Hepatitis C virus IRES efficiency is unaffected by the genomic RNA 3'NTR even in the presence of viral structural or non-structural proteins

Isabelle Imbert, Maria Dimitrova, François Kien, Marie Paule Kieny and Catherine Schuster

INSERM U544, Institut de Virologie, 3 rue Koeberlé, 67000 Strasbourg, France

Correspondence
Catherine Schuster
Catherine.schuster{at}viro-ulp.u-strasbg.fr


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis C virus (HCV) translation is mediated by an IRES structure. Instead of a poly(A) tail, the 3' end of the genome contains a tripartite 3'NTR composed of a non-conserved region, a polypyrimidine tract and a highly conserved stretch of 98 nt, termed the 3'X region. Using a set of bicistronic recombinant DNA constructs expressing two reporter genes separated by the HCV IRES, it was determined whether the HCV 3'NTR sequence, in the presence or absence of HCV proteins, played a role in the efficiency of HCV IRES-dependent translation ex vivo. Bicistronic expression cassettes were transfected into hepatic and non-hepatic cell lines. These results show that neither the entire 3'NTR nor the 3'X sequence alters IRES-dependent translation efficiency, whatever the cell line tested. A potential effect of the 3'NTR on IRES-dependent translation in the presence of HCV proteins was investigated further. Neither non-structural nor structural HCV proteins had any effect on the efficiency of IRES in this system. In addition, in order to mimic HCV genome organization, monocistronic expression cassettes containing the IRES and a Core–DsRed fusion gene were constructed with or without the 3'NTR. In this context, no effect of the 3'NTR on IRES translation efficiency was observed, even in the presence of HCV proteins. These data demonstrate that HCV translation is not modulated by the viral genomic 3'NTR sequence, even in the presence of HCV structural or non-structural proteins.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis C virus (HCV) belongs to the family Flaviviridae, genus Hepacivirus, and is the major aetiological agent of non-A, non-B hepatitis (Choo et al., 1989). The HCV genome consists of a single-stranded, positive-sense RNA of approximately 9·6 kb in length. The open reading frame (ORF) encodes a unique polyprotein and is flanked by 5'- and 3'NTRs. These sequences are the most conserved regions of the HCV genome, which reflects their importance for both virus replication and translation (reviewed by Bartenschlager & Lohmann, 2000). The 5'NTR contains the IRES, which mediates viral RNA translation by a cap-independent mechanism (Tsukiyama-Kohara et al., 1992). The IRES recruits the 40S ribosomal subunit to an internal initiation codon in the mRNA without scanning phenomenon and without the need for additional canonical or non-canonical translation factors (Pestova et al., 1998). This is a unique feature among eukaryotic RNAs and viral IRESs, and it resembles translation initiation in prokaryotic cells where ribosomes recognize directly the Shine–Dalgarno sequence (Shine & Dalgarno, 1974). After translation, the HCV precursor polyprotein is cleaved co- and post-translationally by cellular and viral proteases to yield mature structural (Core, E1 and E2) and non-structural (NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins (reviewed by Reed & Rice, 2000).

The HCV 3'NTR, recognized by the viral RNA-dependent RNA polymerase, is composed of three distinct regions: a short non-conserved sequence of variable length (42–287 nt), a polypyrimidine tract, poly(U/UC) (20–200 nt) and a sequence of 98 nt, termed 3'X (Tanaka et al., 1995; Kolykhalov et al., 1996). The 3'X region is very conserved and is structured in three stem–loops (Blight & Rice, 1997). Yanagi et al. (1999) and Kolykhalov et al. (2000) have shown that both the 3'X conserved region and the polypyrimidine tract are essential for the in vivo infectivity of the virus.

