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1 CNRS-UMR 8161, IBL, Université de Lille I et Lille II, Institut Pasteur de Lille, 59021 Lille cedex, France
2 Laboratoire de Virologie, Centre Hospitalier Universitaire-Hôpital Sud, 80054 Amiens cedex, France
3 Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan
Correspondence
Czeslaw Wychowski
czeslaw.wychowski{at}ibl.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) as well as several extrahepatic diseases (Houghton, 1996). An estimation of about 170 million people infected with HCV worldwide has been reported (Poynard et al., 2003; Thomas, 2000).
HCV is an enveloped single-strand, positive-sense RNA virus and its genome encodes a unique open reading frame that is flanked by two structured non-translated regions in 5' and 3' ends of HCV genome (5'NTR and 3'NTR). Mediated by an internal ribosome entry site (Tsukiyama-Kohara et al., 1992), the translation of HCV RNA genome results in polyprotein synthesis that is processed by cellular and viral proteases into at least 10 structural and non-structural (NS) proteins (Grakoui et al., 1993; Hijikata et al., 1991). In the viral particle, HCV genomic RNA is complexed with the highly basic capsid protein. On its surface, the viral particle bears two envelope glycoproteins E1 and E2 that are anchored in the lipid bilayer. Both these proteins have been shown to accumulate in the endoplasmic reticulum (ER), where the particles are probably assembled (Op De Beeck et al., 2001). A small integral membrane protein, p7, has been reported to function as an ion channel (Griffin et al., 2003; Pavlovic et al., 2003). Among the NS proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, which coordinate the intracellular processes of the virus life cycle, only proteins NS3 through to 5B are sufficient to support the HCV RNA replication (Lohmann et al., 1999). In addition to the polyprotein, a new HCV protein with an unknown function has also been reported. The so-called F-protein (frameshift) or ARFP (alternative reading frame protein) is generated by ribosomal frameshifting into an alternative reading frame within the capsid-coding sequence (Roussel et al., 2003; Varaklioti et al., 2002; Walewski et al., 2001; Xu et al., 2001).
Despite intensive research efforts, the HCV life cycle and host–virus interactions have been difficult to investigate due to the lack of efficient cell culture and small animal models. Nevertheless, significant progress has been made by using heterologous expression systems. Infectivity of some cDNA-derived HCV RNAs has been demonstrated in chimpanzees upon intrahepatic inoculation (Kolykhalov et al., 1997; Yanagi et al., 1997, 1999). Also, the development of a functional cell-based replication system allowing efficient replication of HCV subgenomic RNAs (replicons) has provided an important tool for studying the HCV RNA replication or for evaluating potential antiviral compounds (Blight et al., 2000; Lohmann et al., 1999). Several surrogate systems have also been developed to palliate the difficulties in studying interactions between several candidate HCV receptors and the HCV glycoproteins. Thus, retrovirus-based pseudoparticles (pp) or HCVpp has provided the first insight into HCV entry (Bartosch et al., 2003; Hsu et al., 2003). But the major breakthrough arose recently with the propagation of virus in a human liver hepatoma cell line, Huh-7 (Wakita et al., 2005), by transfecting these cells with an RNA transcribed from a full-length cDNA cloned initially from a patient with a fulminant hepatitis and infected with a genotype 2a isolate (Kato et al., 2001). Unfortunately, it was reported by this group that the efficacy of the infection was low. Subsequently, different papers reported a robust production of infectious virus obtained with a homologous chimeric FL-J6/JFH-1 (Lindenbach et al., 2005) or obtained into Huh-7.5.1 cells (Zhong et al., 2005), derived from a cell line (Huh-7.5) having a defect in the RIG-I pathway (Sumpter et al., 2005).
