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1 UMR 5234 CNRS, IFR66, Université Victor Segalen Bordeaux 2, 146, rue Léo Saignat, 33076 Bordeaux cedex, France
2 UPRES EA 23873, Laboratoire de Virologie, Université Pierre et Marie Curie, CERVI, Hôpital Pitié-Salpêtrière, 75651 Paris Cedex 13, France
3 Laboratoire de Virologie, IFR66, Université Victor Segalen Bordeaux 2, 146, rue Léo Saignat, 33076 Bordeaux cedex, France
Correspondence
Michel Ventura
michel.ventura{at}reger.u-bordeaux2.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work. 
Details of oligonucleotides and plasmids and a phylogenetic tree derived from complete 5' UTR sequences are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Numerous factors including gender, age of infection, alcohol consumption, exposure to other hepatotoxins and perturbation of lipid metabolism have been identified as determinants of pathogenesis. Viral factors may also be critical determinants of steatosis in chronic hepatitis C, particularly HCV genotype (Rubbia-Brandt et al., 2004). Genetic variability of the RNA genome has made it possible to distinguish six HCV genotypes and over 70 subtypes (Simmonds et al., 2005). Each of these genotypes displays particular features such as resistance to interferon/ribavirin treatments. Thus, genotype 1-infected patients respond less efficiently to therapy than those infected with genotype 2 and 3 viruses. Conversely, patients with HCV genotype 3 infection and chronic hepatitis C are more likely to be subjected to a liver steatosis than those infected with HCV genotype 1 (Lonardo et al., 2006). Viral determinants of this differential progression are as yet poorly understood. It has been suspected that sequence variations in the envelope E1 and E2 glycoproteins might be involved in differences in pathogenesis between genotypes 1 and 3 (Shaw et al., 2003). More recently, in vitro models suggested involvement of the core protein as a viral factor associated with lipid accumulation in genotype 3 infection (Abid et al., 2005; Hourioux et al., 2007). However, the genotype 1 core protein has been shown to induce steatosis in transgenic mice (Moriya et al., 1997) and to be associated with lipid droplets (Shi et al., 2002), suggesting that there is a multifactorial contribution of viral determinants in this mechanism.
HCV has a positive-strand RNA genome in which two untranslated regions (UTRs) enclose a large open reading frame. The 341-nucleotide 5' UTR in association with the first nucleotides of the core coding sequence contains an internal ribosome entry site (IRES) that directs cap-independent translation of the viral RNA (Rijnbrand et al., 1995; Wang et al., 1993). The 3' UTR is composed of a short variable region, a poly U–UC tract of variable length and a highly conserved 98-nucleotide segment (3' X). The two latter domains are essential for viral infectivity in vivo (Yanagi et al., 1999) and RNA replication of HCV in the HCV replicon system (Friebe & Bartenschlager, 2002; Yi & Lemon, 2003). Variability in the sequences of HCV is distributed unevenly along the RNA genome. While the coding part of the HCV genome is weakly conserved, the non-coding regions exhibit higher similarity among different isolates. The 5' part of the plus-strand RNA is involved in protein translation through its IRES sequence (He et al., 2003; Reynolds et al., 1996), while its counterpart located at the 3' end of the minus strand is implicated in genome replication (Friebe & Bartenschlager, 2002). Moreover, the first 125 nucleotides of the HCV 5' UTR seem to be involved in replication of the RNA genome (Friebe et al., 2001), whereas nucleotides 41–341 are directly associated with IRES function. Thus, sequence modifications arising within this region may impair the efficiency of either viral translation or replication, leading to distinct pathological properties.
In this study, we describe nucleotide changes in the 5' UTR sequence that are specific to HCV genotype 3 and we investigate their impact on the levels of translation and replication of the genomic RNA.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Genotype 3 5' UTRs were obtained from patient sera. The 5' UTR HCV sequences were extracted from HCVDB (Combet et al., 2004). After a CLUSTAL alignment, positions with variable nucleotides were selected and then sorted with Microsoft Excel as a function of HCV genotype.
Plasmid constructs and RNA templates.