It has been suggested that HCV RNA translation is regulated by at least four distinct elements. Firstly, in vitro studies suggest that several cellular proteins (PTB, hnRNP L and La) can stimulate HCV translation (Ito & Lai, 1999; Anwar et al., 2000; Hahm et al., 1998; Ali & Siddiqui, 1997). However, the elements required for the functionality of the IRES have not been identified clearly. Secondly, the sequence of the IRES is a prominent factor for translation efficiency. Indeed, some mutations result in dramatic changes in IRES activity (Kamoshita et al., 1997; Collier et al., 1998; Honda et al., 1999; Laporte et al., 2000; Lerat et al., 2000). Thirdly, HCV proteins have been described to affect HCV IRES efficiency. Indeed, the HCV Core protein (Shimoike et al., 1999; Zhang et al., 2002) and the non-structural proteins NS4A and NS4B were shown to decrease HCV IRES translation (Kato et al., 2002). Finally, the X region at the 3' end of the HCV genome has been shown to enhance IRES-dependent translation weakly (Ito et al., 1998; Michel et al., 2001), although the mechanism for this enhancement has not been elicited yet and remains controversial. Indeed, Fang & Moyer (2000) have demonstrated that the presence or absence of the 3'NTR sequence did not affect translation efficiency. Moreover, Murakami et al. (2001) have observed recently that the complete 3'NTR downregulated HCV translation in vitro. This inhibition was removed when the poly(U/UC) or stem–loop III regions of the 3'NTR were deleted.

Our study focuses on two of these potential translation regulation mechanisms.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cell culture and transfection.
Human hepatocellular carcinoma cell lines HepG2 and Hep3B as well as HEK 293 (human embryonic kidney) were obtained from the ATCC. The Huh-7 cell line was kindly provided by R. Bartenschlager (University of Heidelberg, Germany). HepG2 and Huh-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % FCS, 50 µg gentamicin ml-1, 0·1 mM non-essential amino acids, 2 mM L-glutamine and 1 mM sodium pyruvate. Hep3B cells were grown in Eagle's MEM with Earle's salts and 2 mM L-glutamine containing 1 mM sodium pyruvate, 0·1 mM non-essential amino acids, 1·5 g sodium bicarbonate l-1, 50 µg gentamicin ml-1 and 10 % FCS. HEK 293 cells were maintained in DMEM supplemented with 10 % FCS, 50 µg gentamicin ml-1, 0·1 mM non-essential amino acids and 2 mM L-glutamine. LS1056-52(2) cells were maintained in MEM{alpha} supplemented with the same components as for the HEK 293 cell line plus 100 µg G418 ml-1 and 75 µg hygromycin ml-1 (Gibco-BRL). Medium, serum and cell culture additives were purchased from Gibco-BRL.

Monolayers of 293 cells or stable 293-derived cell lines were cultivated in 60 mm Petri dishes and transfected with 20 µg DNA using calcium phosphate precipitation (Profection Mammalian Transfection system; Promega). Huh-7, Hep3B and HepG2 cells were transfected using the Lipofectamine Plus method (Invitrogen), as indicated by the manufacturer. Plasmids pIV1171 and pIV1172 were co-transfected with an equimolar amount of commercial plasmid pEGFP-C1 (Clontech) as control.

Plasmids
Plasmid pIV1013.
Plasmid pIV1013 was obtained by inserting a dicistronic expression cassette into a pCIneo plasmid vector (Promega). A fragment containing the 5'NTR and the first 390 nt of the Core-encoding sequence of HCV (nt 342–732 in HCV strain H, genotype 1a) was amplified by PCR using p90/HCV FL-Long pU as template (Kolykhalov et al., 1997). The PCR fragment (5'NTR–390 nt Core) obtained was then inserted by ligation into the EcoRI/SalI sites of the pCIneo vector. The resulting plasmid is called pIV1010. The gene encoding DsRed, the red fluorescent protein from pDsRed-N1 (Clontech), was obtained by restriction digestion and cloned in frame with the Core sequence by ligation into the SalI/NotI sites to generate pIV1011. The 3'NTR of the HCV genome was obtained by PCR amplification of the region encompassing nt 9200–9646. The fragment amplified contains the last 178 nt of HCV NS5B and the entire 3'NTR. This fragment was then inserted into the unique NotI site of pIV1011, generating pIV1012. Finally, an EcoRI fragment encoding EGFP (enhanced green fluorescent protein) was excised from plasmid pEGFP-C1 and inserted into the EcoRI site of pIV1012 to create pIV1013.

Plasmid pIV1018.
This plasmid differs from pIV1013 by the presence of a Core sequence restricted to the first 200 nt of the HCV Core sequence.

Plasmids pIV1014 and pIV1019.
To generate plasmids pIV1014 and pIV1019, the NotI fragment containing the 3'NTR was deleted from pIV1013 and pIV1018, respectively.

Plasmid pIV1086.
This plasmid derives from plasmid pIV1018. The NotI site of pIV1018 at the 5' end of the 3'NTR was eliminated by partial digestion and filled by Klenow polymerase such that the plasmid could be linearized by NotI digestion, directly after the HCV 3'NTR.