In this study, a highly efficient in vitro infection system based on Huh-7 cell line was obtained. The transcribed genomic JFH-1 RNA was used to produce infectious virus. The viral titre was initially low; however, successive infections of naïve Huh-7 cells led to a robust production of virus. The sequencing of the viral genome revealed only a few mutations located in the E2 glycoprotein. Furthermore and based on the characterization of a 1a–2a chimeric virus, we showed by site-directed mutagenesis that 2 aa present in the C-terminal part of the capsid-coding sequence were important for the production of high titres. Consequently, a robust HCV particle production was obtained independently of the Huh-7.5.1 cells or JFH-1 recombinant viral genome.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were grown in Dulbecco's modified essential medium (DMEM; Invitrogen) supplemented with 100 nmol non-essential amino acids l–1 and 10 % fetal bovine serum (FBS).
Antibodies.
Rat monoclonal antibody (mAb) 3/11 (Flint et al., 1999), kindly provided by J. McKeating (Institute of Biomedical Research, Birmingham University, UK) was produced in vitro by using a MiniPerm apparatus (Heraeus) as recommended by the manufacturer. Anti-C (ACAP27) mouse mAb was kindly provided by J. F. Delagneau (Bio-Rad, Marne-La-Coquette 92430, France). Anti-E2 mouse mAb AP33 was kindly provided by A. Patel (MRC Virology Unit, Institute of Virology, Glasgow, UK). Goat anti-
-actin polyclonal antibodies were from Santa Cruz. Alexa 488-conjugated and Alexa 555-conjugated goat anti-mouse secondary antibodies were from Molecular Probes.
Plasmid construction.
The plasmids pJFH-1 containing the full-length cDNA of JFH-1 isolate, belonging to subtype 2a (GenBank accession no. AB047639
[GenBank]
), pJFH-1/GND and pJFH-1/
E1-E2 were described previously (Wakita et al., 2005). Individual or combined viral mutations N534K (N6), F172C and P173S (FP
CS) were introduced into the pJFH-1 plasmid by sequential PCR steps as described using the high fidelity deep vent DNA polymerase (New England Biolabs), then assembled by a second PCR amplification (Goffard et al., 2005), followed by restriction digestions and ligation. The resulting plasmids were named pJFH-1/N6 (N534K) and pJFH-1/CS. The plasmid pJFH-1/CS-N6 was obtained by inserting the fragment BsiW1–NotI obtained from pJFH-1/N6 into the plasmid pJFH-1/CS. All constructs were verified by DNA sequencing.
In vitro transcription.
To generate genomic HCV RNA, the plasmid pJFH-1 and derivatives were linearized at the 3' end of the HCV cDNA with the restriction enzyme XbaI (New England Biolabs). Following treatment with Mung Bean Nuclease, the linearized DNAs were then precipitated overnight and resuspended in RNase-free water to a concentration of 1 µg µl–1. In vitro transcripts were generated using Megascript (Ambion) according to the manufacturer's protocol. The in vitro reaction was set up and incubated at 37 °C for 4 h. To degrade the DNA template, DNase I was added and incubated for another 20 min at 37 °C. The in vitro transcripts were then precipitated by the addition of LiCl and the precipitates were recovered by centrifugation. The concentration of each transcript was determined by measurement of the absorbance at 260 nm. In vitro transcribed RNA was delivered to cells by electroporation as described previously (Kato et al., 2003a). Viral stocks were obtained by harvesting cell culture supernatants at 1 week post-transfection. Secondary viral stocks were obtained by additional infections of naïve Huh-7 cells.
Successive infections and titration of HCV RNA by RT-PCR.
Huh-7 cells were seeded at 7x105 cells in T25 flask and inoculated with 2 ml supernatant medium from cells transfected with the infectious JFH-1 RNA. At 24 h, the cells were supplemented with 4 ml complete DMEM. At day 3 post-infection, infected cells were trypsinized and then replated at 2x106 cells in a T75 flask. Indirect immunofluorescence was used to estimate the levels of infectivity of the amplified virus. In addition, for quantification of HCV RNA, the RNA was extracted from the supernatant of infected cells and titrated by quantitative real-time RT-PCR assay (RT-qPCR) (Castelain et al., 2004).
