1 Station de Pathologie Comparée, INRA-CNRS, 30380 Saint-Christol-lès-Alès, France
2 CERVI Laboratoire de Virologie, UPRES EA 2387 Hospital Pitié-Salpétrière, 83 boulevard de l'Hôpital, 75651 Paris Cedex 13, France
3 Unité Mixte CNRS-bioMérieux, École Normale Supérieure, 69000 Lyon, France
Correspondence
Martine Duonor-Cérutti
duonor{at}ensam.inra.fr
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ABSTRACT |
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INTRODUCTION |
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No mammalian cell lines are known that allow efficient replication of this virus (Bertolini et al., 1993; Shimizu et al., 1993
; Shimizu & Yoshikura, 1994
; Tagawa et al., 1995
; Kato et al., 1995
, 1996
; Nakajima et al., 1996
). Infection of primary cultures of either hepatocytes or lymphocytes resulted only in limited virus replication with, in some cases, release of viral particles into the culture medium (Ito et al., 1996
; Iacovacci et al., 1997
).
The absence of a suitable cellular model for HCV propagation has led to the use of various heterologous expression systems. Among these, the baculovirusinsect cell system has proved useful for analysis of the production and maturation of HCV structural proteins (Matsuura et al., 1992, 1994
; Lanford et al., 1993
; Hsu et al., 1993
; Hüssy et al., 1996a
, 1997
) and non-structural proteins (Hirowatari et al., 1993
; Nishihara et al., 1993
; Overton et al., 1995
; Suzuki et al., 1995
; Hwang et al., 1997
). In addition, this system is a valuable tool (i) for the production of large amount of antigens for immunological studies (Chiba et al., 1991
; Chien et al., 1992
; Inoue et al., 1992
; Basset et al., 1999
) and (ii) for the investigation of viral protein interactions and assembly (Hüssy et al., 1996b
; Wang et al., 1997
). Using recombinant baculovirus, Baumert et al. (1998)
showed that pseudovirus particles could be generated.
The 5'NTR is a highly conserved region, 341349 nt in length, thought to fold into a complex secondary and tertiary structure comprising four major domains, I to IV, a pseudoknot and a helical structure (Brown et al., 1992; Honda et al., 1996
; Smith et al., 1995
; Le et al., 1995
; Wang et al., 1995
). The 5'NTR of HCV contains several AUG codons (three to six, depending on the genotype), two of which (at positions 85 and 215) are highly conserved between HCV and pestiviruses. This structure functions as an internal ribosome entry site (IRES), probably as a type II element (Reynolds et al., 1995
). It also binds different cellular factors such as polypyrimidine-tract-binding (PTB) protein (Ali & Siddiqui, 1995
) and proteins of 25, 87 and 120 kDa (Fukushi et al., 1997
; Yen et al., 1995
), which are thought to be important (except for p87) for IRES function. The core protein is an RNA-binding protein with RNA-binding domains localized to the N-terminal 75 amino acids (Santolini et al., 1994
), and a specific interaction between the core protein and the 5' NTR has been reported (Shimoike et al., 1999
).
Recombinant baculoviruses able to transcribe wild-type HCV RNAs, RNAs mutated in the 5'NTR or with this region deleted were used to analyse the role of this domain in the assembly process. We present evidence of the involvement of the 5'NTR in the self-assembly of HCV structural proteins in insect cells.
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METHODS |
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Construction of plasmids.
Two baculovirus transfer vectors, p118, derived from pGm8022 (Chaabihi et al., 1993; a kind gift of M. H. Ogliastro), and pGmAc118, were used to generate recombinant viruses. pGmAc118 has a BglII linker inserted at a deletion in the polyhedrin gene sequence spanning nucleotides 10 to +483 (position +1 is the first nucleotide of the polyhedrin ATG start codon). This vector presents a deletion in the polyhedrin promoter sequence in order to decrease the expression level of the foreign gene (Gaymard et al., 1996
). Different constructs (Fig. 1
) were made using the cDNA of the HCV-H strain, genotype 1a (Inchauspé et al., 1991
; Simmonds et al., 1993
).
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(ii) p118-5'NTR-E1.
A 1369-bp BamHI fragment containing the 5'NTR and the sequence encoding the capsid protein and 149 aa of the glycoprotein E1 was inserted into the unique BglII site of p118-stop plasmid (p118 with a stop codon downstream of the BglII site), giving a plasmid called p118-5'NTR-E1.