Plasmids pIV1171 and pIV1172.
Plasmids pIV1171 and pIV1172 derive from plasmid pIV1086 and pIV1019, respectively, and were obtained by deletion of the EGFP gene.

Plasmid pIV1060.
To construct pIV1060, the 3'NTR of plasmid pIV1018 was substituted by the 3'X region, which was obtained by PCR amplification of nt 9549–9646 from p90/HCV FL-Long pU. This fragment was then inserted into the unique NotI site of pIV1019, restricted previously by NotI.

A schematic view of the plasmids is presented in Fig. 1.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1. Schematic representation of the bicistronic cassettes. All numbers refer to nucleotide positions in the HCV strain H sequence (genotype 1a) in p90/HCV FL-Long pU (Kolykhalov et al., 1997).

 
Recombinant virus vectors.
Recombinant adenovirus AdIV1053 encodes all six HCV non-structural proteins of HCV strain H, genotype 1a (M. Dimitrova, I. Imbert, M. P. Kieny and C. Schuster, unpublished results).

Recombinant vaccinia virus (Copenhagen strain) vvIV205 expresses the HCV strain H structural protein sequence (Core–E1–E2) (Kien et al., 2003).

Preparation of adenovirus and titration.
Virus propagation and titration by indirect immunofluorescence of the adenoviral DNA-binding protein were carried out as described by Lusky et al. (1998). Titres of infectious virus progeny were determined as infectious units (IU) by quantitative DNA-binding protein immunofluorescence (Reich et al., 1983).

Establishment of an inducible cell line expressing HCV non-structural proteins.
A continuous human cell line expressing all HCV non-structural proteins upon induction by doxycycline (Dox) was generated using the Tet-On Gene Expression system (Clontech). In brief, pIV1056 was generated as follows: first, a PCR fragment encoding the N-terminal region of NS2 (nt 2769–3110) from p90/HCV FL-Long pU cut previously by NheI/MluI was inserted into the MluI/NheI-digested pBI-L vector to generate pIV1002. A second PCR fragment encoding the C terminus of NS5B (nt 7601–9377) cut by NotI/ClaI was inserted into the NotI/ClaI sites of plasmid pIV1002 to generate pIV1005. pIV1056 was then obtained by homologous recombination in BJ5183 Escherichia coli of a ClaI–EcoRI restriction fragment (nt 708–9909) of the original HCV cDNA clone (p90/HCV FL-Long pU) with pIV1005 linearized by NheI digestion. The resulting plasmid, pIV1056, contains the HCV non-structural protein-encoding sequence inserted downstream of the inducible promoter of the pBI-L expression vector. This promoter consists of a tetracycline-responsive element and a minimal cytomegalovirus (CMV) promoter. Therefore, it can be activated by a reverse tetracycline-controlled transactivator (rtTA) when Dox is added to the cell culture medium. The Tet-293 cell line (human embryonic kidney cells), which stably expresses rtTA (purchased from Clontech), was co-transfected with pIV1056 and pTK-Hyg (Clontech) at a ratio of 1 : 20 using calcium phosphate precipitation (Profection Mammalian Transfection system; Promega). Clonal selection was performed in complete MEM{alpha} culture medium supplemented with G418 and hygromycin, as described above. After selection, clone LS1056-52(2) expressing the non-structural proteins under the control of Dox was selected and confirmed for non-structural protein expression.

EGFP and DsRed expression.
At 72 h post-transfection, cells were harvested. EGFP and DsRed expression was determined by FACScan analysis (Becton Dickinson). Patterns of fluorescence were acquired for 200–1000 cells expressing EGFP and DsRed simultaneously after fixation in PBS (Invitrogen) containing 1·5 % paraformaldehyde. Each transfection or infection was performed in triplicate and repeated at least three times.

Antibodies.
Polyclonal rabbit antisera LaIV72 and LaIV73 against NS2 were produced by immunization of rabbits with recombinant E. coli-expressed fusion protein GST–NS2. Polyclonal rabbit antibodies (pAbs) to NS3 and NS5B were kindly provided by R. Bartenschlager. The NS5A-specific monoclonal antibodies (mAbs) 2D9F4 and anti-Core were kindly provided by C. Jolivet (BioMérieux, Lyon, France). The E1-specific mAb A4 was provided by H. B. Greenberg (Department of Medicine, Stanford University School of Medicine, USA) and mAb anti-E2 (H47) was kindly provided by J. Dubuisson (Institut de Biologie de Lille, France).

