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J. Biol. Chem., Vol. 277, Issue 20, 17713-17721, May 17, 2002

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Alternate Translation Occurs within the Core Coding Region of the Hepatitis C Viral Genome^*

Agoritsa Varaklioti, Niki Vassilaki, Urania Georgopoulou, and Penelope Mavromara^§

From the Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vassilisis Sofias Avenue, 115 21 Athens, Greece

Received for publication, July 9, 2001, and in revised form, February 28, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The majority of hepatitis C virus (HCV) isolates contain an open reading frame (ORF) overlapping with the core coding sequences in the +1 frame, which was assumed to be untranslated. We present evidence supporting the expression of this ORF (designated core+1 ORF) via novel translation mechanisms. First, fusion of the luciferase gene with the HCV-1 core+1 ORF followed by in vitro translation resulted in the synthesis of a chimeric protein (core+1-luciferase) that exhibited ~54% luciferase activity relative to the positive control (core-luciferase). Second, antisera raised against two different synthetic core+1 peptides recognized the previously identified p16 (but not p21) core protein band expressed from HCV-1, indicating the presence of epitopes from the core+1 ORF within the p16 protein. Third, HCV-positive sera specifically recognized lysates of Escherichia coli cells expressing recombinant core+1 protein, suggesting the presence of anti-core+1 antibodies in HCV-infected patients. Finally, luciferase tagging experiments designed to assess for 1 frameshifting combined with site-directed mutagenesis experiments supported the presence of +1/1 ribosomal frameshift translation mechanisms within the core coding region. In conclusion, our data provide evidence for novel translation mechanisms within the core coding region and demonstrate the expression of the core+1 ORF, at least for some HCV isolates.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hepatitis C virus (HCV)¹ is the major cause of non-A, non-B acute and chronic hepatitis, which frequently leads to liver cirrhosis and hepatocellular carcinoma (1-3). HCV is a member of the Flaviviridae family, with a positive, single-stranded RNA genome of ~10 kb. The genome encodes a single polyprotein that is proteolytically cleaved to produce three structural (core, E1, and E2) and at least six nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (4, 5). The core protein is located at the N terminus of the polyprotein and is predicted to have a length of 191 amino acids and a molecular mass of 23 kDa (p23) (6-9). Additional processing of p23 produces the mature core protein (p21), consisting of 173-182 amino acids (10). A shorter form of the core protein (p16) with an apparent molecular mass of 16 kDa has also been reported (11, 12). This form was first detected during in vitro expression studies of the prototype HCV-1 isolate and has been largely attributed to a specific Arg-to-Lys mutation in codon 9 of the core coding sequences (11).

Besides its apparent role in viral assembly (5, 13), the core protein has multiple independent activities, thus playing a pivotal role in viral pathogenesis (14, 15). According to recent reports, the core protein interacts with an increasing number of cellular proteins and modulates the expression of several cellular or viral promoters. Thus, the core protein can either activate or inhibit programmed cell death (16, 17), modulate signal transduction pathways of the host cells (18, 19), suppress the host immune response (20), affect lipid metabolism (21, 22), and has a transforming potential (23).

Interestingly, computer-assisted analysis of the HCV genome has revealed the presence of an additional out-of-frame open reading frame (ORF) overlapping the core gene in the +1 frame (24, 25). This novel ORF is open for 124-160 codons in most of the HCV strains (25). Thus, a putative polypeptide of ~14-17 kDa could be potentially synthesized by an alternate translation mechanism. Comparison of the complete genome sequences from different variants of HCV has shown a strong conservation within the core coding region at both the amino acid and nucleotide levels (24, 25). More importantly, it has been reported that synonymous substitutions at the third position are highly suppressed in the core coding region (24). On the other hand, this region lacks an obvious translation start codon, which may help to explain why this sequence conservation was attributed to structural constraints of the viral genome rather than the presence of a functional gene.

In the course of experiments involving the construction of chimeric GST-core hybrid proteins, we found that a chimeric GST protein containing mostly HCV sequences encoded by the +1 ORF was reactive to HCV-positive human sera. This construct was the result of a random PCR-induced single nucleotide deletion mutation near the GST-core junction. This prompted us to examine the possibility that this ORF (designated core+1 ORF) represents a functional ORF. To this end, we undertook three different approaches: (a) protein tagging experiments using the luciferase protein as a tag, (b) directly monitoring the expression of the core+1 ORF in vitro with specific anti-core+1 antibodies combined with site-directed mutagenesis experiments, and (c) screening of sera from HCV-infected patients for the presence of circulating anti-core+1 antibodies. From these studies, we provide evidence supporting the expression of the core+1 ORF, at least for some HCV isolates, via novel translation mechanism(s).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Construction of Plasmids-- All plasmids described in this study were constructed by PCR using various primer pairs and the following conditions: 35 cycles at 94 °C for 60 s, 65 °C for 30 s, and 72 °C for 2 min, with a final extension step at 72 °C for 10 min. For all DNA plasmids described, the DNA sequences were confirmed twice by sequencing (Amersham Biosciences sequencing kit and Applied Biosystems sequencer).