The following plasmids used either as vectors for protein expression or as RNA templates were designed. (i) The pIRF bicistronic reporter vector comprises the firefly luciferase (FLuc) gene followed by the HCV 5' UTR from genotype 1a and the Renilla luciferase (RLuc) gene, all inserted into the vector pcDNA3.1/Zeo (Invitrogen) under the control of cytomegalovirus (CMV) and T7 promoters (Laporte et al., 2000). (ii) The vector pCV-UTR4 containing the 5' UTR from pCV-H77 (genotype 1a) in pGEM9Zf (Astier-Gin et al., 2005) was used to derive all mutants. Mutations in the SL-A1 stem–loop were introduced by PCR and subsequent cloning in the pGEM-T vector. The forward primers were dG3 and G3d (Supplementary Table S1, available in JGV Online) and the reverse primer was 5'341T7. Mutations in the SL-E1 stem–loop were introduced by site-directed mutagenesis using the Quickchange site-directed mutagenesis kit (Stratagene) using primers described in Supplementary Table S1. pCV-SL-A1-SL-E1 G3 was obtained by exchanging the EcoRI–AgeI fragment of pCV-SL-E1G3 (HCV H77 origin) with the homologous fragment of HCV genotype 3 clone 4 (HCV-geno3-Cl4). All plasmid clones described were verified by sequencing. (iii) The minigenome pGEM-T/5UTR-H2AE-3UTR construct, consisting of a gene encoding a polyprotein composed of hygromycin phosphotransferase (HygroR), the foot-and-mouth disease virus 2A protein and the enhanced green fluorescent protein (EGFP), flanked by the HCV 5' and 3' UTRs, was described previously (Dumas et al., 2007).
Cell culture.
Naive human hepatoma Huh7 cell line was grown in Dulbecco's modified Eagle medium supplemented with 10 % heat-inactivated fetal calf serum and gentamicin (50 µg ml–1) at 37 °C in a 5 % CO2 atmosphere. The Huh7/rep5.1 cell line supporting the subgenomic HCV replicon rep5.1 was maintained under G418 selection pressure. The Huh7/NS3-5B cell line, constitutively expressing the non-structural (NS) proteins from HCV strain J4L6, and cured Huh7 cells were described previously (Dumas et al., 2007).
In vitro transcription.
RNAs corresponding to the 3' ends of the different minus-strand RNAs were synthesized by in vitro transcription of DNA obtained by PCR amplification from plasmids listed in Supplementary Table S2. The sequences of the sense primers are given in Supplementary Table S2. The reverse primer (5'341-T7) introduced the T7 promoter in the correct orientation. To generate transcripts for in vitro translation, bicistronic plasmids linearized with XhoI served as templates for in vitro transcription of uncapped RNAs from the T7 promoter. When the ex vivo translation assay was performed, RNA transcripts were capped beforehand by adding a cap analogue (Ambion) during the transcription reaction. To produce the minigenome RNA, the T7 promoter was first introduced upstream of the 5UTR-H2AE-3UTR sequence in the corresponding plasmid by PCR, using Taq Phusion polymerase (Ozyme) and primers T7-5UTR and 3UTR-Stop (Supplementary Table S2). To generate the corresponding transcripts, in vitro transcriptions were performed with the Megascript kit (Ambion). DNA templates were digested with DNase for 15 min. After phenol/chloroform extraction, the RNAs were precipitated with 2-propanol. The purity and integrity of RNAs were determined by analysis on a 4 % polyacrylamide gel containing 7 M urea in TBE buffer (90 mM Tris/borate, pH 8.0, 1 mM EDTA) or by SDS-agarose gel electrophoresis depending on their size.
Transfection of RNA minigenomes.
Twenty-four hours before transfection, cells were seeded at 105 cells per well into 24-well plates. RNA transfection was performed by using 1 µg RNA combined with 3 µl DMRIE-C in 200 µl OptiMEM medium (Invitrogen) according to the manufacturer's protocol. The replication factor R was used to express the results: the number of expressed EGFP molecules per cell can be calculated as the sum of the number of EGFP molecules translated from the input RNA plus the number of EGFP molecules translated from the replicated RNA, the latter depending on the input RNA. This can be written simply as follows: E=T[RNA]+TR[RNA], where E is the number of EGFP molecules, T the translation factor, R the replication factor and [RNA] the RNA input concentration. The replication factor can be calculated as followed: R={E/(T[RNA])}–1. E represents the percentage of fluorescence for analysed cells and T[RNA] the percentage of non-replicating cells, i.e. Huh7 cells. The equation can be rewritten as:
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Translation and luciferase assay.
