INTRODUCTION

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Hepatitis C virus (HCV) is a distinct member of the family Flaviviridae, comprising a group of enveloped viruses to which the flaviviruses like Yellow fever virus and the pestiviruses Border disease virus, Classical swine fever virus (CSFV), and Bovine viral diarrhea virus (BVDV) belong (33). In the majority of cases, HCV causes a persistent infection that is frequently asymptomatic or associated with only mild symptoms. However, persistently infected patients are at high risk to develop a chronic liver disease that can lead to liver cirrhosis and eventually hepatocellular carcinoma (39). In the absence of efficient therapies, chronic hepatitis C has become the main indication for liver transplantation.

HCV possesses a single-stranded RNA genome of positive polarity. This plus-strand RNA carries a single long open reading frame that encodes a polyprotein of ~3,010 amino acids. Mature viral proteins are generated by a series of proteolytic cleavages that are mediated by host cell signal peptidases and two viral proteinases (for a review, see reference 4). The structural proteins core, envelope protein 1 (E1), and E2 are located in the amino-terminal one-third of the polyprotein, and the nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B are located in the remainder. Of the latter, NS3 to NS5B are required and sufficient for RNA replication (28). NS3 possesses proteinase as well as nucleoside triphosphatase (NTPase) and helicase activities and NS5B is the RNA dependent RNA polymerase (RdRp) (3, 6, 17, 23, 26, 49). The function of the hydrophobic polypeptide NS4B is unknown. NS5A may be involved in the resistance to the antiviral activity of alpha interferon, and it is probably required for some steps in RNA replication (8, 15, 16, 24). This process most likely is mediated by a replicase complex that is tightly associated with intracellular membranes (38). Replication proceeds via a minus-strand RNA intermediate that serves as the template for the production of excess copies of plus-strand RNA molecules (28).

Translation of the HCV polyprotein is mediated via the 5' nontranslated region (NTR) that carries an internal ribosome entry site (IRES) (50, 52). Such RNA elements have first been discovered with the poliovirus (PV), the prototype member of the Picornaviridae (36). Analogous to HCV, PV encodes a single polyprotein that is translated in a cap-independent manner via a highly structured 5' NTR. Genetic studies indicated that the cloverleaf-like structure located at the extreme 5' end of the PV genome is not required for translationbut has a modulatory effect (48)and that it acts as an independent RNA element that is sufficient for replication (1, 2, 45). However, several reports suggest that sequences within the PV IRES are required for RNA multiplication, too (9, 21, 47).

Numerous studies have convincingly demonstrated the existence of an IRES in the HCV 5' NTR (reviewed in reference 44). Unlike the case of PV, this RNA element permits the direct binding of the 40S ribosome subunit in the absence of additional translation factors in a way that the initiator AUG of the long open reading frame is directly positioned in the P-site of the ribosome (37). Moreover, the secondary, and probably also the tertiary, structures of the HCV and PV IRESs as well as their activities in cell extracts of various sources are very different (30, 31). Based on these and several other differences, the IRESs of PV and HCV were classified as type 1 and type 3 elements, respectively (25).

Computer predictions and structure probing revealed four distinct RNA domains in the HCV 5' NTR (Fig. 1) (44). The short stem-loop 1 is formed by residues 5 to 20 and it is not required for IRES activity. In fact, its deletion was found to enhance RNA translation, suggesting a regulatory role (20, 22, 42, 43). The importance of stem-loop 2 for IRES function is discussed controversially, but most studies describe an enhanced translation when this structure is present (14, 20, 40, 42). Stem-loop 3 represents the core of the IRES. It has the highest degree of structural conservation and it participates in the formation of an RNA pseudoknot that is important for IRES activity (51). The smaller stem-loop 4 that harbors the initiator AUG codon is not essential for translation of the RNA. In fact, the stability of this stem-loop inversely correlates with translation efficiency (19).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   A schematic presentation of the basic replicon construct used in this study is at the top. The 5' NTR indicated with a thick line is positioned upstream of either the gene encoding the neomycin phosphotransferase (neo) or the luciferase (luc) of the firefly Photinus pyralis. The EMCV IRES (EI) directs translation of the NS3 to NS5B region that is flanked at the 3' end by the 3' NTR (thick line). *, positions of cell culture-adaptive mutations. A schematic presentation of the secondary structure of the HCV 5' NTR according to a previous study (18) is drawn below. Framed numbers refer to stem-loops 1 to 4. The borders of the HCV IRES are indicated with dotted lines. Numbers and arrows refer to the 3' boundaries of the HCV sequences of 5' NTR chimeras. , initiator AUG codon of the HCV polyprotein with the A residue at nucleotide position 342 located in stem-loop 4.