Western blot analysis.
Protein samples were resolved by SDS-PAGE, transblotted onto Hybond-P membranes (Amersham Pharmacia Biotech) and probed with anti-NS pAbs (1 : 50 dilution for anti-NS2 and 1 : 2500 for anti-NS3 and -NS5B) and mAbs (1 : 1000 dilution for anti-NS5A, -Core, -E1 and -E2).


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
HCV IRES activity is not affected by the presence of the 3'NTR
Studies reported previously have shown that HCV IRES translation activity was enhanced two- to threefold by the presence of the HCV 3'X region in an in vitro translation system (Michel et al., 2001), while Fang & Moyer (2000) did not observe any effect of the entire HCV 3'NTR on IRES activity. Murakami et al. (2001) have observed a suppressor effect of the poly(U/UC) and/or stem–loop III of the 3'NTR region on translation efficiency. Only one of the above studies was performed ex vivo (in Huh-7 cells) and demonstrated that IRES activity was stimulated two- to threefold by the 3'X region (Ito et al., 1998).

To address and clarify the controversial effect of the 3'NTR of HCV on the biological activity of the IRES, we have designed a series of ex vivo experiments. We constructed a set of bicistronic expression cassette under the control of a CMV promoter. These cassettes contain two reporter genes, one encoding EGFP and the other encoding DsRed. Between the two reporter genes, we have inserted the entire IRES of HCV followed by the first 390 (pIV1013 and pIV1014) or 200 nt (pIV1018 and pIV1019) of the Core protein sequence since some authors (Shimoike et al., 1999; Wang et al., 2000; Zhang et al., 2002) have observed an effect of the Core sequence (or protein) on IRES activity. The Core-encoding sequence was inserted in frame with the DsRed sequence. At the 3' end of the expression cassette, the entire HCV 3'NTR was either present (pIV1013 and pIV1018) or absent (pIV1014 and pIV1019) (Fig. 1). This reporter system allowed us to rapidly monitor the expression efficiency of the two reporter genes and hence to optimize the bicistronic cassette transfection conditions. The bicistronic plasmid constructions were transfected into various cell lines to assess potential differences in IRES activity between hepatic (Huh-7, HepG2 and Hep3B) or non-hepatic (HEK 293) cell lines. At 72 h post-transfection, the effect of the HCV 3'NTR on the activity of the IRES was assessed. A quantitative marker of IRES activity was calculated as the ratio of DsRed to EGFP fluorescence by FACScan at 72 h post-transfection. DsRed protein expression shows that HCV IRES is functional in the bicistronic context. Fig. 2 shows that IRES activity in the presence of the 3'NTR was similar to that of the construction depleted of this sequence: compare the ratio obtained with pIV1013 versus pIV1014 and pIV1018 versus pIV1019, respectively. Similar results were obtained in human embryonic HEK 293 cells and in hepatic cell lines (Fig. 2). The presence of the 3'NTR corresponding to RNA IV1013 and IV1018 was detected by RT-PCR analysis on total RNAs from transfected cells (data not shown).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2. Relative efficiency of HCV IRES in HEK 293 (grey bars) and in several HCV hepatic cell lines: Huh-7 (open bars), Hep3B (hatched bars) and HepG2 (solid bars). Plasmids pIV1013 and pIV1018 contain the entire 3'NTR; plasmids pIV1014 and pIV1019 are deleted of this sequence. Plasmid pIV1060 contains the 3'X region. EGFP and DsRed protein expression was measured at 72 h post-transfection. The mean±SD of three independent triplicate experiments is shown.

 
Because two studies had described a twofold enhancer effect of the 3'X region on HCV IRES activity in vitro and in Huh-7 cells (Ito et al., 1998; Michel et al., 2001), we also tested in different hepatic cell lines the activity of a bicistronic cassette containing only the 3'X region (Fig. 2). Cells were transfected in parallel with the plasmid containing the 3'X region (pIV1060) and with pIV1019. As for the complete 3'NTR, we did not observe any enhancement effect on IRES-dependent translation by the 3'X region, independently of the cell line tested (Fig. 2). As no effect on translation efficiency could be observed for pIV1060 in hepatoma cell lines, this construct was not tested in HEK 293. These data indicate that neither the 3'X sequence nor the entire 3'NTR affects HCV IRES-dependent translation efficiency in the cell lines tested.