In vitro translations in micrococcal nuclease-treated rabbit reticulocyte lysates (RRL) (Promega) were performed according to the supplier's instructions, after 1 h incubation at 30 °C, in a final volume of 10 µl containing 500 ng RNA. Luciferase activities were assayed on 10 µl reaction mixture by using the Dual-Luciferase reporter assay system (Promega).
When assessing ex vivo translation, 8x104 naive Huh7 or Huh7/NS3-5B cells were seeded into 24-well plates and transfected in triplicate 24 h later with 500 ng of the corresponding capped bicistronic RNA as described above. At 24 h post-transfection, cells were harvested and 20 µl lysate was used for the luminometric assay. HCV IRES relative translational activity is represented by the ratio RLuc/FLuc.
RNA-dependent RNA polymerase (RdRp) assay.
The RdRp assay was carried out using various 3'-end minus-strand RNAs described above and recombinant HCV NS5B-
21 of strain H77 (genotype 1a) and isolate J4L6 (genotype 1b), both expressed in Escherichia coli and purified as described previously (Reigadas et al., 2001). HCV NS5B-21 of genotype 3 was from Replizyme. The assay and quantification of labelled RNA products were performed as described by Reigadas et al. (2001).
Flow cytometry analysis.
Parental or transfected Huh7 cells were dissociated by trypsinization and resuspended at a concentration of 0.5x106 to 1x106 cells ml–1 in PBS containing 2 mM EDTA. Samples were analysed by flow cytometry in a FACS Calibur (Becton Dickinson).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Some of these nucleotides are involved either in a loop structure, such as 13, 14 and 203 on the positive strand (Fig. 1a) and 13, 14, 97 and 203 on the negative strand (Fig. 1b), or in a stem structure, such as 8, 70, 97 and 224 on the positive strand and 8 and 70 on the negative strand, or even on a single-strand RNA such as 224 on the negative strand. The C8U change on the positive RNA strand is supposed to maintain the domain I structure, while it should destabilize the corresponding structure of the negative strand. The G224A change within the positive strand, always associated with the U175C change for genotype 3, should lead to a base impairment in domain III, resulting in an overall structure destabilization. Moreover, even if they are less marked in the organization of secondary structures than in the positive strand, the changes observed at position 224 of the negative strand and modifications denoted in loop structures may lead to an overall reorganization of various domains and modify their interactions with effecting factors. Since all these modifications might be expected to have an effect on the viral life cycle by altering either translation or replication of the viral RNA, we analysed their impact on both mechanisms.
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. It included the three modifications specific to genotype 3 in that region (positions 8, 13 and 14) and other changes found in genotype 3 isolates but present in some other genotypes (see Methods and Supplementary Table S1). This approach thus differs clearly from a previous study (Collier et al., 1998) that did not include the consensus modifications described above. As expected, these two patients infected with genotype 3 showed the seven changes described above in their 5' UTR sequences. We analysed the translation efficiency of this genotype by inserting the genotype 3 IRES DNA sequence (nt 1–370) into the pIRF vector, in place of the genotype 1a IRES, between the FLuc and RLuc genes. In a first step, the efficiency of the IRES of genotypes 1a, 1b and 3 was tested in vitro as described in Methods using RRL. The mean ratios obtained for three independent experiments did not differ significantly, as reported in Table 2.
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), we used capped RNA transcripts corresponding to different bicistronic DNAs to transfect Huh7 cells and measured the resulting luciferase activities. As shown in Table 2, they were lower than those obtained in vitro for all three genotypes, but there was no significant difference between them. Moreover, since previous studies have reported a potential impact of NS proteins on HCV IRES efficiency (He et al., 2003; Kalliampakou et al., 2005), the same experiment performed in Huh7 cells was performed in parallel in Huh7/NS3-5B cells. Results included in Table 2 showed clearly that the IRES activity was not increased in Huh7/NS3-5B cells compared with Huh7 cells. They also indicated that IRES activity was slightly affected in the presence of NS proteins for the IRESs of genotype 1 compared with that of genotype 3, with a significant decrease observed for the IRES of subtype 1b. These experiments were also carried out with the same reporter system in a monocistronic context, giving identical results (data not shown).