The 5' border of the HCV IRES was mapped between residues 38 and 46, i.e., just upstream of stem-loop 2 (18, 41, 52). Interestingly, in most studies, the 3' border was mapped to within the core coding region, although it is not clear whether this RNA sequence participates in IRES structure or serves as a spacer to prevent unfavorable base pairings between the IRES and downstream sequences (discussed in reference 44). Core protein is not required for IRES function (53).

While the role of the 5' NTR for translation has been studied in detail, its importance for RNA replication so far was not addressed. This was due to the lack of an efficient cell culture system. We have recently developed subgenomic HCV RNAs (replicons) that amplify to high levels in the human hepatoma cell line Huh-7 (28). These RNAs are composed of the following elements: the HCV 5' NTR up to the 3' end of the IRES that directs translation of the gene encoding the neomycin phosphotransferase (NPT), the IRES of the encephalomyocarditis virus (EMCV) directing translation of the NS3-5B region, and the authentic HCV 3' NTR (Fig. 1). Upon transfection of Huh-7 cells with these bicistronic RNAs and selection with G418, cell lines were established that carried high amounts of self-replicating HCV RNAs. Moreover, we and others have recently identified cell culture adaptive mutations that increase RNA replication to a level that is sufficient for detection in transient assays (8, 24, 27). The most efficient replicon we developed contains two mutations in NS3 and one in NS5A that increase RNA replication synergistically. Upon replacement of the neo sequence by the gene encoding the firefly luciferase, replication of this cell culture-adapted RNA can be monitored in a transient assay by measurements of the activity of the reporter gene (24).

In this study, we analyzed the sequences at the 5' end of the HCV genome required for RNA replication. By using the replicon cell culture system, we show that the 5' terminal 125 nucleotides of the HCV genome are sufficient for RNA replication, albeit at a low level that is strongly enhanced when the complete 5' NTR is included. These data suggest that the signals required for RNA replication overlap with those necessary for translation.

	MATERIALS AND METHODS

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Cell cultures. Cell monolayers of the human hepatoma cell line Huh-7 (35) were routinely grown in Dulbecco's modified mininal essential medium (Life Technologies, Karlsruhe, Germany) supplemented with 2 mM L-glutamine, nonessential amino acids, 100 U of penicillin, 100 µg of streptomycin, and 10% fetal calf serum.

Plasmid constructions and sequence analysis. Standard recombinant DNA technologies were used for all constructions (46). The basic plasmids pFK-I₃₈₉neo/NS3-3'/5.1 and pFK-I₃₈₉luc/NS3-3'/5.1, carrying two cell culture adaptive mutations in NS3 and one in NS5A, have been described recently (24). Deletions were introduced into the 5' NTR by PCR-based mutagenesis using primers S 24-40-Hind or S 5-20-Hind and A neo-3'Pme (Table 1). PCR fragments were restricted with HindIII and AscI and inserted together with an AscI-XhoI fragment derived from the parental neo or luc replicon construct into the HindIII- and XhoI-restricted vector carrying the luc or neo replicon sequence. For the construction of RNAs with an HCV-PV chimeric 5' NTR, plasmid pFKnt1-PI-lucEI3420-9605/5.1 was generated carrying the following elements (in the 5'-to-3' direction): an SbfI restriction site, the T7 RNA polymerase promoter, a PmeI restriction site, the PV IRES from nucleotide 108 to 746 of the PV genome, the luciferase gene, the EMCV IRES, and the HCV sequence from NS3 up to the 3' end of the genome. This plasmid was obtained by insertion of the hybridized oligonucleotides S T7 nt1-Hind-Eco and A T7 nt1-Hind-Eco into pFK-I₃₈₉luc/NS3-3'/5.1 after restriction with EcoRI and HindIII. The resulting construct was linearized with PmeI and SfiI and ligated with a PmeI-SfiI fragment that carried the PV IRES fused to the luciferase gene (V. Lohmann and R. Bartenschlager, unpublished data) and an SfiI fragment corresponding to nucleotides 3622 to 8499 of the HCV genome. Constructs carrying 5, 12, or 24 nucleotides of the HCV 5' NTR downstream of the T7 promoter were obtained by insertion of the complementary sense and antisense oligonucleotides listed in Table 1 via the SbfI and PmeI restriction sites. To obtain plasmids that were used for synthesis of replicons carrying longer HCV sequences at their 5' ends, PCR fragments were generated by using the sense primer S T7 nt1-Sbf and either of the following antisense oligonucleotides: A23-43-Pme, A61-84-Pme, A102-125-Pme, A271-296-Pme, and A310-341-Pme (Table 1). After restriction with SbfI and PmeI, the fragments were inserted into pFKnt1-PI-lucEI3420-9605/5.1, resulting in plasmids that allowed synthesis of replicons with 43, 84, 125, 296, or 341 nucleotides of the HCV 5' NTR upstream of the PV IRES. The 55-nucleotide-long spacer element was obtained by hybridization of the oligonucleotides S60-sp-Pme and A60-sp-Pme and treatment with DNA polymerase in the presence of high NTP concentrations. The double-stranded DNA fragment was restricted with PmeI and inserted into the various luc replicon constructs described above. This construction resulted in the insertion of a total of 63 nucleotides between the 5' NTR sequences of HCV and the PV IRES. Plasmids carrying the neo replicons and the chimeric 5' NTRs were obtained by insertion of a PmeI-ApaI fragment isolated from pFKnt341-sp-PI-lucEI3420-9605/5.1 and an ApaI/NotI fragment isolated from pBSK-PI-neo (Lohmann and Bartenschlager, unpublished) into each of the luc replicon constructs described above. Constructs with deletions in stem-loop 2 of HCV were generated by PCR using primers S T7nt1-SbfI and A12561-104-Pme or A12575-91-Pme. After restriction of the purified fragments with SbfI and PmeI, they were inserted into pFKnt341-sp-PI-luc-EI3420-9605/5.1 or pFKnt341-sp-PI-neo-EI3420-9605/5.1.