Plasmid pHPI-668 is based on the pGEX-3X expression vector (Amersham Biosciences) and was made to express a chimeric GST-core+1 ORF fusion protein. The core coding region (nt 390-920) was obtained by PCR using, as template, p36-27, which contains the prototype HCV-1 cDNA sequence (nt 268-1052) cloned into the pBluescript KS vector (kindly provided by M. Beach). The oligonucleotides 5'-CCGGAATTCCGTAACACCAACCGTCGCCCA-3' and 5'-CTCGAATTCCACTAGGTAGGCCGAAG-3' (underlined sequences represent EcoRI sites) were used as sense and antisense primers, respectively. The PCR fragment was digested with EcoRI and cloned into the pGEX-3X expression vector. Plasmids pHPI-756 and pHPI-996 contain the core coding sequences (nt 341-920) from prototype HCV-1 and HCV-1a (strain H) (kindly provided by G. Inchauspe), respectively, cloned into the pGEM-3zf(+) vector (Promega) under the control of the SP6 promoter. For the construction of pHPI-756, a double-step PCR was performed using plasmid p36-27 as template. The reason for the double-step PCR was to ensure the presence of the 10 consecutive adenine residues at nt 363-372 of the prototype HCV-1 core region, inasmuch as DNA sequencing analysis of this region gave inconclusive results. For the first PCR, we used 5'-GTGCTTGCGAATTCCCCGGGA-3' as the sense primer and the 5'-ACGTTTGTTTTTTTTTTGAG-3' as the antisense primer. For this PCR only, different conditions were used: 35 cycles at 94 °C for 60 s, 50 °C for 30 s, and 72 °C for 120 s, with a single final extension step at 72 °C for 10 min. The product of the first PCR was used in the second PCR as the sense primer along with 5'-CTCGAATTCCACTAGGTAGGCCGAAG-3' as the antisense primer. Plasmid p36-27 was used again as template. The conditions for the second PCR were exactly the same, except that the annealing step was at 62 °C for 30 s. The final PCR product was digested with EcoRI and cloned into the EcoRI cloning site of the pGEM-3zf(+) vector under the control of the SP6 promoter. Plasmid pHPI-996 was also obtained by PCR using pRc/CMV/HCV-H as template and primers 5'-GTGCTTGCGAATTCCCCGGGA-3' (sense) and 5'-CTCGAATTCCACTAGGTAGGCCGAAG-3' (antisense). The PCR product was subsequently digested with EcoRI and cloned into the EcoRI cloning site of the pGEM-3zf(+) vector under the control of the SP6 promoter. Plasmids pHPI-725, pHPI-736, and pHPI-737 contain the luciferase gene fused at the 0, +1, or -1 frame, respectively, with a mutated HCV-1 core coding region (9 A residues). The HCV cDNA sequences encoding the IRES and part of the mutated core coding sequences (nt 9-630) were obtained by PCR using plasmid pHPI-888 as template. The sense primer 5'-CGCCGGATCCTGATGGGGGCGACA-3' was used for all three plasmids. The antisense primers were 5'-AGACAGGATCCAATCCCGCC-3' (oligonucleotide A) for pHPI-736, 5'-CACGGGGAGACAGGATCCATCCCGCCCACC-3' (oligonucleotide B) for pHPI-725, and 5'-CACGGGGAGACAGGATCCACCCGCCCACCC-3' (oligonucleotide C) for pHPI-737 (underlined sequences represent BamHI sites). The PCR products were digested with BamHI and inserted into the BamHI cloning site of the pGEM-luc vector (Promega). Plasmid pHPI-888 is based on the pGEM-3zf(+) vector and contains cDNA sequences (nt 9-1054) from the prototype HCV-1 isolate. Nucleotide sequence analysis of pHPI-888 revealed the presence of a single nucleotide (A³⁶³) deletion in the core coding region.

Plasmids pHPI-766, pHPI-767, and pHPI-768 contain the wild-type core coding region (nt 9-630) from the prototype HCV-1 isolate fused to the luciferase gene. As before, to ensure for the presence of the wild-type nucleotide sequences within the nt 363-372 region, we used a double-step PCR cloning approach. For the first PCR, plasmid pHPI-888 was used as template, and oligonucleotides 5'-AGTGTTGGGTCGCGAAAGGCC-3' (the NruI site located at nt 271 of the 5'-untranslated region is underlined) and 5'-ACGTTTGTTTTTTTTTTGAG-3' were used as sense and antisense primers, respectively. Subsequently, the PCR product was used as the sense primer, and oligonucleotide 5'-CCAAGGGTACCCGGGCTGAG-3' (the KpnI site located at nt 585 in the core coding region is underlined) was used as the antisense primer for the second PCR. The template was again plasmid pHPI-888. The PCR product of the second PCR was digested with NruI and KpnI and used to replace the corresponding sequences from plasmid pHPI-736, yielding pHPI-766. Plasmids pHPI-767 and pHPI-768 were made by replacing the ScaI-KpnI fragment (ScaI is a vector site located 5' to the HCV sequences) of the pHPI-725 or pHPI-737 plasmid with the corresponding sequences from pHPI-766.

Plasmids pHPI-748, pHPI-749, and pHPI-750 contain the entire IRES and part of the core coding sequences (nt 9-630) from the HCV-1a (H) strain fused to the luciferase gene in all three frames. Cloning was performed by PCR using oligonucleotide 5'-CGCCGGATCCTGATGGGGGCGACA-3' as the sense primer and oligonucleotide A (for pHPI-748), oligonucleotide B for (pHPI-749), and oligonucleotide C (for pHPI-750) as the antisense primers. The PCR products were digested with BamHI and inserted into the BamHI cloning site of the pGEM-luc vector. Plasmid pHPI-1309 contains the core coding sequences (nt 385-920) from the prototype HCV-1 isolate with a start codon in the +1 frame. The core coding region was obtained by PCR using pHPI-755 as template and primers 5'-CCGGAATTCGTAATGCCAACCGTCGCCCACAGGACGTCAAGTTCC-3' (sense) and 5'-CTCGAATTCCACTTAGTAGGCCGAAGC-3' (antisense) (underlined sequences represent the EcoRI sites, and the AUG start codon and the TTA stop codon are in boldface). The PCR product was digested with EcoRI and cloned into the EcoRI site of pGEM-3zf(+) under the control of the SP6 promoter.

Finally, plasmid pHPI-CS contains codons 1-18 of the HCV core sequence cloned into the HindIII-BamHI sites of the pGEM-3zf(+) vector under the control of the SP6 promoter. For the cloning, the following two primers were used: sense primer C1s, 5'-AGCTTATGAGCACGAATCCTAAACCTCAAAAAAAAAACAAACGTAACACCAACCGTCG-3'; and antisense primer C2s, 5'-GATCCGCGACGGTTGGTGTTACGTTTGTTTTTTTTTTGAGGTTTAGGATTCGTGCTCATA-3'.

Site-directed mutagenesis was performed using the QuikChange^TM site-directed mutagenesis Kit (Stratagene) and plasmid pHPI-755 as template. The pHPI-755 plasmid contains the HCV-1 core coding sequences (nt 341-920) cloned into the EcoRI cloning site of the pGEM-3zf(+) vector under the control of the T7 promoter. Plasmid pHPI-774 is a product of site-directed mutagenesis at nt 342 and 343 of the prototype HCV-1 core coding region (mutation of A³⁴²  T and T³⁴³  A) using primers 5'-CGTGCACCTAGAGCACGGATCCTAAACCTC-3' (sense) and 5'-GAGGTTTAGGATCCGTGCTCTAGGTGCACG-3' (antisense). This double substitution converts the start codon of the core coding region (ATG) into a stop codon (TAG). The primers also insert a BamHI enzyme site, mutating A³⁵¹  G, allowing us to check the mutation by digestion.