Although bicistronic constructs have been used repeatedly to study the translation efficiency of two genes transcribed under the control of a single promoter, it is clear that this does not reflect the exact genome organization of HCV. To ensure that the IRES position and the few extra nucleotides positioned at the 3' extremity of the HCV sequence in our constructs do not mask an effect of the 3'NTR, plasmids pIV1171 and pIV1172 were generated (Fig. 1). Hence, these monocistronic expression cassettes contain the HCV IRES under the control of the CMV promoter followed by the first 200 nt of Core fused to DsRed, preceding or not the HCV 3'NTR. These two plasmids were linearized by NotI digestion, which results in the liberation of the 3' extremity of the HCV genome with only six extra nucleotides. Four of the six extra nucleotides were then removed by mung bean nuclease digestion. Plasmids pIV1171 and pIV1172, linearized by NotI and blunted using mung bean nuclease, were co-transfected with control plasmid pEGFP-C1 into Huh-7 cells. At 72 h post-transfection, EGFP and DsRed expression in a same cell were quantified by flow cytometry. The results obtained in the monocistronic context show that the DsRed reporter gene was translated with the same efficiency in the presence or absence of the 3'NTR (Fig. 3). This demonstrates that the absence of modulation of translation efficiency by the 3'NTR observed previously was not due to the internal position of HCV IRES or to the presence of a stretch of additional nucleotides at the 3' end of our constructs.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3. Absence of modulation of HCV IRES-dependent translation by the 3'NTR in a monocistronic context. Prior to transfection into Huh-7 cells, plasmids pEGFP-C1 and pIV1171 were linearized by NotI digestion and blunted with mung bean nuclease. pEGFP-C1 and pIV1172 were used as controls. The mean±SD of three independent triplicate experiments is shown.

 
Effect of the 3'NTR on IRES activity in the presence of HCV proteins
Munroe & Jacobson (1990) have described that the presence of a poly(A) tail stimulates the binding of the 40S ribosomal subunit to the mRNA cap via transient or stable mRNA circularization. This closed-loop model is based on a physical interaction helped by canonical factors, eIF4G and eIF4E, and a non-canonical factor, poly(A)-binding protein (Tarun & Sachs, 1996). Since, Pestova et al. (1998) have shown that efficient HCV IRES translation does not require the presence of any canonical or non-canonical factors. It may be possible, therefore, that a putative enhancement of HCV IRES-dependent translation by the 3'NTR requires the presence of one or more HCV proteins. These proteins may establish a bridge between the IRES and the 3'NTR sequences. Such an interaction could create a switch between translation and replication, as has been observed for poliovirus (PV) (Gamarnik & Andino, 1998).

(a) HCV non-structural proteins expressed transiently by a recombinant adenovirus
To address this question, we have investigated a potential role of HCV non-structural proteins on IRES activity. A recombinant adenovirus expressing all six HCV non-structural proteins of HCV strain H genotype 1a under the control of a CMV promoter was generated. Expression of the non-structural proteins in cells infected with recombinant adenovirus AdIV1053 was detected by Western blot (Fig. 4a). At 24 h post-transfection of Huh-7 cells with pIV1018 or pIV1019, the six non-structural HCV proteins were provided in trans following infection with recombinant adenovirus AdIV1053 or AdIV1043 (as control) at an m.o.i. of 100. Under these conditions, no cytopathic effect was observed in Huh-7 cells. After 72 h, IRES activity was evaluated. We observed that expression of HCV non-structural proteins did not influence translation efficacy in the presence or absence of the entire 3'NTR (compare pIV1018 to pIV1019) (Fig. 5a). The same results were obtained with pIV1086NotI compared to pIV1019NotI. Similar results were obtained after transfection with pIV1171 and pIV1172, as described previously (data not shown).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4. Detection of HCV proteins by Western blotting. (a) Non-structural protein expression in Huh-7 cells infected over 72 h by AdIV1053 and AdIV1043 (as control). (b) Non-structural protein expression in LS1056-52(2) cells induced (+) or not (-) by Dox. (c) Core, E1 and E2 protein expression in Huh-7 cells infected with vvIV205 or vv wt (as control) over 18 h.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5. Effect of HCV proteins on IRES efficiency. (a) Ratios of DsRed and EGFP protein fluorescence measured at 96 h post-transfection with the given plasmids. Infection with recombinant adenovirus AdIV1053 encoding all six HCV non-structural proteins occurred at 24 h post-transfection at an m.o.i. of 100. (b) IRES activity was assessed at 72 h post-transfection with the given plasmids in cell line LS1056-52(2) expressing all HCV non-structural proteins. (c) HCV IRES activity in Huh-7 cells transfected with the given plasmids and infected with vvIV205 (expressing Core, E1 and E2) or vv wt (as control). The mean±SD of three independent triplicate experiments is shown.