HCV RNA genome sequencing.
Five microlitre aliquots of the RNA solutions were subjected to reverse transcription with random hexamer and moloney murine leukemia virus reverse transcriptase (Superscript II; Invitrogen) at 42 °C for 1 h. PCR primers of 20-mer designated on the sequence of JFH-1 were used to amplify five fragments of HCV cDNA (nt 129–626, 467–2367, 2285–4665, 4594–7003 and 6950–9634) to cover most of the HCV genome. One microlitre of the cDNA was subjected to PCR with TaKaRa LA Taq polymerase (Takara Biochemicals), and PCR conditions consisted of 30 cycles each with a denaturing cycle at 95 °C for 30 s, an annealing cycle at 60 °C for 30 s and an extension cycle at 72 °C for 2 min. The sequence of each amplified DNA was determined.
Titration of HCV cell culture (cc).
Huh-7 cells were seeded at 8x104 cells per well in a 24-well plate. The next day, cell supernatants of transfected or infected Huh-7 cells were serially diluted in DMEM and used to infect naïve Huh-7 cells. The inoculum was incubated for 2 h at 37 °C, washed with DMEM and then overlaid with complete DMEM. The viral titre was then determined at 3 days post-infection by indirect immunofluorescence staining of the capsid protein and expressed as focus-forming unit per millilitre (f.f.u. ml–1) as described previously (Zhong et al., 2005).
Western blot analysis.
Cells were lysed in a buffer containing 50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5 % (v/v) Igepal and a mixture of protease inhibitors (Complete; Roche). Protein content of pre-cleared cell lysates was determined by the BCA method as recommended by the manufacturer (Sigma), using BSA as a standard. Total proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes (Hybond-ECL; Amersham) by using a Trans-Blot apparatus (Bio-rad) and revealed with a specific mAb followed by goat anti-mouse or anti-rat IgG conjugated to peroxidase (Jackson Immunoresearch) and donkey anti-goat conjugated to peroxidase. The immune complexes were visualized by enhanced chemiluminescence detection (ECL; Amersham) as recommended by the manufacturer.
Indirect immunofluorescence microscopy.
Infected Huh-7 cells grown on coverslips were fixed in 4 % paraformaldehyde. Immunostaining was performed as described previously (Rouille et al., 2006) using anti-C ACAP27 mouse mAbs and anti-E2 3/11 rat mAb, followed by species-specific-conjugated secondary antibodies. Image acquisition was carried out using an Axiophot 2 microscope (Zeiss) equipped with a camera. For confocal microscopy, double-label staining was performed with anti-E2 mouse mAb AP33 (IgG1) and anti-C mouse mAb ACAP27 (IgG2a) followed by Alexa 488-conjugated goat anti-mouse IgG2a and Alexa 555-conjugated goat anti-mouse IgG1. Fluorescent signals were collected with a Leica SP2 confocal microscope equipped with a PL APO x100/1.4 immersion objective.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Transfected cells were then passaged every 5–7 days in order to maintain subconfluent cultures during the experiment. Immunofluorescence staining for capsid and E2 proteins revealed that the percentage of positive cells increased from 30 % at day 2 to 80 % at day 12, after two passages (Fig. 1a). These results suggest that the virus spread within the cell culture allowing the untransfected cells to be infected. Western blot analyses of transfected cells showed that the capsid and E2 proteins were still detected after 33 days (Fig. 1b) and even after 90 days (data not shown). Similar results were obtained for NS3 (data not shown). Virus released in the supernatant of transfected cells was then used to inoculate naïve Huh-7 cells. Immunofluorescence staining revealed that a low percentage of cells was positive (Fig. 1c), but after several passages, all cells were infected (data not shown). Controls for transfection and infection were also performed with JFH-1/E1-E2 and JFH-1/GND. No infection was observed with these constructs as described previously (data not shown) (Wakita et al., 2005). Titration of HCV RNA by RT-qPCR assay revealed that after the first passage of transfected cells the level of detection of HCV RNA was low (Table 1). However, the extracellular HCV RNA increased slowly in the supernatant of transfected cells reaching a maximal level of 2x106 genome equivalent (GE) ml–1 after four passages (P4). In the same time, the infectious viral titre was only approximately 103 f.f.u. ml–1. These results indicate that our Huh-7 cells can replicate JFH-1 RNA and can be infected with HCV particles. However, in transfected cells maintained in culture, the production of infectious viral particles remained low.