RNA synthesis from the 3' end of the minus-strand RNA is lower for genotype 3 than for genotype 1
Modifications in the nucleotide sequence of the HCV 5' UTR plus-strand RNA lead to changes in the 3' end of the minus-strand RNA, where initiation of plus-strand RNA synthesis probably occurs. To determine whether nucleotide changes specifically associated with genotype 3 modify in vitro RNA synthesis, RdRp assays were performed using recombinant HCV NS5B polymerases from genotypes 1a, 1b and 3a and a 341 nt complementary sequence of the 5' UTR designated (–)IRES. As shown in Table 3, the level of RNA synthesis from (–)IRES of HCV genotype 1a was about eight times higher than that obtained with (–)IRES from HCV genotype 3 with the three enzymes. As we have described already, the first nucleotide found at the 5' end of the HCV genome isolated from humans or chimpanzees could be either G or A for genotype 1, but is always A for genotype 3 (C or U for minus strand). To determine whether the low level of RNA synthesis observed with (–)IRES 3 wild-type (WT) RNA was due to the 3' U nucleotide, we changed it to C in order to obtain the (–)IRES 3 CGG. Moreover, to allow comparisons, (–)IRES RNA 1a with a 3' U instead of C was included in the experiment. When the amount of RNA produced by NS5B (1a) from (–)IRES 1a WT was taken as 100 %, the rate of RNA synthesis from (–)IRES 1a UGG was only 12 % of that from (–)IRES 3 WT (UGG) and was increased to 38 % when (–)IRES 3 CGG was used as the template (Table 4). This suggests that nucleotide changes in other regions of the (–)IRES RNA from genotype 3 could account for the low level of RNA synthesis of this genotype.
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). On the minus-strand RNA, the first three nucleotide changes are located in a domain folded in a hairpin structure composed of a six-base stem and a four-base apical loop named SL-A1 (Fig. 1b). Secondary structure prediction using RNA Draw software indicates that this region of HCV genotype 3 also folds in a hairpin, but that the stem is shortened to five nucleotides and the sequence of the apical loop differs at two of the four positions. We introduced some of these changes into the wild-type (–)IRES 1a and used the corresponding RNA mutants in RdRp assays. Mutants were designed in such a way that a SL-A1 stem–loop was maintained and that the secondary structure of the other domains of the RNA fragment was not modified, as determined by computer predictions. The results described in Table 4 indicate that the exchange of the SL-A1 domain of HCV strain H77 1a with that of HCV-geno3-Cl4 [(–)IRES SL-A1 G3] decreased RNA synthesis to 55 %. Surprisingly, mutation of one nucleotide in the apical loop, either alone at position 13 or 14 or at position 13 together with changes at positions 8 and 17 [(–)IRES G3d], reduced the level of RNA synthesis. In contrast, replacement of a GC pair by an AU pair [(–)IRES G3c] in the SL-A1 stem or deletion of a GC pair [(–)IRES G3c GC] did not reduce the amount of RNA synthesized.
As the level of RNA synthesis obtained with (–)IRES SL-A1 G3 was slightly higher than that obtained with (–)IRES 3 CGG, we hypothesized that genotype 3-specific changes in other regions may also interfere with RNA synthesis. Structure prediction indicates that the U70C and A97G mutations do not modify the secondary structure of the SLB-1 stem–loop. In addition, we have shown previously that mutations or deletions in the SLB-1 stem–loop (including its entire deletion) did not decrease RNA synthesis by HCV NS5B in vitro (Astier-Gin et al., 2005). Concerning the SL-E1 stem–loop (Fig. 1b), the following three mutations were introduced in the (–)IRES 1a WT sequence: (i) the specific nucleotide change at position 203 (A203U or A203C) in the apical loop, giving the (–)IRES A203C RNA template, (ii) the substitution C224U to obtain the (–)IRES 1a C224U sequence, which is predicted to lengthen the stem of the SL-E1 hairpin, and (iii) an additional mutant [(–)IRES SL-E1 G3] with the two genotype 3-specific substitutions (A203C and C224U) plus a third A to G mutation at position 175. This latter change is not specific to genotype 3, as it is also observed in some HCV isolates from genotypes 4 and 5, but it is present in all genotype 3 isolates sequenced so far. The level of RNA synthesis obtained with this latter RNA as template was decreased by 43 % compared with the wild-type (–)IRES 1a (Table 4). An even greater reduction in RNA synthesis by viral polymerase was observed when a single point mutation was introduced at position 224, whereas the A203C change only slightly affected in vitro RNA replication. Finally, the introduction of the sequence of the SL-A1 domain of HCV-geno3-Cl4 into (–)IRES SL-E1 G3 [(–)IRES 1a SL-A1-SL-E1 G3] reduced RNA synthesis to 24 % of the (–)IRES 1a WT. Taken together, our data indicate that introduction of nucleotide changes specific to genotype 3 in the SL-A1 and SL-E1 regions reduces in vitro RNA synthesis from the 3' end of the minus-strand RNA of genotype 1a HCV.