 
(b) Inducible expression of HCV non-structural proteins in a stable human cell line
The potential effect of stable expression of HCV non-structural proteins was evaluated further. For this purpose, we generated a stable human cell line expressing HCV non-structural proteins, LS1056-52(2). Plasmid pIV1056 used for construction encodes all six non-structural proteins under the control of a Tet-On inducible promoter. Protein expression was detected by Western blotting (Fig. 4b). Plasmids pIV1086 and pIV1019, linearized by restriction with NotI (pIV1086NotI and pIV1019NotI), were transfected into LS1056-52(2) cells. Induction with Dox was performed during 96 h. At 72 h post-transfection, expression of the reporter EGFP and DsRed proteins was assessed by flow cytometry. Results shown in Fig. 5(b) confirm that HCV non-structural proteins do not modulate HCV IRES activity whether in the presence or absence of the 3'NTR.

(c) HCV structural proteins expressed transiently by a recombinant vaccinia virus
To analyse a potential effect of the HCV structural proteins on IRES efficiency regulated by the 3'NTR, we used a vaccinia virus vector encoding the Core, E1 and E2 proteins of HCV genotype 1a (termed vvIV205) and wild-type vaccinia virus as control (vv wt). Linearized bicistronic expression cassettes pIV1086NotI and pIV1019NotI were transfected into Huh-7 cells. At 48 h post-transfection, cells were infected with either vvIV205 or vv wt (as control) at 5 p.f.u. per cell for 18 h. Expression of the structural proteins (Core, E1 and E2) was detected by Western blot (Fig. 4c). Using this m.o.i., an immunofluorescence analysis with mAb anti-Core showed that 90 % of cells expressed Core without visible cytopathic effect. Expression of the reporter genes was assessed at 72 h post-transfection to analyse the effect of Core, E1 and E2 expression on IRES-dependent translation activity with or without the 3'NTR. Results obtained show that expression of HCV structural proteins does not modify IRES activity in the presence or absence of the 3'NTR (Fig. 5c). Similar results were obtained after transfection with pIV1171 and pIV1172, as described previously (data not shown).


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we investigated the role of the 3'NTR in the regulation of HCV IRES-dependent translation in the presence or absence of HCV proteins. Our data show that neither the 3'NTR nor the 3'X region affects HCV IRES-dependent translation efficiency. This lack of effect was assessed unambiguously in three HCV hepatic cell lines (HepG2, Hep3B and Huh-7) as well as in a non-hepatic cell line, HEK 293. The absence of modulation of IRES-dependent translation by the 3'NTR, as observed in our study, contradicts results reported by three other groups (Ito et al., 1998; Michel et al., 2001; Murakami et al., 2001). Several reasons may explain this discrepancy. Firstly, previous studies have used cell-free expression systems, whereas we chose to perform all experiments in cultured mammalian cells. Indeed, experiments described by Michel et al. (2001) were performed in rabbit reticulocyte lysates depleted of ribosomes in order to obtain optimal cap-poly(A) synergy; the two other groups used standard in vitro translation systems. In addition, eukaryotic mRNA translation enhancement by a poly(A) tail is strictly dependent on the concentration of potassium and magnesium ions. Among the three reports cited above, only Murakami et al. (2001) used physiological ion concentrations, whereas the two other studies used much lower concentrations. A second explanation for the discrepancy might depend on the HCV genotype studied. Indeed, we have used sequences from genotype 1a HCV isolates, while two of the three previous studies have used a genotype 1b isolate (Ito et al., 1998; Michel et al., 2001) and the third studied a genotype 2b strain (Murakami et al., 2001). Even though the 5' and 3'NTR sequences are very conserved, they contain nucleotide substitutions that might be important for the specific interaction with cellular or viral proteins or for long-range RNA–RNA interactions. Hence, specific nucleotide interactions may form tertiary structures that may account for the specific regulation of IRES activity by the 3'NTR. This hypothesis is strengthened by the fact that IRES-dependent translation efficiency depends on the HCV genotype (Honda et al., 1999; Collier et al., 1998). For example, the IRES activity of a genotype 2b virus has been reported to be more efficient than that of a genotype 1b strain (Tsukiyama-Kohara et al., 1992).