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. To follow the release of infectious virus particles in the supernatant, the viral RNA was extracted from supernatant and titrated by RT-qPCR. The successive infections, performed on Huh-7 cells with JFH-1 (noted I1–I6), led to a progressive release of viral genomes in the supernatant of inoculated cells, which reached a maximal level of 2.9x108 GE ml–1 after six successive infections (Table 1
). Substantially more viral RNA was released into supernatant fluids of infected cells in I5 or I6 than in transfected cells. Interestingly, infectious titres ranging between 105 and 106 f.f.u. ml–1 were obtained at I5 and I6, indicating that a robust infection could be obtained with Huh-7 cell line after successive infections.
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A and UC). The third mutation, at position 1942 (UA), led to a change in amino acid from Asn 534 to a Lys (N534K), which is a potential site of N-glycosylation (N6 site) (Fig. 3a). Thus, to verify that the core glycosylation was modified in E2 of JFH-1 produced in I6, the E2 glycoprotein resulting of the first transfection was compared with that resulting of the last infection (I6). As confirmed by immunoblotting, a slight shift in the E2 migration was observed (Fig. 3b, lanes I6 and wt). Furthermore, no differences in the migration profiles of E2 were observed after deglycosylation by PNGase F treatment, indicating that the difference observed in the molecular mass of E2 is due to the glycosylation. Due to the higher infectious titre obtained with this final virus, we can speculate that the lack of a glycan at position N6 of E2 might favour a better interaction with an HCV receptor. Alternatively, we cannot exclude that this mutation improves the assembly and/or release of infectious particles.
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, infectious virus in the supernatant of transfected cells was initially low. Indeed, the RNA and viral titres of the JFH-1/N6 (N534K) were initially comparable to the original JFH-1 virus. However, after only two successive amplifications in naïve cells, the JFH-1/N6 virus spread faster than the wild-type JFH-1 virus leading to a better production of infectious particles (Table 1). Together, these data indicate that the N534K mutation facilitates the amplification of JFH-1 virus in Huh-7 cells.
Release of infectious particles is improved by mutations in the capsid-coding sequence
Recently, a chimeric virus containing the genotype 1a capsid-coding sequence in the context of the full-length 2a sequence [JFH-1/C(+)6-1a2a] was constructed in order to analyse the expression of F protein (D. Delgrange, T. Wakita & C. Wychowski, unpublished data). Interestingly, higher levels of infectious particles were detected in the supernatant of cells transfected with the virus JFH-1/ C(+)6-1a2a (1.5x108 GE ml–1, 105 f.f.u. ml–1), suggesting that some residues present in the genotype 1a capsid protein might improve the infectivity of JFH-1. Taking advantage of this result and of previous published data with FL-J6/JFH-1 chimeric construct (Lindenbach et al., 2005), the capsid-coding sequences of genotype 1a, 2a (FL-J6) and 2a (JFH-1) were aligned to identify residues that might potentially improve the JFH-1 infectivity. A sequence alignment was performed as presented in Fig. 4 and differences in the amino acid sequence of JFH-1 capsid were identified at positions 20, 48, 81, 145, 151, 152, 172 and 173. We were particularly interested by the differences in amino acids at positions 172 and 173 because they correspond to drastic mutations in the context of JFH-1. Furthermore, another study relating to the genotype 2a capsid protein has shown that some modifications in the C-terminal 31 aa of core protein were important for its processing and/or its morphogenesis (Kato et al., 2003b). Consequently, the mutations F172C and P173S (FPCS) were introduced by site-directed mutagenesis in JFH-1 capsid-coding sequence to determine whether the release of infectious viral particles could be improved (Fig. 5a). HCV RNA was quantified and the secretion of particles was analysed by successive passages of the transfected cells or by successive infections on naïve Huh-7 cells as initially described in this study (Table 1). High viral titres of JFH-1/CS were obtained faster and were higher than those obtained with JFH-1. These data suggest that the replacement of FP to CS residues confers an advantage for the virus and these modifications might improve the morphogenesis and/or the release of viral particles.