Plasmid pHPI-775 is a product of site-directed mutagenesis at nt 357 of the prototype HCV-1 core coding region (mutation of A³⁵⁷  T) using primers 5'-ATGAGCACGGATCCTTAACCTCAA-3' (sense) and 5'-TTGAGGTTAAGGATCCGTGCTCAT-3' (antisense). This substitution converts Lys (AAA) into a stop codon (TAA). The primers also insert a BamHI enzyme site, mutating A³⁵¹  G, allowing us to check the mutation by digestion. Plasmid pHPI-777 is a product of site-directed mutagenesis at nt 453 of the prototype HCV-1 core coding region (mutation of C⁴⁵³  A) using primers 5'-GTTTACTTGTTGACGCGCAGGGG-3' (sense) and 5'-CCCCTGCGCGTCAACAAGTAAAC-3' (antisense). This substitution converts Cys (TGC) of the core+1 ORF into a stop codon (TGA). The primers also insert a HincII enzyme site, mutating C₄₅₃  A, allowing us to check the mutation by digestion.

Bacterial Expression-- For bacterial expression of the recombinant core+1 fusion proteins, Escherichia coli XL1-Blue cells were transformed with plasmid pGEX-3X or pHPI-668 and grown at 37 °C in LB medium containing 50 µg/ml ampicillin to an absorbance of 0.6 at 600 nm. Isopropyl--D-thiogalactopyranoside (Sigma) was added to a final concentration of 0.7 mM, and the bacterial cells were grown for an additional 3 h at 37 °C and then pelleted by centrifugation and stored at -20 °C.

Antibodies-- For the production of polyclonal antibodies against the core+1 ORF, peptide R1 (consisting of amino acid sequence CCRAGALDWVCARRERLPSGRNLEV) was chemically synthesized using the branched method, and peptide R2 (consisting of amino acid sequence AGGRDGSCLPVALGLA) was chemically synthesized and conjugated with keyhole limpet hemocyanin. Both peptides were used to immunize rabbits. Specifically, 100 µg of each peptide were mixed separately with 750 µl of complete Freund's adjuvant (Sigma) and injected into New Zealand White rabbits. The rabbits were boosted three times with the same antigens mixed with incomplete Freund's adjuvant (Sigma) at an interval of ~2 weeks each. The antisera were collected 2 weeks after the last boost and used in Western blot analysis and enzyme-linked immunosorbent assays. The monoclonal antibody against the core protein was obtained from Chemicon International, Inc. and Biogenesis.

Western Blot Analysis-- Proteins from E. coli lysates transformed with pHPI-668 and the corresponding empty vector were subjected to 10% SDS-PAGE and transferred onto nitrocellulose membrane (Schleicher & Schüll). The membrane was incubated with blocking buffer (5% nonfat dry milk and 0.05% Tween 20 in phosphate-buffered saline) for 2 h at room temperature. Subsequently, the membrane was incubated overnight at 4 °C either with human sera diluted 1:100 or with anti-GST antiserum in 1% nonfat dry milk and Tween 20 in phosphate-buffered saline. Subsequently, horseradish peroxidase-conjugated anti-human or anti-rabbit immunoglobulin, respectively, was used as the secondary antibody. Incubation with the secondary antibody was carried out at room temperature for 2 h. Recombinant proteins were detected using 4-chloro-1-naphthol solution as substrate.

In Vitro Transcription and Translation-- For all plasmids lacking an IRES, the TNT reticulocyte lysate coupled in vitro transcription/translation reaction kit (Promega) was used in a standard 50-µl reaction according to the manufacturer's protocol. For all the luciferase-expressing plasmids containing the HCV IRES element, Flexi rabbit reticulocyte lysates (Promega) supplemented with KCl at 120 mM and Mg(OAc)₂ at 0.5 mM were used. 1 µg of each DNA was linearized with SalI and transcribed in vitro with SP6 RNA polymerase (Promega) according to the manufacturer's instructions. In vitro translation experiments were carried out on uncapped RNAs in a total volume of 25 µl using [³⁵S]Met (Amersham Biosciences). 5 µl of the translation products were analyzed by 12% SDS-PAGE, transferred onto nitrocellulose membranes, and detected by autoradiography. 5 µl of the same translation products were assayed for chemiluminescence using a Turner TD-20/20 luminometer and a Promega luciferase assay kit according to the manufacturer's protocol. Each in vitro transcription/translation reaction was performed in triplicate.

RNA Sequencing-- 10 µg of plasmid pHPI-CS were linearized with BamHI and transcribed in vitro with SP6 RNA polymerase according to the manufacturer's instructions. The RNA was dephosphorylated using shrimp alkaline phosphatase (Roche Molecular Biochemicals) for 2 h at 37 °C. Subsequently, the RNA was end-labeled using [-³²P]ATP and T4 polynucleotide kinase (MBI Fermentas). After elution from a 5% denaturing polyacrylamide gel, the -labeled RNA was incubated with RNases A, T2, CL3, and T1 for 4 min at 37 °C in buffer containing 10 mM HEPES (pH 7.4), 1 mM EDTA, and 1 µg of tRNA and subsequently loaded onto a 10% denaturing polyacrylamide gel.