In vitro transcription, electroporation, and selection of G418-resistant cell lines. These methods have been described in detail elsewhere (27). In brief, plasmid DNA was restricted with AseI and ScaI (New England Biolabs, Bad Schwalbach/Taunus, Germany) and after purification by phenol extraction and ethanol precipitation was dissolved in RNase-free water. For an in vitro transcription reaction, 5 µg of restricted plasmid DNA was added to a buffer containing 80 mM HEPES, pH 7.5, 12 mM MgCl₂, 2 mM spermidine, 40 mM dithiothreitol (DTT), 3.125 mM of each NTP, 50 U of RNasin (Promega, Mannheim, Germany), and 30 U of T7 RNA polymerase (Promega) in a total volume of 50 µl. After 2 h at 37°C, 15 U of T7 RNA polymerase was added, and the reaction mixture was incubated for another 2 h. Transcription was terminated by the addition of 6 U of RNase-free DNase (Promega) and 30 min of incubation at 37°C. DNA was extracted with acidic phenol and chloroform, precipitated with isopropanol, and dissolved in RNase-free water. The RNA concentration was determined by measurement of the optical density at 260 nm, and the integrity was checked by denaturing agarose gel electrophoresis. For the electroporation of selectable replicons, 0.3 to 100 ng of in vitro transcript adjusted with total RNA from naïve Huh-7 cells to a final amount of about 9 µg was mixed with 400 µl of a suspension of 10⁷ Huh-7 cells per ml in a cuvette with a gap width of 0.4 cm (Bio-Rad, Munich, Germany). After one pulse at 960 µF and 270 V with a Gene pulser system (Bio-Rad), cells were immediately transferred to 8 ml of complete Dulbecco minimal essential medium (DMEM) and seeded in a 10-cm-diameter culture dish. After 24 h, medium was replaced by complete DMEM supplemented with 500 µg of G418 (Geneticin; Life Technologies)/ml. Three to 4 weeks later, colonies were stained with Coomassie brilliant blue (0.6 g/liter in 50% methanol, 10% acetic acid). To determine the efficiency of colony formation of a given construct, serial dilutions of in vitro transcripts were transfected in parallel. Representative results of multiple independent transfections are shown.

Transient-replication assays with luciferase replicons. Huh-7 cells were transfected by electroporation as described above using 5 µg of a replicon carrying the firefly luciferase gene. After addition of 9 ml of complete DMEM, aliquots of the cell suspension were seeded in 3-cm-diameter culture dishes and harvested at various time points. To determine the luciferase activity, cells were washed three times with phosphate-buffered saline, scraped off the plate into 350 µl of ice-cold lysis buffer (1% Triton X-100, 25 mM glycylglycine, 15 mM MgSO₄, 4 mM EGTA, 1 mM DTT). Then, 100 µl of lysate was mixed with 360 µl of assay buffer (25 mM glycylglycine, 15 mM MgSO₄, 4 mM EGTA, 1 mM DTT, 2 mM ATP, 15 mM K₂PO₄, pH 7.8) and, after addition of 200 µl of a 200 µM luciferin stock solution, measured in a luminometer (Lumat LB9507 from Berthold, Freiburg, Germany) for 20 s. Values obtained with cells harvested 4 h after electroporation were used to correct for the transfection efficiency.