Only one study performed in a cellular context (in Huh-7 cells) showed that the 3'X region enhanced IRES activity by a factor of two (Ito et al., 1998). However, Ito et al. (1998) did not use the entire HCV 3'NTR. Moreover, stimulation of IRES activity by the 3'X region observed in this study was weaker than that obtained with the IRES of other viruses, such as encephalomyocarditis virus (EMCV) or PV. In these latter cases, IRES activity was enhanced four- to sixfold and up to tenfold by the poly(A) tail (Michel et al., 2001). The effect is even weaker when compared to eukaryotic mRNA cap-dependent translation stimulation, which can be upregulated up to 120 times by the poly(A) tail (Munroe & Jacobson, 1990). In addition, the 3'X stimulation effect observed on HCV IRES activity was not specific for the (HCV IRES/3'NTR) system, since a similar enhancer effect of the HCV 3'X region was observed on cap-dependent translation and on an unrelated IRES-dependent translation (i.e. EMCV) (Ito et al., 1998; Michel et al., 2001).

The absence of regulation of HCV IRES activity by the 3'NTR demonstrated in our study was also observed by Fang & Moyer (2000) in an in vitro translation system. Recently, the impact on translation efficiency of a series of mutations on the 3'NTR was analysed. The results reported showed that complete deletion of the variable region, the poly(U/UC) tract or the 3'X region did not modify HCV IRES activity (Friebe & Bartenschlager, 2002). These experiments were performed in Huh-7 cells using an HCV replicon system (which is based on self replication of subgenomic HCV RNA) (Lohmann et al., 1999, 2001) and with a bicistronic reporter construct. More recently, Kong & Sarnow (2002) have shown that the HCV 3'NTR modulates neither the translation nor the stability of a chimeric mRNA.

When PV protein synthesis reaches a threshold, PV IRES translation activity is inhibited in order to favour RNA replication (Gamarnik & Andino, 1998). HCV genomic RNA serves as template for translation of the polyprotein as well as for replication of positive-strand RNA. These two phenomena are antagonistic and so HCV must have developed regulating mechanisms to switch between translation and replication. This led us to investigate whether HCV proteins could modulate IRES-dependent translation. To test this hypothesis, we complemented the bicistronic construct in trans either with a recombinant adenovirus or via a stable cell line expressing all six non-structural HCV proteins. For expression of structural proteins, we used a vaccinia virus vector expressing Core, E1 and E2. Even if some non-structural proteins were shown to bind to the 3'NTR (at least NS3 and NS5B) (Cheng et al., 1999; Oh et al., 1999; Banerjee & Dasgupta, 2001) and despite the attractiveness of this hypothesis, our data do not support a role of HCV structural or non-structural proteins in regulating IRES-dependent translation efficiency.

In conclusion, we have demonstrated that the HCV 3'NTR does not affect IRES-dependent translation efficiency. This feature is similar to that of certain plant viruses, such as turnip yellow mosaic virus and Alfalfa mosaic virus. For these viruses, the presence or absence of a 3'NTR does not affect cap-dependent translation (Gallie & Kobayashi, 1994). Schematically, the 3'NTRs of RNA-positive eukaryotic viruses vary in their ability to regulate translation. The first and main group contains viruses, such as reoviruses, for which the 3'NTR can stimulate cap- or IRES-dependent translation. Cap-dependent translation of these viruses can be stimulated by a closed-loop complex via a viral protein (NSP3) that binds to the 3'NTR (Vende et al., 2000). In the second group of viruses, the 3'NTR can downregulate translation. This is the case for West Nile virus (Li & Brinton, 2001). In the third group, the 3'NTR does not affect translation. We have shown that HCV belongs to this latter group.

Lastly, additional viral RNA regions not included in the bicistronic cassettes may be involved in modulating translation efficiency. Further studies are needed to identify and analyse the role of cis-additional RNA elements and protein components that may be involved in the regulation of HCV translation.