Immunoprecipitation Analysis-- 20 µl of the translation products were mixed with 250 µl of triple detergent buffer consisting of 50 mM Tris (pH 8), 150 mM NaCl, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1% Nonidet P-40, and 0.5% sodium deoxycholate. The reactions were incubated overnight at 4 °C either with 5 µl of monoclonal antibody or with 10 µl of polyclonal antibodies. 50 µl of protein A-Sepharose (Sigma) were added, and the reactions were incubated for 2 h at 4 °C. After microcentrifugation, the Sepharose beads were washed three times with buffer consisting of 50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA, 0.25% gelatin, and 0.02% sodium azide. The immunoprecipitates were subsequently subjected to 12% SDS-PAGE, transferred onto nitrocellulose membranes, and detected by autoradiography.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Evidence for Alternate Translation within the HCV Core Coding Region-- As a first attempt to investigate the possibility of alternate translation within the HCV core coding region, the luciferase gene was fused with the first half of the core coding sequences (630 nt) in the 0, +1, and -1 frames relative to the AUG initiation codon of the polyprotein, and the expression of these constructs was analyzed in a cell-free system using rabbit reticulocyte lysates. To facilitate the construction of these plasmids, we designed three different sets of primers for PCR cloning of the HCV cDNA sequences into a luciferase gene-expressing vector (pGEM-luc). Each set of primers uses the same 5'-oligonucleotide, but different oligonucleotides complementary to the 3'-core coding sequences (Fig. 1A). Oligonucleotide A was designed to place the luciferase gene in the 0 frame relative to the AUG initiation codon. Oligonucleotide B contains a single nucleotide insertion mutation (T⁶²⁷) that places the luciferase gene in-frame with the overlapping ORF in the +1 frame relative to the core coding sequences (core+1 ORF). Oligonucleotide C contains a deletion of the adenine residue at nt 626, which places the luciferase gene in-frame with the -1 frame relative to the AUG initiation codon. Additionally, all three oligonucleotides contain a single base substitution at nt 630 (C to A) to create a BamHI cloning site. Three groups of plasmids were constructed using the above primers.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Tagging experiments with the luciferase gene. A, shown is a schematic representation of the constructs used for the tagging experiments. The entire HCV IRES (nt 9-340) and part of the core coding sequences (nt 341-630) were cloned into the pGEM-luc expression vector under the control of the SP6 promoter. Nucleotide sequences at the junction of the core and luciferase coding regions are illustrated underneath. The AUG initiation codon of the luciferase gene is boxed. Oligonucleotide A (Oligo-A) was used to fuse the luciferase gene in-frame with the AUG initiator codon (0 frame) in plasmids pHPI-766, pHPI-748, and pHPI-736. Oligonucleotide B (Oligo-B) was used to fuse the luciferase gene in the +1 frame relative to the AUG initiator codon (+1 frame) in plasmids pHPI-767, pHPI-749, and pHPI-725. Oligonucleotide C (Oligo-C) was used to fuse the luciferase gene in the 1 frame relative to the preceding core coding sequence (1 frame) in plasmids pHPI-768, pHPI-750, and pHPI-737. The underlined nucleotide indicates an insertion of a thymidine residue, and the inverted triangle indicates a deletion of an adenine residue. B, the different IRES-core-luciferase fusion constructs (as described under "Experimental Procedures") were transcribed in vitro, and equal amounts of uncapped RNAs were used to program in vitro translation reactions using Flexi rabbit reticulocyte lysates. 5 µl of the products were resolved by SDS-PAGE, and the results from autoradiography are shown. Products are indicated by the arrow. 5 µl of the same translation products were measured for luciferase activity according to the Promega protocol, and the resulting values are illustrated in the graph. Each bar represents the average luciferase activity of triplicate in vitro translation samples. Error bars indicate the S.D. values of triplicate samples. For clarity, bars corresponding to the same series of constructs have been similarly shaded.

The first group contains the core coding sequences from the prototype HCV-1 isolate, and it was designed to test for a possible expression of the core+1 ORF. Plasmid pHPI-766, constructed using oligonucleotide A, contains the luciferase gene fused in-frame with the preceding core coding sequences and served as a positive control. This plasmid was expected to produce a chimeric core-luciferase protein with an apparent molecular mass of 72 kDa, which is ~10 kDa larger than the mass of the native luciferase protein. Plasmid pHPI-767, constructed using oligonucleotide B, contains the luciferase gene fused in-frame with the core+1 ORF. This plasmid was predicted to produce a chimeric luciferase protein only if the core +1 frame was expressed. The size of this putative protein would depend on the location of the translation initiation codon of the core+1 ORF. Finally, pHPI-768 plasmid, made using oligonucleotide C, carries the luciferase gene fused in-frame with the -1 frame relative to the core coding sequences and served as a negative control. Because the 1 frame contains multiple stop codons, no functional luciferase was predicted to be produced. As anticipated, in vitro translation of pHPI-766 resulted in the synthesis of a chimeric luciferase protein with an apparent molecular mass of 72 kDa. High enzymatic activity also indicated that this construct was capable of supporting the expression of an active chimeric core-luciferase protein (Fig. 1B, lane 4). As expected, only background levels of luciferase activity and no protein were detected from the expression of the pHPI-768 plasmid (Fig. 1B, lane 6). However, when pHPI-767 was used, a fusion protein with an apparent molecular mass of 72 kDa was again produced. This protein exhibited ~54% luciferase activity relative to that from the pHPI-766 construct (Fig. 1B, lane 5). Notably, normal translation from the RNA of this plasmid led to a translation stop shortly after the luciferase gene region. As expected, the chimeric luciferase protein expressed from pHPI-767 reacted strongly with anti-core+1 antibodies (R1 and R2; see below), whereas no reaction was observed with the chimeric luciferase protein expressed from pHPI-768 (data not shown). Therefore, the results presented here provide the first direct evidence supporting the expression of the core+1 ORF from prototype HCV-1.