In vitro translation. Extracts from HeLa cells were prepared as described elsewhere (32). The preparation of Huh-7 cell extracts was performed in the same way, with minor modifications. Prior to harvest, cells were washed twice with a buffer containing 140 mM NaCl, 5 mM KCl, 10 mM HEPES (pH 7.4), and 1 g of glucose/liter, detached from the culture dish by treatment with trypsin, and washed three times. All further steps were as described for HeLa cell extracts (32). Rabbit reticulocyte lysates were purchased from Promega. For in vitro translation in HeLa cell extracts, 4.5 µl in vitro-transcribed RNA (corresponding to 0.5 µg) was mixed with 8 µl of master mix (75 µl of HeLa cell extract, 11 µl of 2 M KAc, 1.5 µl of 50 mM MgAc, 6 µl of 30 mM MgCl₂, 15 µl of ³⁵S-protein labeling mixture [NEN Life Science, Köln, Germany], and 25 µl of translation buffer that was obtained by mixing the following components: 40 µl of 100 mM ATP, 6 µl of 40 mM GTP, 40 µl of 1 M creatine phosphate (Sigma), 10 µl of creatine phosphokinase (10 mg/ml; Sigma), 76 µl of 1 M HEPES buffer (pH 7.6), 80 µl of 100 mM DTT, 20 µl of calf liver tRNA (5 mg/ml; Roche Molecular Biochemicals, Mannheim, Germany), 50 µl of 1 mM amino acid mixture without methionine (Promega), 10 µl of 100 mM spermidine (Sigma), and 168 µl of RNase-free water. After incubation for 14 to 16 h at 30°C, reactions were terminated by the addition of protein sample buffer (200 mM Tris-HCl, pH 8.0, 5 mM EDTA, 3.3% sodium dodecyl sulfate, 2% 2-mercaptoethanol, 10% sucrose, and 0.1% bromophenol blue). For in vitro translations in cell extracts of Huh-7 cells, 0.5 µl of RNA (corresponding to 0.5 µg) was mixed with 16.5 µl of extract, 3.5 µl of translation buffer, 1 µl of ³⁵S-protein labeling mixture, 0.5 µl of RNasin (40 U/µl), and 3 µl of salt mix (900 mM KAc, 7.3 mM MgCl₂, 3.3 mM Mg-acetate). After 2 h at 30°C, translations were terminated by the addition of protein sample buffer. In vitro translation reaction mixtures in rabbit reticulocyte lysates were composed of 8.75 µl of lysate, 0.25 µl of RNasin, 0.25 µl of amino acid mixture without methionine, 1.5 µl of ³⁵S-protein labeling mixture, and 1.75 µl of RNA (corresponding to 0.5 µg). Reactions were terminated after 1 h at 30°C as described above. Proteins in all translation reaction mixtures were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and HCV-specific bands were quantified by phosphorimaging.

Determination of translation efficiency in transfected cells. Five micrograms of in vitro-transcribed luciferase replicon RNA was mixed with 15 µg of an EMCV-IRES-lacZ in vitro transcript and used for electroporation of Huh-7 cells as described above. Cells were seeded in 3-cm-diameter culture dishes, and duplicates were harvested 4 h after transfection. Lysates were used for the determination of luciferase activities as described above. To correct for different transfection efficiencies, beta-galactosidase (-Gal) activities were determined in the same lysates as follows: 30 µl of cell lysate was mixed with 3 µl of magnesium solution (100 mM MgCl₂, 4.5 M 2-mercaptoethanol), 66 µl of ONPG solution (4 mg of o-nitrophenyl--D-galactopyranoside [Sigma]/ml dissolved in Na-phosphate buffer, pH 7.5), and 201 µl of Na-phosphate, pH 7.5. After a 5-h incubation at 37°C, reaction mixtures were centrifuged for 10 min at 13,000 × g and optical densities of cleared supernatants were measured at 420 nm.

	RESULTS

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Experimental approach. The design of the bicistronic replicon RNAs used in this study is shown in Fig. 1. The 5' NTR of HCV was fused either to the neo gene or to the gene encoding the firefly luciferase (luc) that were expressed as fusion proteins carrying 16 amino acids of the core protein at their amino terminus. The EMCV IRES was used to direct translation of the HCV NS3-5B region into which three cell culture-adaptive mutations were introduced. These are located in NS3 (E1202G and T1280I) and in NS5A (S2197P), and they enhance RNA replication synergistically (24). Upon transfection of Huh-7 cells with the neo replicon, RNA replication could be measured by determining the number of G418-resistant colonies obtained after selection with Geneticin. In the case of the luc replicon, RNA replication was analyzed by measuring the luciferase activity at various time points after transfection in comparison to cells transfected with a replication-defective RNA. We have shown that both the number of G418-resistant colonies obtained with a given replicon and the luciferase activity determined 24 to 96 h posttransfection are a direct correlate of the efficiency with which a replicon multiplies in cells (24). The advantage of the neo replicons was the higher sensitivity of detection because 1 µg of the parental replicon rep 5.1 yielded ~5 × 10⁵ colonies and, therefore, mutants with a low capability of replication still could be detected. The advantage of the luc replicons was the determination of RNA replication in a transient assay and, therefore, a rapid test of various mutants. It should be noted that both assays yield congruent results (24).