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). We next wanted to determine whether the JFH-1/CS mutant might affect the subcellular localization of the capsid protein, leading to some colocalization with the envelope proteins. As shown in Fig. 5(b), no differences were observed between the two viruses in the intracellular distribution of the capsid protein and E2 glycoprotein. For both clones, E2 colocalized with ER markers, whereas capsid protein was associated with lipid droplets (data not shown). In addition, no differences were observed in the capsid protein when analysed by Western blotting (Fig. 5c). Consequently, no detectable differences in the capsid processing and intracellular localization were observed that could explain the enhanced production of viral particles.
N534K, F172C and P173S mutations improve the infection of JFH-1 in Huh-7 cells
In order to produce a higher infectious JFH-1 virus in cell culture, we introduced three mutations N534K, F172C and P173S into JFH-1 (JFH-1/CS-N6). Moreover, to visualize more efficiently the infectivity of this mutant on cell culture, Huh-7 cells were transfected with the in vitro transcribed JFH-1, JFH-1/N6, JFH-1/CS and JFH-1/CS-N6 RNAs (Fig. 6a) and the supernatants obtained at 3 days post-transfection were used in the infection of naïve Huh-7 cells (Fig. 6b). The profile of E2 produced by each virus was also analysed before and after treatment with PNGase F and was consistent with predicted results (Fig. 3b). Our results suggest that the JFH-1/CS-N6 virus expands more rapidly and reaches higher titres than the JFH-1, JFH-1/N6 and JFH-1/CS viruses (Fig. 6b and Table 1). As for JFH-1/CS, no differences were observed in the intracellular distribution of the capsid protein and E2 glycoprotein of JFH-1/CS-N6 (data not shown). Furthermore, we also observed that after a single round of amplification the JFH-1/CS-N6 virus displayed an accelerated cytopathic effect, which was faster than what we observed for the JFH-1/CS virus (data not shown). These data suggested that the combined mutations located in the capsid- and E2-coding sequences resulted in an enhanced virus infectivity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Different groups have developed robust cell culture systems for the propagation of HCV, and the data led to the conclusions that the Huh-7 cell culture is important for the propagation of the virus and that each Huh-7 cell line can display different permissiveness to the virus. In this study, we established a strong production of infectious HCV particles by using successive infections of naïve Huh-7 cells or by introducing specific mutations into the JFH-1 genome.