We then sought to explore the possibility of a -1 ribosomal frameshift event within the core coding region of the same HCV isolate. Because the -1 frame of the core gene contains multiple stop codons, premature termination would prevent the detection of a potential -1 frameshift event. To overcome this limitation, we constructed a new series of plasmids that contained a single nucleotide deletion mutation within the core coding region (A³⁶⁴). This mutation introduced a change in the reading frame after the first 9 amino acids of the core ORF, thus placing the upstream AUG codon out-of-frame with respect to the core ORF and in-frame with the core+1 ORF. Consequently, plasmid pHPI-736, which was made with oligonucleotide A, placing the luciferase gene in-frame with the core ORF, now had the luciferase gene out-of-frame relative to the AUG initiation codon. This plasmid was therefore predicted to produce a functional luciferase protein only if a -1 frameshift event occurred. On the other hand, plasmid pHPI-725, which was made using oligonucleotide B, placing the luciferase gene in-frame with the core+1 ORF, now had the luciferase gene in-frame with the AUG initiation codon. This plasmid was expected to give rise to a hybrid luciferase protein containing the first 10 amino acids of the core protein followed by the 86 amino acids of the core+1 ORF in-frame with the luciferase gene. Finally, plasmid pHPI-737, which was made using oligonucleotide C, was our negative control plasmid because the luciferase gene was fused in the third frame containing the multiple stop codons. As shown in Fig. 1B (lane 1), when plasmid pHPI-725 was used to program in vitro translation, a fully active luciferase protein with an apparent molecular mass of 72 kDa was produced. This construct reproducibly gave the highest levels of luciferase activity. Interestingly, plasmid pHPI-736 also produced an active luciferase protein with a similar size (72 kDa) and an enzymatic activity of ~22% compared with our positive control construct (Fig. 1B, lanes 2 and 1, respectively). As predicted, anti-core+1 antibodies R1 and R2 reacted strongly with the chimeric luciferase protein from pHPI-725 and failed to react with the translation product from pHPI-736 (data not shown). No detectable signal was obtained from the translation products of our negative control construct plasmid pHPI-737 (Fig. 1B, lane 3). These data provide strong evidence for the presence of a -1 ribosomal frameshift mechanism within the core coding sequences and predict that the slippery site would be within the nucleotide sequences encoding the N terminus of the core protein.

Finally, we sought to assess the expression of the core+1 ORF from another HCV isolate. Thus, a third set of plasmids containing the core coding sequences from the HCV-1a (H) strain cloned into the luciferase-expressing vector was constructed. Plasmid pHPI-748 (equivalent to the pHPI-766 construct) contains the luciferase gene fused in-frame with the core coding sequences. Plasmid pHPI-749 (equivalent to pHPI-767) contains the luciferase gene fused in-frame with the core+1 ORF, and pHPI-750 (equivalent to pHPI-768) contains the luciferase gene in-frame with the 1 frame relative to the AUG initiator codon. As shown in Fig. 1B, translation of plasmid pHPI-748 resulted in the expression of a fully active luciferase protein with an apparent molecular mass of 72 kDa (lane 7), whereas no luciferase expression was detected from the negative control plasmid pHPI-750 (lane 9). However, plasmid pHPI-749, with the luciferase gene fused in-frame with the core+1 ORF, was not able to support detectable levels of chimeric luciferase protein (Fig. 1B, lane 8), suggesting that the HCV-1a (H) isolate does not support efficient expression of the core+1 ORF under the experimental conditions of this study.

Overall, these results provide evidence for the presence of novel translation mechanism(s) within the HCV-1 core coding sequences. However, the reasons for the apparent lack of detectable expression of the core+1 ORF from the HCV-1a (H) isolate are not currently clear.

Evidence for Expression of the Core+1 ORF in Vitro-- Previous in vitro translation studies have shown that the core coding region from the HCV-1a (H) isolate expresses predominantly the 21-kDa core protein (p21), whereas the same cDNA sequence from the prototype HCV-1 isolate produces predominantly a faster migrating form of the core protein that is ~16 kDa in size (p16), in addition to p21 (11). The p16 protein is assumed to represent a C-terminal truncated form of the core, and its synthesis has been correlated to an Arg-to-Lys change in codon 9 (11). However, the possibility that p16 is in fact a different protein generated from an alternative reading frame of the HCV core coding region was not addressed at that time.

To explore this possibility and to further characterize the nature of the p16 protein, we raised specific antibodies against the core+1 ORF using two different peptides, R1 and R2, corresponding to the HCV sequences encoded by nt 448-522 and 613-660, respectively. The reactivity of these antibodies against the translation products of the core coding region from the HCV-1a (H) or prototype HCV-1 isolate was analyzed by immunoprecipitation experiments in a coupled in vitro transcription/translation system. Consistent with previous studies, the major translation product from plasmid pHPI-996 (HCV-1a (H)) was a protein of 21 kDa (p21), whereas plasmid pHPI-756 (HCV-1) resulted in the production of two major products of 21 and 16 kDa (p21 and p16, respectively) (Fig. 2B, lanes 1 and 5). Immunoprecipitation experiments carried out with the in vitro translation products indicated that the p21 protein from both plasmids pHPI-996 (HCV-1a (H)) and pHPI-756 (HCV-1) reacted strongly with the monoclonal antibody against the core (Fig. 2B, lanes 2 and 6), but failed to react with the R1 and R2 polyclonal antibodies (lanes 3 and 4 and lanes 7 and 8, respectively). These results are in agreement with previous studies and provide strong evidence for the specificity of the R1 and R2 polyclonal sera. In contrast, the p16 protein was recognized by the anti-core monoclonal antibody (Fig. 2B, lane 6) and the two specific anti-core+1 antisera (lanes 7 and 8), suggesting that the 16-kDa protein band contains epitopes from both the core and core+1 proteins. Thus, in contrast to the hypothesis made in previous studies, the p16 protein more likely represents a chimeric protein containing amino acids encoded by both the core and core+1 ORFs.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Expression studies using an in vitro transcription/translation system. A, shown is a schematic representation of the constructs used in the in vitro expression studies. HCV nucleotide sequences spanning from nt 342 to 920 were cloned into the pGEM-3zf(+) expression vector under the control of the SP6 promoter. Plasmid pHPI-756 corresponds to the prototype HCV-1 cDNA sequences, and plasmid pHPI-996 corresponds to the HCV-1a (H) nucleotide sequences. B, equal amounts of both plasmid DNAs were used to program a coupled in vitro transcription/translation reaction using the TNT reticulocyte lysates. Products of the in vitro reactions were either directly separated by SDS-PAGE (lanes 1 and 5) or immunoprecipitated by various antibodies (lanes 2-4 and 6-8). Details of the transcription/translation reactions as well as the conditions used in the immunoprecipitation experiments are described under "Experimental Procedures." p21 and p16 protein bands are indicated by arrows. Lane 1, translation product with the HCV-1a (H) DNA; lanes 2-4, immunoprecipitation products with anti-core monoclonal antibody and anti-core+1 polyclonal antibodies R1 and R2, respectively; lane 5, translation product with the prototype HCV-1 DNA; lanes 6-8, immunoprecipitation products with the anti-core monoclonal antibody and anti-core+1 polyclonal antibodies R1 and R2, respectively. Protein molecular mass markers (in kilodaltons) are indicated on the right.

