function in the internal ribosome binding mechanism of cellular mRNAs.     

Stability of a stem-loop involving the initiator AUG controls the efficiency of internal initiation of translation on hepatitis C virus RNA.

The initiation of translation on the positive-sense RNA genome of hepatitis C virus (HCV) is directed by an internal ribosomal entry site (IRES) that occupies most of the 341-nt 5' nontranslated RNA (5'NTR). Previous studies indicate that this IRES differs from picornaviral IRESs in that its activity is dependent upon RNA sequence downstream of the initiator AUG. Here, we demonstrate that the initiator AUG of HCV is located within a stem-loop (stem-loop IV) involving nt -12 to +12 (with reference to the AUG). This structure is conserved among HCV strains, and is present in the 5'NTR of the phylogenetically distant GB virus B. Mutant, nearly genome-length RNAs containing nucleotide substitutions predicted to enhance the stability of stem-loop IV were generally deficient in cap-independent translation both in vitro and in vivo. Additional mutations that destabilize the stem-loop restored translation to normal. Thus, the stability of the stem-loop is strongly but inversely correlated with the efficiency of internal initiation of translation. In contrast, mutations that stabilize this stem-loop had comparatively little effect on translation of 5' truncated RNAs by scanning ribosomes, suggesting that internal initiation of translation follows binding of the 40S ribosome directly at the site of stem-loop IV. Because stem-loop IV is not required for internal entry of ribosomes but is able to regulate this process, we speculate that it may be stabilized by interactions with a viral protein, providing a mechanism for feedback regulation of translation, which may be important for viral persistence.     

A segment of the 5'' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation.

Picornavirus RNAs are uncapped messengers and have unusually long 5' nontranslated regions (5'NTRs) which contain many noninitiating AUG triplets. The translational efficiency of different picornavirus RNAs varies between different cell-free extracts and even in the same extract, such as micrococcal nuclease-treated rabbit reticulocyte lysates. The effect of the poliovirus 5'NTR on in vitro translation was compared with that of the 5'NTR of encephalomyocarditis virus by the use of synthetic mRNAs, micrococcal nuclease-treated HeLa cell extracts, and rabbit reticulocyte lysates. Artificial mono- and dicistronic mRNAs synthesized with T7 RNA polymerase were used to investigate whether the 5'NTR of encephalomyocarditis virus RNA contains a potential internal ribosomal entry site. The sequence between nucleotides 260 and 484 in the 5'NTR of encephalomyocarditis RNA was found to play a critical role in the efficient translation in both mono- and dicistronic mRNAs. Our data suggest that an internal ribosomal entry site resides in this region.     

HIV-2 genomic RNA contains a novel type of IRES located downstream of its initiation codon

Eukaryotic translation initiation begins with assembly of a 48S ribosomal complex at the 5' cap structure or at an internal ribosomal entry segment (IRES). In both cases, ribosomal positioning at the AUG codon requires a 5' untranslated region upstream from the initiation site. Here, we report that translation of the genomic RNA of human immunodeficiency virus type 2 takes place by attachment of the 48S ribosomal preinitiation complex to the coding region, with no need for an upstream 5' untranslated RNA sequence. This unusual mechanism is mediated by an RNA sequence that has features of an IRES with the unique ability to recruit ribosomes upstream from its core domain. A combination of translation assays and structural studies reveal that sequences located 50 nucleotides downstream of the AUG codon are crucial for IRES activity.     

Domains I and II in the 5' nontranslated region of the HCV genome are required for RNA replication.

Hepatitis C virus (HCV), a hepacivirus member of the Flaviviridae family, has a positive-stranded RNA genome, which consists of a single open reading frame (ORF) and nontranslated regions (NTRs) at the 5' and 3' ends. The 5'NTR was found to contain an internal ribosomal entry site (IRES), which is required for the translation of HCV mRNA. Moreover, the 5'NTR is likely to play a key role in the replication of viral RNA. To identify the cis-acting element required for viral RNA replication, chimeric subgenomic replicons of HCV were generated. Dissection of the replication element from the translation element was accomplished by inserting the polioviral IRES between the serially deleted 5'NTR of HCV and ORF encoding neomycin phosphotransferase. The deletions of the 5'NTR of HCV were performed according to the secondary structure of HCV. Replicons containing domains I and II supported RNA replication and further deletion toward the 5' end abolished replication. The addition of domain III and the pseudoknot structure of the 5'NTR to domains I and II augmented the colony-forming efficiency of replicons by 100-fold. This indicates that domains I and II are necessary and sufficient for replication of RNA and that almost all of the 5'NTR is required for efficient RNA replication.     