With the aim of analysing the size of RNA produced from the wild-type and different RNA mutants, RdRp assays were performed by using 32P-UTP as the labelled nucleotide. In order to visualize better any possible slight differences in the migration pattern of RNA products, each lane was loaded with the same amount of labelled RNA (50 000 c.p.m.). Results illustrated in Fig. 2 show that, in all cases, the major RNA product was of the template size. No arrest band was observed after a 2 h synthesis with the exception of (–)IRES 3 WT and 3 CGG RNAs, which gave small amounts of a shorter product indicated by an asterisk. In all cases, slower-migrating bands were also observed, the major one migrating at a position corresponding to an RNA twice as large as the template. For the wild-type (–)IRES RNA, we have shown previously that this product corresponds to two successive copies of the template (Reigadas et al., 2001) as a result of a new initiation event (Astier-Gin et al., 2005). Products of higher molecular mass were also visible in very small amounts. They may correspond to three (or more) successive copies of the template.
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) (Dumas et al., 2007). The 5UTR-H2AE-3UTR minigenome was used to analyse the replicative activity of the HCV replication complex in Huh7 cells. The 2A protein was included to cleave between HygroR and EGFP protein. The functionality of this system was assessed by transfection of naive Huh7 or Huh7/NS3-5B cells with the RNA minigenome obtained through in vitro transcription and culture in the presence of various concentrations of hygromycin. Whereas naive Huh7 cells died rapidly, the Huh7/NS3-5B cells were able to grow in the presence of hygromycin and their replicative activity could be followed for several weeks and analysed by measuring the EGFP expression by flow cytometry. Fig. 3(b) indicates that the percentage of cells expressing EGFP increased until 1 month and correlated with the hygromycin concentration. The stronger the selective pressure, the larger the number of fluorescent cells, produced as a consequence of minigenome replication. Replication of the RNA minigenome in Huh7/NS3-5B cells was also demonstrated by the presence of minus-strand RNA 96 h after transfection of the corresponding plus strand (Dumas et al., 2007).
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). Extension of cell culture beyond 1 month in the presence of 100 µg hygromycin ml–1 showed that the genotype 1 minigenome could be replicated efficiently (Fig. 3d). In contrast, the genotype 3 minigenome led either to low expression of EGFP protein in Huh7/NS3-5B cells in one experiment or to the absence of any EGFP expression, as no cells were recovered after 1 month, in three other experiments. In these latter three cases, the Huh7/NS3-5B cells were not able to grow in the presence of hygromycin. Because we have shown that the level of translation from the genotype 3 IRES was identical to that of genotype 1, this result suggests that the level of replication of the genotype 3 minigenome was insufficient to allow Huh7 cells to grow in the presence of hygromycin. Thus, the minigenome with the 5' UTR sequence from genotype 3 was replicated less efficiently than that from genotype 1, confirming the results obtained in vitro. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The consistent difference in the nucleotide sequence between the 5' UTRs of genotype 3 and other genotypes raises the question of the origin of this HCV genotype. In the phylogenetic analysis obtained from CLUSTAL alignments, genotype 3 should originate from the root giving rise to genotypes 2 and 6, having a closer relationship to genotype 6 (Supplementary Fig. S1). Previous studies that have analysed full-length HCV sequences have shown that genotypes 3, 5 and 6 were derived from a common root, with genotype 3 remaining more closely related to genotype 5 (National Institutes of Health, 2002; Magiorkinis et al., 2006; Simmonds et al., 2005). Our results seem to indicate that genotype 3 is more distant from the other genotypes than has currently been reported.