In an attempt to further characterize the p16 protein, three separate mutations were introduced into the HCV-1 core coding sequences, and the expression of the p21 and p16 proteins was analyzed in vitro as described above (Fig. 3A). First, the AUG initiator codon for the core was mutated to a UGA stop codon, resulting in plasmid pHPI-774. Second, a stop codon was introduced 6 amino acids downstream of the AUG initiator codon and in-frame with the core reading frame, yielding pHPI-775. Finally, plasmid pHPI-777 contains a nucleotide substitution at nt 453 that introduces a stop codon (TGA) into the core+1 ORF (Fig. 3A). As shown in Fig. 3B (lane 1), plasmid pHPI-755, which contains the wild-type core coding sequences of the HCV-1 isolate, produced both forms (p21 and p16) of the core protein. On the other hand, plasmids pHPI-774 and pHPI-775, both containing a termination codon at the beginning (first or sixth amino acid) of the conventional frame of the core coding sequences, failed to synthesize the 21-kDa as well as the 16-kDa core proteins (Fig. 3B, lane 2). The phenotype of both mutations is in agreement with previous studies (12), suggesting that the expression of the p16 protein is dependent on the AUG initiator codon of the polyprotein and that both the p21 and p16 proteins share common amino acids. Interestingly, however, plasmid pHPI-777, which contains a stop codon (TGA) in the core+1 ORF, failed to express the 16-kDa protein, whereas the 21-kDa protein was normally synthesized (Fig. 3B, lane 4). These data are in agreement with the observed reactivity of the p16 protein to anti-core+1 antibodies (Fig. 2) and strongly suggest that the synthesis of the p16 protein is dependent on the translation of both the core and core+1 ORFs.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Mutagenesis experiments. A, shown are nucleotide sequences within the core coding regions of plasmids pHPI-755, pHPI-774, pHPI-775, and pHPI-777. Underlined nucleotides represent mutated nucleotides. The AUG initiator codon of the core coding region is indicated in italics. B, equal amounts of all plasmid DNAs were used to program a coupled in vitro transcription/translation reaction. [35S]Met translation products of the in vitro reactions were directly separated by SDS-PAGE. p21 and p16 protein bands are indicated by arrows. Lane 1 represents the translation product with the wild-type HCV-1 DNA.

Finally, to exclude the possibility that the expression of the core+1 ORF is the result of slippage of the SP6 polymerase in the A-rich region of the HCV core sequence (nt 364-373), we performed direct/enzymatic sequencing of the in vitro synthesized HCV RNA of this region. As shown in Fig. 4, following digestion with the indicated RNases, the stretch of 10 A residues is clearly visible, with no apparent deletion or insertion that would lead to the synthesis of a frameshifted product.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   RNA sequencing of the transcript containing codons 1-18 of the HCV core sequence. Shown are the results from partial digestion with RNase A, which cuts after C and U (lane 1); RNase T2, which cuts specifically after A (lane 2); RNase CL3, which cuts after C and U (lane 3); and RNase T1, which cuts after G (lane 4). The locations of the 10 adenines and the 2 flanking cytosines are indicated by arrows.

Evaluation of HCV-positive Human Sera for the Presence of Anti-core+1 Antibodies-- Patients with chronic HCV infection produce antibodies against most HCV proteins. If the core+1 ORF is expressed during HCV infection, it is likely that some HCV-positive patients will have antibodies recognizing epitopes of the core+1 ORF, in addition to other anti-HCV antibodies. To test this possibility, serum samples from both HCV-infected and healthy individuals were evaluated for their reactivity against recombinant core+1 proteins expressed in E. coli. For this purpose, a cDNA fragment containing the core coding region (nt 390-920) of the prototype HCV-1 isolate was cloned into the pGEX-3X expression vector, resulting in plasmid pHPI-668 (Fig. 5A). This plasmid was designed to produce a GST-core+1 fusion protein with a calculated molecular mass of 41 kDa. At first, the expression of the pHPI-668 plasmid was analyzed by Western blotting using anti-GST antiserum. As expected, E. coli extracts harboring the pHPI-668 plasmid expressed the 41-kDa protein, whereas bacterial lysates harboring the plasmid vector expressed the vector-encoded GST protein (27 kDa) (Fig. 5B, lanes 1 and 2). The 41-kDa protein was also reactive with the R1 and R2 anti-core+1 polyclonal antibodies (data not shown). Next, using the same E. coli extracts, we screened a panel of previously characterized sera from HCV-positive individuals (34 samples) and controls (15 samples) by immunoblot assays. Representative results are shown in Fig. 5B. Interestingly, most of the HCV-positive human sera (77%) recognized a ladder of protein bands with apparent molecular masses of 41 to 29 kDa only in the pHPI-668-transformed E. coli lysates (Fig. 5B, lanes 4, 6, 8, and 10), suggesting the presence of circulating antibodies against the core+1-encoding determinants in the HCV-infected individuals. No reaction was observed with the corresponding bands in E. coli extracts harboring the pGEX-3X vector (Fig. 5B, lanes 3, 5, 7, and 9). Furthermore, all of the HCV-negative serum samples from healthy individuals failed to react (data not shown). These results indicate that the reactivity of the HCV-positive human sera to extracts from E. coli cells expressing the GST-core+1 protein was not caused by nonspecific binding of serum immunoglobulins to E. coli proteins or to the GST part of the recombinant antigen. However, because of the presence of multiple instead of a single sero-reactive band, we constructed an additional recombinant core+1 antigen based on the pMal-c2 expression vector. Screening of human sera against the maltose binding protein-core+1-expressing E. coli extracts verified the positive reactivity of the HCV-positive sera (data not shown). As before, however, the serum reactivity was mainly against a broad protein band, suggesting instability problems inherent to the recombinant core+1 antigen in E. coli.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Screening of human sera for the presence of anti-core+1 antibodies. A, construction of the GST-recombinant core+1 plasmid. Nucleotides 390-920 from the HCV core coding sequences were cloned in the +1 frame into the pGEX-3x expression vector, resulting in plasmid pHPI-668. The GST-core+1 fusion protein has a calculated molecular mass of 41 kDa. B, Western blot analysis of the GST-recombinant core+1 plasmid. Plasmid pHPI-668 and the pGEX-3X control vector were transformed into E. coli cells; and after induction with isopropyl--D-thiogalactopyranoside, the cell lysates were resolved by SDS-PAGE. Bacterial lysates transformed with the pGEX-3X vector alone (lanes 1, 3, 5, 7, and 9) and with plasmid pHPI-668 (lanes 2, 4, 6, 8, and 10) were tested by Western blotting for their reactivity against anti-GST antiserum and human sera from HCV-positive patients. Protein molecular mass markers (in kilodaltons) are indicated on the left, and the migration of the recombinant proteins is indicated by the arrow. C, schematic diagram of plasmid pHPI-1309, which contains the core+1 coding region under the control of the SP6 promoter with a start codon in the +1 frame. D, immunoprecipitation experiments performed with the core+1 protein. Lanes 1-3, immunoprecipitations using human sera from HCV-positive patients; lanes 4-6, immunoprecipitations using human sera from HCV-negative individuals. The location of the core+1 protein is indicated by the arrow.