(iii) pGmAc-5'NTRm-NS2.
A DNA fragment of 256 bp containing a modified 5'NTR with mutations of the five silent AUG codons (5'NTRm) was reconstituted using a set of 14 overlapping oligonucleotides. Mutations were chosen in order to create convenient sites for subsequent cloning steps, but not to preserve the secondary structure of the 5'NTR (Table 1). A 2987-bp NheIBglII fragment containing 93 bp of the wild-type 5'NTR and the sequence encoding C, E1, E2 p7 and 158 aa of NS2 was introduced downstream of the mutated 5'NTR in the transfer vector pGmAc118. A BglII linker inserted in the NS2 gene introduced an in-frame stop codon, resulting in plasmid pGmAc-5'NTRm-NS2
.
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Protein analysis.
Infected cells were collected and washed with cold PBS and resuspended in reducing sample buffer (Laemmli, 1970). After boiling (100 °C for 5 min), protein samples were resolved by 12·5 % SDS-PAGE under denaturing conditions (Laemmli, 1970
). The proteins were then transferred onto a nitrocellulose filter (BAS 85, 0·45 µm; Schleicher & Schuell) with a semi-dry electroblotter apparatus (Ancos). The nitrocellulose membrane was stained with Ponceau red (Ponceau-S; Sigma) and subsequently blocked with a solution of Tris-buffered saline (TBS; 0·05 M Tris/HCl, pH 7·4, 0·2 M NaCl) containing 0·05 % Tween 20 and 5 % dry skimmed milk (TBS-sat). HCV capsid protein was detected using the mouse monoclonal antibody 27D5C5 (1 : 500 in TBS-sat) as the primary antibody and a rabbit anti-mouse IgGhorseradish peroxidase conjugate as the secondary antibody (1 : 1000 in TBS-sat; Sigma). Glycoprotein E1 was detected by using a rabbit polyclonal anti-E1 hyperimmune serum (1 : 200 in TBS-sat) as the primary antibody and a goat anti-rabbit IgGhorseradish peroxidase conjugate or a mouse anti-rabbit IgGalkaline phosphatase conjugate as the secondary antibody (1 : 10 000 in TBS-sat; Sigma). Glycoprotein E2 was detected by using a rabbit polyclonal anti-E2 hyperimmune sera (1b8356 or WU105, 1 : 500 in TBS-sat) as the primary antibody and a goat anti-rabbit IgGhorseradish peroxidase conjugate or a mouse anti-rabbit IgGalkaline phosphatase conjugate as the secondary antibody. Blots were also probed with anti-HCV-positive human serum (1 : 500 in TBS-sat) containing antibodies against the three structural proteins and revealed using an anti-human Igalkaline phosphatase conjugate. Immunoreactive bands were visualized by using either 1-amino-3-ethyl carbazol or NBT/BCIP as chromogenic agents.
Evaluation of glycosylation.
Tunicamycin at a final concentration of 10 µg ml1 was added to the cell culture medium at 7 h post-infection (p.i.). Infected cells were collected at 52 h p.i. and protein samples were analysed by Western blotting as described above. As a control, cells were infected under the same conditions but without tunicamycin.
Isolation of total RNA and Northern blot hybridization.
Isolation of total RNA was performed using Trizol reagent (Gibco-BRL) according to the manufacturer's instructions followed by chloroform extraction and isopropanol precipitation. Total RNA was resuspended in RNase-free water and quantified by spectrophotometry. After addition of RNA sample buffer (Promega), 10 µg of each sample was electrophoresed on a 1 % non-denaturing agarose gel and transferred to a nylon membrane (Roche). Blots were hybridized with a 417-bp probe (encompassing 205 bp at the 3'-end encoding the capsid protein and 212 bp at the 5'-end encoding glycoprotein E1) radiolabelled with 32P using a random-primed DNA-labelling kit (Roche).
Isopycnic centrifugation on sucrose gradients.
At 48 or 72 h p.i., cells were resuspended in cold low-salt buffer (10 mM HEPES/NaOH, pH 7·9, 10 mM KCl, 1·5 mM MgCl2, 0·5 % NP40) with protease inhibitors (O-Complete; Roche) and were disrupted with a potter (Bioblock Scientific). After low-speed centrifugation (10 min at 4 °C and 10 000 g), the supernatant was layered onto a linear 2060 % (w/w) sucrose gradient (in TEN buffer; 50 mM Tris/HCl, pH 7·4, 100 mM NaCl, 5 mM EDTA) and subjected to isopycnic centrifugation for 24 h at 4 °C and 150 000 g. Fractions (500 µl) were collected from the bottom and analysed by Western blotting. In some experiments, 1 % NP40 was added to the crude protein extracts before layering onto the sucrose gradient.