Sequences upstream of the IRES are essential for RNA replication. A number of studies have shown that the first ~40 nucleotides of the 5' NTR are not required for IRES function. Based on this observation and the fact that sequences at the 5' end of a viral RNA genome are very important determinants for replication, we anticipated that the same might be true for HCV. The region upstream of the IRES contains stem-loop 1 that is formed by nucleotides 5 to 20 and that is separated from stem-loop 2 by a 23-nucleotide-long spacer (Fig. 1). To analyze the importance of these elements for RNA replication, two constructs were generated in which either the spacer or stem-loop 1 were deleted (Fig 2A). We did not attempt to produce a replicon that lacks the very first nucleotides because this mutation was expected to be lethal (11). To exclude the possibility that these deletions affected IRES function, the neo replicon RNAs were first used for in vitro translations in cell extracts from different sources: rabbit reticulocytes, HeLa cells, and Huh-7 cells, which are the only ones that support replication of these RNAs. Owing to the bicistronic design of the replicons, translation efficiency of the 5' (neo) cistron that was under control of the HCV IRES could be determined independently from the translation efficiency of the downstream cistron (NS3-5B) that was under control of the EMCV IRES. Translation from the latter was assumed to be unaffected by mutations in the 5' region of the molecule, and therefore, NS3 served as an internal control for the quality of the translation reaction. As shown by the representative result in Fig. 2B, within a given cell extract, the ratios of the amounts of the core-NPT fusion protein and NS3 were similar in all cases, irrespective of the overall expression levels that were highest in rabbit reticulocyte lysates. The analogous result was found when HeLa cell extracts were programmed with the corresponding luc replicons or with replicon RNAs in which all heterologous sequences were deleted (not shown). The latter were obtained by fusing the core coding sequence via a ubiquitin cleavage site in frame to NS3. These results suggested that the translation efficiency was not affected by the deletions and independent of the reporter gene. Moreover, the findings were in agreement with previous observations showing that the HCV IRES is active in a multitude of host cells (10). It should be noted that apart from some unprocessed precursors, only NS3 was clearly visible in the in vitro translations. This was probably due to the absence of microsomal membranes that are required for efficient polyprotein cleavage in vitro or for stabilization of the cleavage products (3). However, the appearance of NS3 was sufficient to determine the translation efficiency from the EMCV IRES.

View larger version (46K): [in this window] [in a new window]   FIG. 2.   Effect of sequences upstream of HCV IRES on translation of replication-competent bicistronic replicon RNAs. (A) Schematic presentation of the 5' NTRs of the various replicons. (B) In vitro translation of replicon RNAs in extracts of rabbit reticulocytes (left), Huh-7 (middle), and HeLa (right) cells. Lysates were programmed with either an RNA encoding the NPT under control of the poliovirus IRES (lane neo) or the replicon RNAs given above the lanes. An RNA encoding only the NS3 under control of the EMCV-IRES was used to generate authentic NS3 as a size marker. The background was determined with translation reaction mixtures without in vitro transcript (no RNA). Note that the replicon RNAs direct the expression of a C-NPT fusion protein that carries 16 amino acid residues of the core protein at its amino terminus. Numbers to the left refer to the sizes of molecular mass standards in kilodaltons; proteins are specified to the right. Numbers below the lanes refer to the translation levels of the C-NPT fusion protein after normalization for the amount of NS3. Translation efficiency obtained with the replicon carrying the full-length HCV IRES (389) was set at 1. Representative results are shown. (C) Translation efficiencies after transfection of Huh-7. Cells were transfected with either of the replicon constructs specified in the bottom together with an RNA encoding -Gal under control of the EMCV IRES. Four hours after transfection, cells were lysed and reporter activities were determined as described in Materials and Methods. Luciferase activity obtained with the parental replicon 389 was set at 100%. Mean values of three independent experiments, and the error ranges are given.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Sequences upstream of stem-loop 2 are essential for RNA replication. (A) Result of transient-replication assay with replicons carrying firefly luciferase gene. Huh-7 cells were transfected with RNAs (given at the bottom), and luciferase activities were measured 24, 48, and 72 h later. Values are normalized for transfection efficiency as determined from the luciferase activities measured 4 h after transfection (set at 100%). The experiment is a representative example of three repetitions. Values are means of quadruplicates and standard deviations. The result obtained with an RNA with a full-length HCV 5' NTR and an inactive NS5B RdRp is shown on the right (389/GND). (B) Selectable replicons carrying deletions in the first 40 nucleotides of the HCV 5' NTR are unable to confer G418 resistance. Huh-7 cells were transfected with either 1 ng of the selectable replicon carrying the first 389 nucleotides of the HCV genome (389) or with each 100 ng of the deletion mutants. Twenty-four hours later, cells were subjected to G418 selection, and after about 3 weeks, colonies were fixed and stained. Transfection of 100 ng of the replicon with the unaltered 5' NTR and an inactive RdRp in which 10 amino acid residues spanning the active site were deleted served as a negative control (389/GDD).