Synergism of the 3'-untranslated region and an internal ribosome entry site differentially enhances the translation of a plant virus coat protein

The use of internal ribosome entry sites (IRESs) is one of the unorthodox mechanisms exploited by viruses to initiate the translation of internal genes. Herein, we report a plant virus exploiting an IRES and its 3'-untranslated region (UTR) to express its internal genes, notably the 3'-proximal viral coat protein gene. Hibiscus chlorotic ringspot virus (HCRSV), a positive-strand non-polyadenylated RNA virus, was demonstrated to harbor a unique 100-nucleotide (nt) IRES, located 124 nt upstream of the coat protein gene, that could function in wheat germ extract, rabbit reticulocyte lysate, and mammalian cells. In comparison with other known IRESs of picornaviruses and eukaryotic mRNAs, this 100-nt IRES is distinctively short and simple. The IRES activity was tested in homologous and heterologous bicistronic constructs, and the expression of the 3'-proximal gene was enhanced when the 3'-UTR was present. When the IRES element was bisected, each half still possessed IRES activity and could initiate internal translation on its own. Site-directed mutagenesis and deletion analyses revealed that the primary sequence within the 5' half was crucial for IRES activity, whereas the primary sequence of the second half and a GNRA motif were non-essential. To our knowledge, this is the first report describing a mechanism whereby an IRES, located in the 3' portion of the virus genome, co-operates with the 3'-UTR to enhance gene expression differentially.     

Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

Sequences in the 5' and 3' termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5' end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5' NTR required for RNA replication. We show that deletions introduced into the 5' terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5' NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5' NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5' NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5' NTR of HCV RNA essential for replication of this virus.     

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

The hepatitis C virus (HCV) internal ribosome entry site (IRES) is a highly structured RNA element that directs cap-independent translation of the viral polyprotein. Morpholino antisense oligonucleotides directed towards stem loop IIId drastically reduced HCV IRES activity. Mutagenesis studies of this region showed that the GGG triplet (nucleotides 266 through 268) of the hexanucleotide apical loop of stem loop IIId is essential for IRES activity both in vitro and in vivo. Sequence comparison showed that apical loop nucleotides (UUGGGU) were absolutely conserved across HCV genotypes and the GGG triplet was strongly conserved among related Flavivirus and Pestivirus nontranslated regions. Chimeric IRES elements with IIId derived from GB virus B (GBV-B) in the context of the HCV IRES possess translational activity. Mutations within the IIId stem loop that abolish IRES activity also affect the RNA structure in RNase T(1)-probing studies, demonstrating the importance of correct RNA folding to IRES function.     

Replication of hepatitis A viruses with chimeric 5' nontranslated regions.

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.     

A Phylogenetically Conserved Stem-Loop Structure at the 5′ Border of the Internal Ribosome Entry Site of Hepatitis C Virus Is Required for Cap-Independent Viral Translation

Hepatitis C virus (HCV) initiates translation of its polyprotein under the control of an internal ribosome entry site (IRES) that comprises most of the 341-nucleotide (nt) 5′ nontranslated RNA (5′NTR). A comparative analysis of related flaviviral sequences suggested that an RNA segment for which secondary structure was previously ill defined (domain II, nt 44 to 118) forms a conserved stem-loop that is located at the 5′ border of the HCV IRES and thus may function in viral translation. This prediction was tested by a mutational analysis of putative helical structures that examined the impact of both covariant and noncovariant nucleotide substitutions on IRES activity in vivo and in vitro. Results of these experiments provide support for predicted base pair interactions between nt 44 to 52 and 111 to 118 and between nt 65 to 70 and 97 to 102 of the HCV 5′NTR. Substitutions at either nt 45 and 46 or nt 116 and 117 resulted in reciprocal changes in V1 nuclease cleavage patterns within the opposing strand of the putative helix, consistent with the predicted base pair interactions. IRES activity was highly dependent on maintenance of the stem-loop II structure but relatively tolerant of covariant nucleotide substitutions within predicted helical segments. Sequence alignments suggested that the deduced domain II structure is conserved within the IRESs of pestiviruses as well as the novel flavivirus GB virus B. Despite marked differences in primary nucleotide sequence within conserved helical segments, the sequences of the intervening single-stranded loop segments are highly conserved in these different viruses. This suggests that these segments of the viral RNA may interact with elements of the host translational machinery that are broadly conserved among different mammalian species.     

The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated

A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt), both of which include the common 3' exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.