Particles present in the cell culture supernatant were isolated as follows: after low-speed centrifugation (10 min at 4 °C and 10 000 g) the supernatant was pelleted (12 h at 4 °C and 100 000 g). This pellet was then resuspended in TEN (containing protease inhibitors) and subjected to isopycnic sucrose gradient centrifugation. Under these conditions, the supernatant was concentrated 20- to 100-fold. Fractions (500 µl) were collected and analysed as described below.
Electron microscopy.
Antigen-positive fractions isolated by isopycnic centrifugation were examined directly with a Zeiss EM 10C/CR electron microscope after negative-staining with uranyl acetate. For morphological studies, cells were harvested at 48 or 72 h p.i. by centrifugation at 1000 g for 5 min, washed with PBS and fixed with 2 % glutaraldehyde in 0·1 M cacodylate buffer, pH 7·4. After post-fixation with 2 % osmium tetroxide in the same buffer, cells were dehydrated in a graded series of ethanol solutions followed by propylene oxide and embedded in Epon 812 resin (Fluka). Ultrathin sections were stained with uranyl acetate and lead citrate before observation.
For immunogold labelling, cells were fixed with 4 % paraformaldehyde/0·25 % glutaraldehyde in PBS. Two different sample preparation methods were then used. (i) Following dehydration in ethanol solutions, cells were embedded in Unicryl resin (TEBU) and polymerized under a UV source at 4 °C for 2 days; ultrathin sections were cut with a diamond knife in an LKB Ultrotome III apparatus. (ii) Preparation of cryosections was performed as described by Reggio & Boller (1989). Briefly, after washing in PBS/50 mM NH4Cl, cells were embedded in gelatin, kept on ice for 2 h and allowed to infuse for at least 30 min in 2·3 M sucrose (in 0·1 M phosphate buffer, pH 7·2). After rapid freezing of the sample in liquid nitrogen, ultrathin sections were cut using an RMC Ultracryotom MT-7 apparatus. Ultrathin sections prepared as described in (i) or (ii) were collected on nickel grids and pre-incubated for 10 min in NH4Cl (50 mM in PBS) before saturation for 30 min in PBS containing 1 % casein. Preparations were incubated for 2 h with anti-HCV patient serum (1 : 50 in 0·1 % casein/PBS), washed three times with PBS and probed with gold-conjugated anti-human Ig (1 : 50 in 0·1 % casein/PBS). After several washes with PBS and distilled water, sections were stained with uranyl acetate and observed. Cryosections were covered with a film of 70 % methylcellulose in uranyl acetate.
Alternatively, infected cells were immunolabelled after permeabilization with Triton X-100 (13 % in PBS). Cells were incubated with the anti-capsid polyclonal antibody WU105 (1 : 100 in 0·1 % casein/PBS) and gold-conjugated secondary anti-rabbit IgG (10 nm; Biocell) diluted 1 : 50 in 0·1 % casein/PBS before subsequent treatment for morphological analysis, as described before.
Cell lysis control.
Lysis of infected cells was assessed using the Promega LDH detection kit (Cytotox 96 non-radioactive cytotoxicity assay).
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RESULTS |
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TEM observations of cells infected with AcSLP10-5'NTR-E1 showed all of the characteristic features found in cells infected with a recombinant baculovirus, nuclear hypertrophy, the presence of large amounts of virus in the nucleus and the absence of polyhedrin or P10 structures (Fig. 3a). Neither reticulate cytoplasmic retention nor aggregate formation were noticed, and no intracellular proteins were immunolabelled with the anti-HCV-positive human serum (data not shown), confirming the Western blot results.