Construction of HCV replicons with HCV/PV-chimeric 5' NTRs and analysis of RNA translation. To map the minimal sequence in the 5' NTR required for RNA replication, we constructed a series of chimeric replicons in which various portions of the HCV 5' NTR were fused to a heterologous IRES (Fig. 4A). This approach allowed the analysis of HCV replication signals without affecting RNA translation. We chose the IRES of PV for several reasons: (i) it is a well-characterized element that does not require coding sequences for activity, (ii) we found that this IRES was highly active in Huh-7 cells (data not shown), and (iii) both in structural and in functional terms, the HCV IRES is very different from the PV IRES and, therefore, it should not contain sequences or structures that could compensate for deleted sequences of the HCV 5' NTR. Because of the similarities of the structures of the HCV and the BVDV IRES, we did not attempt to use the latter as a functional substitute of the HCV translation signal. Moreover, full activity of the BVDV IRES requires part of the N^pro coding region, presumably to prevent the formation of stable RNA structures downstream of the initiator AUG (7, 12, 34). The EMCV IRES was not chosen because it was already present in the replicon RNA and we wanted to avoid a duplication of such a long sequence. In the first set of experiments, various portions of the 5' end of the HCV genome were directly fused to the PV IRES in the context of the neo and luc replicons (see Materials and Methods). Translation of the resulting RNAs carrying the first 43, 125, 296, or 341 nucleotides of the HCV genome at the 5' end was analyzed both by in vitro translation and in transfected cells. We found that expression of the luciferase gene was severely impaired in transfected cells when the full-length HCV 5' NTR was directly fused to the PV IRES, whereas no reduction of luc translation was observed with the other 5' variants (data not shown). This result indicated an interference between the HCV and the PV RNA elements similar to what was recently found with a recombinant PV that carries an HCV IRES to direct translation of the PV polyprotein (56).

View larger version (19K): [in this window] [in a new window]   FIG. 4.   (A) Schematic presentation of the replicon constructs with HCV-PV chimeric 5' NTRs. HCV sequences in the 5' NTR are drawn as a thick dotted line, the 63-nucleotide-long spacer (sp) element is a black box, and the PV IRES (PVI) is an open arrow. (B) Detailed presentation of the various chimeras. The parental construct with the full-length HCV 5' NTR is framed. The two constructs in which the apical stems of stem-loop 2 were removed were derived from construct 125-sp-PVI (arrows).

View larger version (39K): [in this window] [in a new window]   FIG. 5.   Translation studies of replicons with HCV-PV chimeric 5' NTRs in vitro and in transfected cells. (A) Equal amounts of in vitro transcripts in which various portions of the HCV 5' NTR were fused via the 63-nucleotide spacer to the IRES of PV were used to program HeLa cell extracts as described in Materials and Methods. Translation reactions were terminated by the addition of protein sample buffer, and radiolabeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins are specified on the right, the positions of molecular mass standards (kilodaltons) are indicated on the left. The result obtained after in vitro translation of the parental replicon with the authentic HCV 5' NTR is shown in lane 3 (rep5.1). Note that this RNA encodes a C-NPT fusion protein whereas the neo gene is directly fused to the PV IRES in the case of the replicons with the chimeric 5' NTRs. For comparison, an RNA containing the neo gene under the control of only the PV IRES was translated in parallel (lane 2). Lane 1, background bands obtained with the cell extracts without exogenous RNA. (B) Translation efficiencies of the HCV-sp-PV chimeric RNAs in cells. Huh-7 cells were transfected with luciferase replicons (specified at the bottom) together with an RNA encoding -Gal. Four hours after transfection, cells were lysed and luciferase activities were determined. Values are corrected for transfection efficiencies as determined by measuring -Gal activities in the same cell lysates. Mean values of four independent experiments and the error ranges are given. Values obtained with the parental replicon, 341-sp/wt, were set 100%. The results obtained with the replication defective RNAs that contain or lack the spacer between the HCV 5' NTR and the PV IRES are shown in the right (341-sp/GND and 341/GND, respectively). Only a representative selection of the data obtained with the chimeric RNAs is shown.