Finally, in an attempt to overcome this problem, a truncated form of the core+1 protein lacking the first 10 amino acids of the core region (Fig. 5C) was synthesized in vitro in the presence of [³⁵S]methionine. Immunoprecipitation experiments with human sera from HCV-positive patients revealed a weak reactivity with a single protein band (~16 kDa) corresponding to the core+1 protein, whereas no significant reactivity was observed with sera from the HCV-negative controls (Fig. 5D). It should be noted, however, that in contrast to the previous screening analysis, background levels could be detected in some HCV-negative sera from non-healthy individuals (Fig. 5D, lane 6). Taken together, these results are in agreement with recent studies (26) and support the presence of circulating anti-core+1 antibodies in the HCV-infected individuals, thus implying the expression of this ORF in vivo.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, evidence is presented that supports the expression of the ORF that overlaps the HCV core coding sequences in the +1 frame, designated in this study as the core+1 ORF. Most importantly, we have shown that the previously identified p16 protein band from the HCV-1 isolate contains antigenic determinants of both the core and core+1 polypeptides. Finally, we have provided preliminary evidence supporting the presence of novel translation mechanisms within the core coding region.

The first direct evidence for the expression of the core+1 ORF was obtained from in vitro translation studies of the core region of the HCV-1 isolate using luciferase tagging experiments. Fusion of the luciferase gene with the +1 frame relative to the core coding sequences resulted in the expression of a chimeric luciferase protein that reacted with specific anti-core+1 antibodies. Moreover, the chimeric protein exhibited ~54% luciferase activity relative to the control construct carrying the luciferase gene fused to the core ORF. Because, by design, expression of the luciferase gene from this construct (pHPI-767) occurs only through the translation of the +1 frame, these data provide strong evidence that the core+1 ORF from HCV-1 is a functional ORF.

Further evidence for the expression of the core+1 ORF was obtained by analyzing the reactivity of novel anti-core+1 antibodies (R1 and R2) against the translation products of the core region from the HCV-1 isolate. Interestingly, we showed that the HCV-1 p16 protein contains epitopes not only from the core, but also from the core+1 ORF, inasmuch as R1 and R2 specifically immunoprecipitated the p16 protein band, whereas they failed to react with the p21 core product. Both p21 and p16 were immunoprecipitated, as expected, by the anti-core monoclonal antibody. This was an unanticipated finding because the p16 protein has been assumed to represent a processed form of the p21 core protein (11-12). To explain these data, we considered two likely possibilities. Either the p16 protein is a chimeric protein containing core coding sequences at the N terminus and core+1 amino acid sequences at the C terminus, i.e. p16 and p21 are identical in their N-terminal ends, but differ at their C-terminal ends, owing to the use of the +1 translation frame; or the p16 protein band may consist of two proteins with the same apparent molecular mass: the truncated core protein and the core+1 polypeptide. Because of the basic nature of the core and putative core+1 proteins (predicted pI > 11), two-dimensional gel electrophoresis would not resolve these polypeptides. On the other hand, separation of those polypeptides on Tricine gels reproducibly gave a single protein (data not shown). Moreover, site-directed mutagenesis experiments support the first possibility, inasmuch as insertion of a translation termination codon after the ninth amino acid of the core abolished the expression of both the p21 and p16 proteins, whereas insertion of a stop codon in the core+1 ORF (nt 453) abolished the expression of the p16 protein only.

When the core region of the HCV-1a (H) isolate was used to perform similar experiments, no evidence for detectable core+1 expression was found. Luciferase tagging experiments performed with the core+1 frame from HCV-1a (H) gave no detectable expression of the luciferase gene, indicating no or very low expression of the core+1 ORF. Furthermore, no reactivity of R1 and R2 anti-core+1 antibodies was detected with in vitro translated products of the core region. The latter finding is in agreement with the low levels of expression of p16 from HCV-1a (H). Thus, there appears to be a correlation between the lack of efficient core+1 expression (based on the luciferase tagging experiments) and the synthesis of the p16 protein in the HCV-1a (H) isolate, further supporting the suggestion that expression of the HCV-1 p16 protein is directly related to the translation of the core+1 ORF.

At this time, it is unclear why expression of the core+1 ORF and/or the synthesis of p16 protein is predominantly observed only for the prototype HCV-1 isolate, a finding that raises important questions regarding the biological relevance of p16 in the life cycle of the virus. However, two recent reports may shed some light on this issue. Of particular interest is the study by Suzuki et al. (27) indicating that the p16 protein can be expressed by other isolates, but in an unstable form due to proteasome-induced degradation. Furthermore, Yeh et al. (28) provided evidence for the expression of the p16 protein during natural infection. Notably, the expression of p16 is associated with three different mutations located within codons 9-11 other than the Lys-to-Arg change observed in HCV-1. One critical concern is to verify that the p16 protein produced in those studies is the same protein as the p16 protein produced by the HCV-1 isolate in vitro. Interestingly, Yeh et al. (28) showed that polyclonal antibodies against p16 fail to react with the p21 protein and vice versa. This suggests the presence of different epitopes in the two proteins and, similar to our findings, supports the different nature of the p16 and p21 proteins.