Here we demonstrate, using two different approaches, that specific nucleotide changes do not alter the IRES activity of genotype 3 compared to that of genotype 1, in agreement with other reports (Collier et al., 1998; Motazakker et al., 2007; Saiz et al., 1999). The changes observed in the stems of domains II and III are expected to maintain the IRES structure, and that located in the apical loop of domain III (U203A) does not modify IRES activity. Moreover, the sensitivity of genotype 3 to interferon therapy does not seem to be associated with the specific changes in the IRES structure, as demonstrated recently in two independent studies (Hazari et al., 2005; Motazakker et al., 2007; Yasmeen et al., 2006).
Concerning genotype 3 replication, we observed that the level of replication of the negative RNA strand in vitro is reduced strikingly when compared with that of genotype 1 whatever the genotype of NS5B used (1a, 1b or 3). This low level cannot be ascribed entirely to the presence of a 3'-terminal U, since its replacement by C increased the amount of synthesized RNA by only three times. Besides the 3'-terminal nucleotide, the first 20 nucleotides of the genotype 3 isolate used in this study differ from the genotype 1a (H77) minus-strand 3' end at six positions (4, 8, 11, 13, 14 and 17). Accordingly, our data reveal that mutations in the apical loop of this hairpin (U13G and A14C) reduce in vitro RNA synthesis for genotype 1a, whereas other mutations that destabilize slightly or shorten the stem have no effect (Table 4). Moreover, while mutation at position 203 in the SL-E1 apical loop (A203C) modifies the level of RNA synthesis in vitro only slightly, modification at position 224, also specific to genotype 3 (C224U), dramatically reduces RNA synthesis.
To analyse the effect of these variations in the 5' UTR sequence on replication ex vivo, we designed a minigenome as a reporter transgene in order to render the viral translation and replication steps independent. Indeed, in the replicon system, replication and translation are linked. The arrest of translation following HCV IRES modification leads to a lower level of expression of the replication complex and, consequently, to reduced RNA synthesis. Moreover, decreasing the replication efficiency leads to a decrease in the amount of viral RNA and, thus, in translation activity. In that respect, our minigenome system was able to replicate, as indicated by the replication factor, thanks to the HCV NS proteins provided either by constitutive expression in Huh7/NS3-5B cells or by Huh7/rep5.1 cells supporting the replicon (Fig. 3c). This indicates that the 5' UTR is functional for both genotypes. We did not observe any significant difference in the level of replication in the first 3 days in the absence of hygromycin pressure. The large input of RNA minigenome during the transfection (1 µg) should explain this result by masking, in the context of the replication complex, the smaller difference in the replication level between the two genotypes than that observed through in vitro studies. However, by using hygromycin selection pressure, we showed that the genotype 3 minigenome is replicated less efficiently than that of the genotype 1 after 2 weeks of culture. The decrease in replication activity leads to a reduced level of hygromycin phosphotransferase and then to cellular death in presence of the antibiotic in three experiments, a fourth one showing lower EGFP expression (Fig. 3d). It is always difficult to extrapolate in vitro results to the situation in vivo, particularly as there is as yet no model to study the replication of genotype 3 viruses. Therefore, it is tempting to speculate about the reduced replication rate observed in this work. Decreasing replication should lead to a smaller number of double-stranded RNA molecules in infected cells. Since these cells are the target of the interferon response, it could be envisaged that they should be more sensitive to interferon therapy. Nevertheless, the significance of these results remains to be investigated in more detail by comparing the number of viral RNA molecules in hepatic cells from patients infected with genotype 1 and genotype 3 viruses. This aspect is currently under investigation.
In summary, we have shown that HCV genotype 3 seems to be more distant from the others than is currently assumed, since it clearly differs by a set of seven specific nucleotide changes within its 5' UTR. Moreover, some of these modifications are located in positions previously regarded as well conserved among other HCV isolates and seem to affect the in vitro replication activity of HCV genotype 3.
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ACKNOWLEDGEMENTS |
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Received 6 April 2007;
accepted 19 September 2007.
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