Complete 5' NTR of HCV is required for efficient RNA replication. Having confirmed the functionality of the translation signals in the various chimeras, we next analyzed the replication of these RNAs by using the transient assay described above. The results in Fig. 6A demonstrate that the luc replicons carrying 84 or fewer nucleotides of the HCV genome at the 5' end did not replicate (data for replicons with fewer than 43 nucleotides are not shown). Luciferase activities found with these RNAs were as low as the ones determined with the defective replicon carrying an inactive NS5B RdRp (341-sp/GND). However, a low level of replication was found with the RNA carrying the 125 5'-terminal nucleotides of the HCV genome at its 5' end. A further increase of the length of the HCV portion led to an increase of replication which was at maximum when the full-length HCV 5' NTR was placed at the 5' end of the replicon. The analogous result was found with the neo replicons. After transfection of Huh-7 cells and G418 selection, no colonies were obtained with replicons carrying 84 or fewer nucleotides of the HCV 5' NTR at their 5' end. Some colonies were obtained with the 125-sp-PVI replicon, but the efficiency of colony formation of this RNA was only 9,000 ± 2,000 (mean ± standard deviation) CFU/µg in vitro transcript. This number was ~20-fold-higher with the 296-sp-PVI replicon, and the highest efficiency was found with the RNA carrying the full-length HCV 5' NTR at its 5' end (~10⁶ CFU/µg). These data demonstrate that the first 125 nucleotides comprising stem-loops 1 and 2 are sufficient for HCV RNA replication, but efficiency is significantly enhanced in the presence of the full-length 5' NTR.

View larger version (39K): [in this window] [in a new window]   FIG. 6.   The complete HCV 5' NTR is required for efficient RNA replication. (A) Replicons with chimeric HCV-sp-PV 5' NTRs directing the expression of the luciferase gene were transfected into Huh-7 cells. After 4 (not shown), 24, 48, and 72 h, cells were lysed and luciferase activities were determined. The 4-h value that was set at 100% was used to correct for different transfection efficiencies. A representative result of three independent experiments is shown. Values represent the mean of quadruplicates and the standard deviation. (B) Huh-7 cells were transfected with selectable RNAs in which the neo gene was under the control of the different chimeric 5' NTRs. About 3 weeks after transfection and G418 selection, cells were fixed and stained with Coomassie brilliant blue. The CFU/microgram of in vitro transcript as determined by transfection of serial dilutions of RNA is given below each culture dish. GDD, inactive replicon with a 10-amino-acid residue deletion spanning the active site of the NS5B RdRp (28).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Signals required for replication of plus-strand RNA viruses usually are located in the 5' terminal regions of the template strands, and they act as promoter elements for initiation of minus- and plus-strand RNA synthesis. In order to map the sequences required for HCV RNA replication, we took advantage of the replicon system that allows high-level self-replication of subgenomic replicons in the human hepatoma cell line Huh-7 (28). By using both selectable replicons and a novel transient-replication system that we developed recently (24), we showed that the 5'-terminal 125 nucleotides of the HCV genome are sufficient for RNA replication, and we mapped the 3' border of the minimal replication signal between nucleotides 84 and 125. However, replication was significantly improved upon the addition of further sequences, and maximum replication levels in both assays were obtained with the full-length 5' NTR positioned at the 5' end of the HCV RNAs. This result is different from what has been described for some other plus-strand RNA viruses. For instance, for PV, it was shown that the 5' NTR is composed of two elements: a 5' terminal cloverleaf-like structure spanning nucleotides 1 to 88 that is essential for RNA replication and the IRES spanning at least residues 134 to 556 of the PV genome (for review, see reference 54). Replacement of the PV IRES by the HCV IRES results in viable chimeras in which RNA replication is controlled by the PV 5'-terminal element, whereas translation is mediated by the HCV IRES (29, 57, 58). Thus, sequences of the PV IRES appear to be dispensable for PV RNA replication. However, in agreement with what we describe here for HCV, several recent studies indicate that sequences within the IRES of PV are also involved in RNA replication, suggesting that the signals for translation and replication overlap (9, 21, 47).