Contrary to what was observed in other groups (Lindenbach et al., 2005; Zhong et al., 2005), the robust production of HCVcc in our Huh-7 cell line was only obtained after several successive infections. Initially, the JFH-1 virus released into the culture medium after the first transfection was low, which was consistent with a previous report (Wakita et al., 2005). It is worth noting that several passages of transfected cells did not change the viral titre. However, after repeated infections of naïve Huh-7 cells, analyses of HCV RNA in the medium of infected cells revealed an increase in the particle release, which was correlated with a higher titre of infectious virus. The direct sequencing of HCV RNA genome was used to determine the major modifications appearing in JFH-1. Surprisingly, the only coding mutation identified was an Asn to Lys mutation located at aa 534 (N534K) in E2. This modification, which is characterized as preventing the addition of an N-glycan at the E2 glycosylation site 6, may favour the interaction between HCV E2 glycoprotein and a cellular receptor. Indeed, the introduction of this mutation in JFH-1 leads to a higher infectious titre after only two successive infections. This suggests that the particles produced by JFH-1/N6 are more effective than those produced by JFH-1 for the reinfection of Huh-7 cells. This is also true when the mutation N534K is combined with the modifications FPCS at positions 172 and 173 in JFH-1 (Fig. 6b). Consequently these mutations might lead to a better viral expansion on Huh-7 cells. There is some evidence that CD81 is essential for the entry of HCVpp (Hsu et al., 2003; Lavillette et al., 2005) or HCVcc (Lindenbach et al., 2005; Zhong et al., 2005) into hepatoma cells via an interaction with the HCV E2 glycoprotein (Pileri et al., 1998). Residues critical for the CD81 binding have been identified in the HCV glycoprotein E2. These residues are located at positions 420, 437, 438, 441, 442, 529, 530 and 535 of E2 glycoprotein (Drummer et al., 2006; Owsianka et al., 2006). Moreover, replacement of Thr at the E2 glycosylation site 6 results in moderately increased CD81 binding (Owsianka et al., 2006). This is consistent with the hypothesis that the loss of the N-linked E2 glycosylation site 6 favours a better exposure of E2 to the E2-binding site of CD81 and then the JFH-1 reinfection. In a similar way, Zhong et al., (2006) have identified a mutation located at aa 451 of HCV E2 glycoprotein, which displays an accelerated spreading of the virus. This G451A mutation is located between HVR1 and HVR2 of E2 in a region that has been reported to modulate the accessibility of the CD81-binding site (Roccasecca et al., 2003).
In the present study, the substitution of amino acids FPCS at positions 172 and 173 of the capsid protein leads to an increased viral production. The amino acids Cys and Ser appear to be important determinants for the spreading of the virus and they are probably involved in the morphogenesis and/or the release of the viral particles. The C172 and S173 residues are highly conserved among the HCV isolates. In a former in vitro study, we showed that several amino acids located at the C terminus of the capsid protein were important for the mature p21 protein, and FPCS mutations showed a higher level of the immature capsid protein (p23) (Kato et al., 2003b). The hydrophobic sequence at the C terminus of the capsid protein was described as the signal sequence necessary for the translocation of E1 glycoprotein into the ER lumen (Hijikata et al., 1991). Recently, this signal sequence was also described as a substrate for signal peptide peptidase (SPP) (McLauchlan et al., 2002). During the assembly of the virus, two consecutive membrane-dependent cleavages are responsible for the production of p23 and p21 forms of the capsid protein (Liu et al., 1997). Detected in particles isolated from the blood of infected patients (Yasui et al., 1998), the p21 mature capsid protein is produced by cleavage of the p23 immature capsid protein by a cellular protease identified as SPP (Hussy et al., 1996; Lemberg & Martoglio, 2002; McLauchlan et al., 2002). It may be hypothesized that the production of p23 and then p21 is important for the morphogenesis of the virus and the production of infectious viral particles. In this hypothesis, the immature capsid protein p23 would be necessary for an initial step of the particle formation, for example its accumulation and its oligomerization at the ER membrane where a progressive maturation would be introduced by SPP cleavage. The completion of the maturation of the viral particle could then occur after the budding process. However, immature capsid protein (p23) has not been detected in an in vivo study using JFH-1/CS probably due to the low production or a completion of the cleavage during the preparation of cell extracts. Additional experiments have to be conducted to understand the function of these amino acids in the morphogenesis of the viral particle.
In conclusion, the data presented in this study show that few modifications are sufficient for a more efficient production of HCVcc in Huh-7 cells. These mutations are located in the structural proteins and likely affect the recognition of a cellular receptor and/or the morphogenesis of the viral particle. Extensive modifications introduced in the C terminus of the capsid protein and analyses of the resulting viruses will help the understanding of the role of individual amino acids in particle assembly.
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ACKNOWLEDGEMENTS |
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Received 23 January 2007;
accepted 1 May 2007.
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