The mechanisms of the core+1 expression and/or synthesis of the p16 protein are unknown. However, our data support ribosomal frameshifting into the +1 frame, even though we cannot formally exclude other, less "orthodox" translation mechanisms such as protein splicing and RNA editing that may operate in vivo. Although the luciferase tagging experiments cannot directly address the mechanism responsible for core+1 ORF expression, the apparent molecular mass of the chimeric luciferase protein (encoded by pHPI-767) argues against the possible use of an obvious start codon in the +1 frame, as the first AUG codon is located just 12 amino acids upstream of the luciferase gene. Furthermore, the combined reactivity of p16 to both anti-core and anti-core+1 antibodies provided strong evidence for a translation mechanism that would allow translation from both the 0 and +1 frames, resulting in the synthesis of a chimeric protein. Further support for a +1 ribosomal frameshift mechanism was provided by the in vitro mutagenesis experiments, inasmuch as insertion of a stop codon into either the 0 or +1 frame (6 or 37 amino acids downstream of the AUG initiator codon, respectively), abolished the expression of the p16 protein. This is in contrast to the expression of the p21 protein, which, as expected, was not affected by the insertion of a stop codon in the +1 frame. Moreover, alteration of the AUG initiator codon to a stop codon eliminated the expression of both the p21 and p16 proteins.

Interestingly, the luciferase tagging experiments also support the presence of a 1 ribosomal frameshift event within the core region. The 1 frame relative to the core ORF contains multiple stop codons. Thus, should such a mechanism operate in vivo, it could function as a regulatory switch to control the expression of the core and/or core+1 rather than to culminate in the synthesis of a new protein, as is the case in the putative +1 frameshift event. Negative regulation of core expression through the 1 frameshift event or translation of the core+1 ORF would function in favor of encapsidation or RNA replication. The details of the molecular mechanisms responsible for alternate translation within the core region are currently under investigation.

The data presented here suggest the presence of novel RNA signals within the core coding region responsible for the reading of alternative frames by the ribosomes. According to previous studies, the efficiency of -1 frameshifting depends on the primary sequence of the RNA slippery site or on distal sequences that participate in a secondary structure that is responsible for the pausing of the ribosomes (29). On the other hand, the cis-acting elements responsible for the +1 frameshift mechanism are less well defined (30, 31). Finally, it should be noted that the previously described mutation in codon 9 of the HCV-1 isolate generates a region of 10 consecutive A residues that represents a known 1 slippery site (29). Notably, Yeh et al. (28) detected p16 from clinical isolates containing mutations at nt 366-374 (codons 9-11) that failed to reproduce the 10-A residue region of HCV-1, indicating that the presence of 10 A residues is not obligatory for the efficient expression of the p16 protein.

The critical question that remains to be answered concerns the biological importance of our findings in the context of the HCV life cycle. Our first attempt to address this was to screen human sera from HCV-positive patients against E. coli lysates expressing recombinant core+1 protein or in vitro synthesized core+1 protein. Our data suggest the presence of circulating antibodies against epitopes from the core+1 ORF in most of the HCV-infected individuals, implying that the core+1 ORF may be expressed in vivo during HCV infection. Notably, although the nature of the positive signal for the E. coli antigen is a family of overlapping polypeptides rather than a single protein band, the reaction was specific for the core+1-containing E. coli extracts, inasmuch as no reaction was observed with the negative controls. Furthermore, our data are in agreement with recent studies reporting the identification of antibodies to synthetic peptides representing the core+1 ORF in HCV-infected patients (26).

The genome organization of all the members of the Flaviviridae family is similar and characterized by the presence of the structural proteins at the amino terminus and the nonstructural proteins at the carboxyl terminus of the polyprotein. Notably, however, the structural region of the pestiviruses has two distinguishing characteristics: the presence of an extensive secondary structure at the 5'-untranslated region of their genome that functions as an IRES element as well as the presence of the N^pro gene located upstream of the core coding sequence. N^pro is a nonstructural protease that is autocleaved from the nascent polyprotein (32). Flaviviruses lack both characteristics, whereas the HCV genome encodes an IRES element. In fact, secondary structure modeling of the 5'-untranslated region of HCV and pestiviruses revealed a remarkable folding similarity, and several short stretches with a significant sequence identity have been recognized in the 5'-untranslated region of both viruses (33, 34). Thus, it is tempting to speculate that the HCV core +1 polypeptide might represent the counterpart of N^pro in HCV. Interestingly, a closer examination of the catalytic amino acids of N^pro and other members of the chymotrypsin-like cysteine proteases revealed that the important catalytic amino acids are relatively conserved in the +1 ORF between nt 615 and 656 (35).

In summary, this study provides direct evidence for the presence of alternate translation mechanisms within the core coding region of the HCV-1 isolate. Such mechanisms may be important for translation of the core+1 ORF and/or regulation of core expression itself. How widespread expression of the core+1 ORF and/or the putative +1/1 frameshift mechanism(s) may be remains an open question. The finding of a p16 protein from clinical isolates combined with the observed reactivity of human sera to recombinant core+1 antigens or peptides suggests that the core+1 ORF and/or the alternate translation mechanism within the core may indeed represent novel functions for HCV.

Finally, it should be noted that during the review process of our paper, Xu et al. (36) reported the discovery of a novel HCV protein (designated F protein) synthesized from the core coding region. It was shown that this protein is synthesized from the coding sequence that overlaps the core protein via a ribosomal frameshift translation mechanism. It appears that the F protein is identical to the core+1 protein described in this report.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Graeme L. Conn for critically reading the manuscript and Scott Walker for helpful discussions on the RNA sequencing experiments.

	FOOTNOTES

^* This work was supported by grants from the National Secretariat of Research and Technology. A preliminary report of this study describing the luciferase tagging experiments and the screening of human sera was presented at the Seventh International Meeting on Hepatitis C and Related Viruses, December 3-7, 2000, Gold Coast, Queensland, Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Posttranscriptional Control Group, Dept. of Biomolecular Sciences, UMIST, P. O. Box 88, M60 1QD Manchester, UK.

^§ To whom correspondence should be addressed. Tel.: 30-10-6478-877; Fax: 30-10-6478-877; E-mail: penelopm@hol.gr.

Published, JBC Papers in Press, March 7, 2002, DOI 10.1074/jbc.M201722200

	ABBREVIATIONS

The abbreviations used are: HCV, hepatitis C virus; ORF, open reading frame; GST, glutathione S-transferase; nt, nucleotide(s); IRES, internal ribosome entry site; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

	REFERENCES

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