In spite of the lack of substantial primary sequence homology, the 5' NTRs of HCV and pestiviruses like BVDV share several structural and functional features. Both contain an IRES that is located at the 3' end of the 5' NTR and that is composed of three stem-loop structures. The initiator AUG of the polyprotein coding region resides in the loop of stem-loop 4 and a pseudoknot structure is formed by sequences at the 3' end of stem-loop 3. One important difference is the presence of two stem-loops (Ia and Ib) upstream of the pestiviral IRES, whereas there is only one such structure in case of HCV. However, for neither BVDV nor HCV are these stem-loops required for IRES activity (12, 20, 43). By constructing a series of BVD viruses with chimeric BVDV/HCV or BVDV/EMCV 5' NTRs in which the BVDV IRES was replaced by the one of HCV or EMCV, Frolov and coworkers (13) made the surprising observation that the addition of the tetranucleotide sequence GUAU to the HCV or EMCV IRES was sufficient for efficient BVDV replication in cell culture. Interestingly, chimeric BVD viruses with Ia and Ib upstream of the HCV IRES replicated almost as efficient as the parental BVDV, whereas chimeras with only Ia were unstable. Upon passage of such viruses, pseudorevertants arose in which most of Ia was deleted in a way that the tetranucleotide sequence GUAU was restored (13). Assuming that the sequence at the 3' end of minus-strand RNA acts as the promoter for initiation of plus-strand RNA synthesis, this observation implies that the BVDV replicase complex can recognize the minus-strand complement of the HCV or EMCV IRES to initiate plus-strand RNA synthesis and/or that only the first 4 nucleotides direct the specificity of this process. Alternatively, sequences within the BVDV genome, i.e., downstream of the inserted heterologous IRES sequence may play an important role for RNA replication. As described in this report, the requirements for RNA sequences or structures within the 5' NTR of HCV appear to be more complex because much longer sequences or particular structures within the IRES were necessary for efficient RNA replication. Chimeras carrying only the first 5 nucleotides of HCV (or even the first 84) fused to the PV IRES did not replicate to a level detectable in our assays. This effect was not due to the particular PV IRES or the reporter genes (neo and luciferase) we utilized, because the same results were found with replicons that lacked these sequences. In this case, replication was measured in transient assays with monocistronic replicons in which various portions of the HCV 5' NTR were fused via the spacer sequence to the EMCV IRES that directed translation of the NS3-5B replicase (P. Friebe and R. Bartenschlager, unpublished data). These results imply that the signals required for efficient BVDV replication are simpler than those required by HCV. However, differences in the experimental approaches used in this study and by Frolov and colleagues (13) might also account for the discrepant results. For instance, we had to utilize subgenomic replicons, whereas the work with BVDV was performed with full-length infectious genomes. Moreover, we cannot exclude that the fusion of the HCV 5' NTR sequences to the PV IRES (or the EMCV IRES in case of the monocistronic RNAs) affected the structure or function of the HCV replication signal. In this case, the HCV sequences we identified to be essential for RNA replication would primarily be required for the preservation of the structural and functional integrity of the minimal replication signal rather than being a direct part of itself. Further studies will be required to clarify these issues.

Our results that deletions in the 5' NTR upstream of the HCV IRES reduce RNA translation appear to contradict the observations made with HCV or pestiviral reporter gene assay-based systems (reviewed in reference 44). For instance, in the case of BVDV, it was found that the 5' terminal stem-loops Ia and Ib are completely dispensable for IRES activity (12). In contrast, when analyzed in the context of a self-replicating subgenomic BVDV RNA, mutations in the 5' terminal stem-loop Ia affected both RNA translation and replication (55). For instance, translation in BHK-21 cell extracts was reduced by more than 10-fold when Ia was deleted or replaced by the 5' terminal stem-loop 1 of HCV and these RNAs no longer replicated. However, when a deletion of Ia either alone or together with a deletion of part of Ib was introduced into an infectious full-length BVDV genome, these RNAs still produced infectious virus, although the specific infectivities of the corresponding in vitro transcripts were low (5). Similar to the results obtained with the subgenomic BVDV RNAs, we also observed a reduction of RNA translation with replicons carrying deletions of stem loop 1 or the spacer sequence, although the effect was less drastic (only two- to threefold reduction). Our results therefore suggest that analysis of the HCV IRES in the context of a self-replicating molecule is important to identify possible effects of HCV proteins or sequences outside of the IRES on RNA translation.

The reduction of translation observed with replicons 5-20 and 24-40 that lack either stem-loop 1 or the spacer sequence does not explain the block of replication. First, owing to the bicistronic design of the replicons, translation of the HCV NS3-5B replicase was directed by the EMCV IRES that was not affected by the manipulations at the 5' end of the replicons. Second, the reduction of RNA translation was at maximum threefold (24-40). Although even modest effects on translation activity may result in a significantly greater cumulative reduction of RNA replication, some of the replicons with a twofold-lower translation activity still replicated efficiently (for instance, 296-sp-PVI). Thus, the deletions introduced into the first 40 nucleotides of the HCV 5' NTR primarily affected RNA replication and not translation.

In summary, we have performed the first characterization of sequences in the 5' NTR of HCV that are required for RNA replication. We found that stem-loops 1 and 2 play an important role suggesting that this region constitutes or is part of the minimal promoter for initiation of minus-strand RNA synthesis. By generating chimeric 5' NTRs, we were able to genetically uncouple the signals in the HCV genome required for RNA replication and translation. These results provide the first map of HCV replication signals, and they will guide our further studies in this important area.