Trans-acting effect of the 5'NTR on capsid protein multimerization
The interaction between the 5'NTR and capsid protein was analysed by coexpressing the two baculoviruses AcSLP10-5'NTR-E1 and AcSLP10-C-E1, thus combining a 5'NTR-containing, non-translatable transcript with a 5'NTR-deleted, translation-competent transcript. Isopycnic centrifugation analysis of crude cellular extracts showed a new distribution of the capsid protein and CE1 polypeptide in the gradient (compare Fig. 4a and b). While C and CE1 proteins sedimented at a density of 1·211·26 g ml1 when expressed in cells infected solely with AcSLP10-C-E1, the two proteins sedimented at a density of 1·151·18 g ml1 in double-infected cells also expressing the RNA transcript 5'NTR-C-E1. This shift in the sedimentation profile could be explained by the formation of lipid-associated multimers. This hypothesis was confirmed by treatment of the crude extract with a mild non-ionic detergent (1 % NP-40) prior to analysis of the cellular extract on the sucrose gradient. In this case, immunoreactive material was detected at higher density (1·241·26 g ml1; Fig. 4c
).
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TEM observation of cells infected with the recombinant AcPH-5'NTRm-NS2 showed a perinuclear ring of very dense material probably resulting from the accumulation of HCV structural proteins associated with the ER (Fig. 5d
). Immunolabelling of cryosections of infected cells with the anti-HCV-positive human serum confirmed the colocalization of HCV proteins in this cytoplasmic area. Faint labelling was also observed in the nuclei (not shown).
Analysis of cytoplasmic extracts prepared from cells infected with AcPH-5'NTRm-NS2 showed sedimentation of immunoreactive proteins in fractions with a density of 1·141·18 g ml1 when probed with the anti-HCV-positive human serum (Fig. 6a
). Under these conditions, the major product detected was the capsid protein, essentially as the p21 processed form. Treatment of cell extracts with NP40 (1 % final in the cell lysis buffer) resulted in a shift of the particle density from 1·141·18 g ml1 to 1·231·26 g ml1 (Fig. 6b
). TEM observation of these anti-HCV-positive fractions after negative staining showed icosahedral particles of about 30 nm diameter (Fig. 7a
). The difficulties encountered in observing complete particles may be related to their high intrinsic instability or to their high sensitivity to the purification process.
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To verify that particles were not released in the cell culture medium as a consequence of premature cell lysis, the time-course of secretion of the HCV capsid and of a cytosolic control protein, lactate dehydrogenase (LDH), was followed using Western blot analysis and an LDH detection kit (Fig. 7b). Our results indicated that core protein could be detected in the culture medium as early as 30 h p.i. At this time, cell lysis could not account for release of cellular components. Moreover, detailed observation of ultrathin sections of cells infected with AcPH-5'NTRm-NS2
and labelled with the anti-HCV-positive human serum revealed budding of some enveloped, immunogold-decorated particles (Fig. 7b
). This phenomenon might have occurred via an exocytosis process, as described for other members of the family Flaviviridae (Rice, 1996
).
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DISCUSSION |
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We investigated the effect of the 5'NTR on translation and assembly of HCV genotype 1a structural proteins in baculovirus-infected cells. When the 5'NTR of HCV was maintained in cis, i.e. upstream of the CE1 coding region, no production of structural proteins was observed in insect cells, despite efficient transcription of this region. Deletion of the 5'NTR led to the expression of capsid and E1 proteins with the expected sizes, 21 kDa for the capsid protein and 3035 kDa for the fully processed E1 glycoprotein, as reported previously (Baumert et al., 1998). Moreover, mutation of the five AUG codons present in the 5'NTR restored translation of HCV structural proteins. Taken together, these data suggest that, in insect cells, initiation of translation occurs via a ribosome-scanning process, at the first AUG codon encountered, and not by an internal ribosome entry process. In mammalian expression systems, at least three proteins, proteins of 25 and 120 kDa and the PTB protein, have been shown to bind to the HCV 5'NTR. Inhibition of their binding led to a significant reduction in translation efficiency (Ali & Siddiqui, 1995
; Fukushi et al., 1997
; Yen et al., 1995
). One could then hypothesize that specific cellular factors are required for proper initiation of translation by an internal ribosome entry process and that such factors are lacking in infected insect cells.
The high density (1·211·26 g ml1) of the capsid protein and CE1 polypeptide in sucrose gradients when expressed in the absence of the 5'NTR-CE1 transcript could probably be the result of the association of these proteins with the rough ER (Santolini et al., 1994) and/or of multimer formation. In fact, it has been shown that the core protein is capable of homotypic interaction and multimerization both in vivo and in vitro (Matsumoto et al., 1996
). Further analysis revealed that coproduction of structural proteins with the 5'NTRCE1 transcript in trans or mutated 5'NTR in cis led to a shift in density of the anti-HCV-positive material compared with protein density in the absence of the 5'NTR. Instead of sedimenting at a density corresponding to a multimer or a rough ER-associated protein (1·211·26 g ml1), core protein was found at a density of 1·151·18 g ml1, similar to HCV pseudoparticle density previously reported in a baculovirus expression system (Baumert et al., 1998
). TEM observation showed formation of VLPs labelled with the anti-core-positive antibody in the perinuclear area of the cytoplasm of cells coproducing the C and E1 proteins and the 5'NTRCE1 transcript in trans. Incorporation of partially uncleaved structural polypeptide into the envelope of those particles may be responsible for their morphological heterogeneity. Khromykh et al. (1998)
reported the trans-encapsidation of a Kunjin replicon RNA by Kunjin structural proteins, describing for the first time interaction between RNA and structural proteins of a flavivirus. Our results showed that similar RNAprotein interactions could have functional importance for HCV VLP assembly in our heterologous system. In a previous study, Wang et al. (1997)
reported that HCV genotype 1b RNA representing the 5'NTRC (short RNA transcript) or CE1E2p7 (missing the 5'NTR in cis) region failed to induce the formation of core particles. Such a discrepancy might be explained by differences (i) in the genotype of the HCV cDNA, (ii) in the constructs used for the expression of structural proteins or (iii) in the constructs used to produce the 5'NTR transcript. A minimal size of the RNA (5'NTRCE1) or sequence elements encompassing more than one packaging signal potentially present on the HCV genome may be necessary for efficient interaction with structural proteins. Indeed, this was observed for a murine leukaemia retrovirus (MoLV) which has a primary encapsidation signal in the 5'NTR and an extended encapsidation signal in the gag ORF; this latter is responsible for increased viral RNA packaging efficiency and for viral titre enhancement (Bender et al., 1987
). Moreover, we cannot exclude that the length of the encapsidated genomic RNA molecule might have a direct effect on assembly. When wild-type intact proteins were produced in cells infected with a virus bearing a mutated 5'NTR (AcPH-5'NTRm-NS2
), the same capsid protein sedimentation profile was observed in lysates prepared from cells coinfected with AcSLP10-C-E1 and AcSLP10-5'NTR-E1. The weak representation of the E1 and E2 glycoproteins in the core-positive fractions may reflect their low incorporation in the VLP. This could be due to (i) inefficient processing and/or (ii) retention of these proteins in an aggregated form in the ER, as reported previously (Deleersnyder et al., 1997
). In addition, the formation of native HCV glycoprotein complexes could represent a limiting step in particle morphogenesis, this process being more or less efficient from one genotype to another. Treatment of the pseudoparticles with NP40 resulted in disruption of membranenucleocapsid interactions and the alteration of their sedimentation profile: the anti-HCV-positive fractions were found at a density of 1·241·26 g ml1. This value is in good agreement with the density (1·25 g ml1) reported for human-serum-derived HCV nucleocapsid treated with the same detergent (Miyamoto et al., 1992
). TEM observation of these fractions reveals the presence of few, probably unstable icosahedron-shaped particles about 30 nm in diameter. Finally, weak secretion of VLPs into the cell culture medium was observed for cells infected with AcPH-5'NTRm-NS2
. This release occurred early, before cell lysis became generalized (as estimated by LDH activity of the cell culture supernatant). In addition, genuine budding has been assessed by EM (Fig. 7g, h
). Such extracellular release of HCV VLPs in insect cells has not so far been reported, to our knowledge. As no VLPs could be observed in the supernatant of cells coproducing the CE1 structural region and the 5'NTR-C-E1 RNA transcript, we hypothesize that correct self-assembly can only be achieved when the 5'NTR is located in cis or that the absence of some HCV structural elements results in a lower stability of HCV VLP.
In this study, we present evidence that the 5'NTR of HCV genotype 1a is implicated in the assembly process of HCV structural proteins in insect cells, although we have no evidence for a direct interaction between the 5'NTR and these structural proteins. Nevertheless, the use of this heterologous baculovirus system for a better understanding of the mechanism of HCV assembly may constitute an interesting alternative to mammalian cell systems, especially in regard to the lack of an efficient cellular system for the replication of the virus.
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ACKNOWLEDGEMENTS |
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Received 16 December 2003;
accepted 11 August 2004.
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