   ACKNOWLEDGEMENTS
 
We are grateful to C. M. Rice for providing the p90/HCV FL-Long pU clone (no. PH 13149611755) and to Transgene (Strasbourg, France) for generously supplying mAb anti-DBP and shuttle plasmids to generate recombinant adenoviruses. We thank L. Gloeckler for technical help with FACScan analysis. We are grateful to ‘L'Association pour la Recherche Contre le Cancer’ and ‘La Ligue Régionale Contre le Cancer’ for grants to I. I. and F. K. We thank HCVacc Cluster (no. QLK2-CT99-00356) for a grant to M. D. This work was supported by grants from the ARC (no. 7603), FRM (no. INE20 000407028), BNP Paribas Fondation (no. EPB/GP/CL011011) and the ‘Réseau Fondamental Hépatites' (no. 1A133C).


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ali, N. & Siddiqui, A. (1997). The La antigen binds 5' noncoding region of the hepatitis C virus RNA in the context of the initiator AUG codon and stimulates internal ribosome entry site-mediated translation. Proc Natl Acad Sci U S A 94, 2249–2254.[Abstract/Free Full Text]

Anwar, A., Ali, N., Tanveer, R. & Siddiqui, A. (2000). Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation. J Biol Chem 275, 34231–34235.[Abstract/Free Full Text]

Banerjee, R. & Dasgupta, A. (2001). Specific interaction of hepatitis C virus protease/helicase NS3 with the 3'-terminal sequences of viral positive- and negative-strand RNA. J Virol 75, 1708–1721.[Abstract/Free Full Text]

Bartenschlager, R. & Lohmann, V. (2000). Replication of the hepatitis C virus. Baillière's Best Pract Res Clin Gastroenterol 14, 241–254.[CrossRef][Medline]

Blight, K. J. & Rice, C. M. (1997). Secondary structure determination of the conserved 98-base sequence at the 3' terminus of hepatitis C virus genome RNA. J Virol 71, 7345–7352.[Abstract]

Cheng, J. C., Chang, M. F. & Chang, S. C. (1999). Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3' end of the viral RNA. J Virol 73, 7044–7049.[Abstract/Free Full Text]

Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W. & Houghton, M. (1989). Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359–362.[Medline]

Collier, A. J., Tang, S. & Elliott, R. M. (1998). Translation efficiencies of the 5' untranslated region from representatives of the six major genotypes of hepatitis C virus using a novel bicistronic reporter assay system. J Gen Virol 79, 2359–2366.[Abstract]

Fang, J. W. & Moyer, R. W. (2000). The effects of the conserved extreme 3' end sequence of hepatitis C virus (HCV) RNA on the in vitro stabilization and translation of the HCV RNA genome. J Hepatol 33, 632–639.[CrossRef][Medline]

Friebe, P. & Bartenschlager, R. (2002). Genetic analysis of sequences in the 3' nontranslated region of hepatitis C virus that are important for RNA replication. J Virol 76, 5326–5338.[Abstract/Free Full Text]

Gallie, D. R. & Kobayashi, M. (1994). The role of the 3'-untranslated region of non-polyadenylated plant viral mRNAs in regulating translational efficiency. Gene 142, 159–165.[CrossRef][Medline]

Gamarnik, A. V. & Andino, R. (1998). Switch from translation to RNA replication in a positive-stranded RNA virus. Genes Dev 12, 2293–2304.[Abstract/Free Full Text]

Hahm, B., Kim, Y. K., Kim, J. H., Kim, T. Y. & Jang, S. K. (1998). Heterogeneous nuclear ribonucleoprotein L interacts with the 3' border of the internal ribosomal entry site of hepatitis C virus. J Virol 72, 8782–8788.[Abstract/Free Full Text]

Honda, M., Rijnbrand, R., Abell, G., Kim, D. & Lemon, S. M. (1999). Natural variation in translational activities of the 5' nontranslated RNAs of hepatitis C virus genotypes 1a and 1b: evidence for a long-range RNA–RNA interaction outside of the internal ribosomal entry site. J Virol 73, 4941–4951.[Abstract/Free Full Text]

Ito, T. & Lai, M. M. (1999). An internal polypyrimidine-tract-binding protein-binding site in the hepatitis C virus RNA attenuates translation, which is relieved by the 3'-untranslated sequence. Virology 254, 288–296.[CrossRef][Medline]

Ito, T., Tahara, S. M. & Lai, M. M. (1998). The 3'-untranslated region of hepatitis C virus RNA enhances translation from an internal ribosomal entry site. J Virol 72, 8789–8796.[Abstract/Free Full Text]

Kamoshita, N., Tsukiyama-Kohara, K., Kohara, M. & Nomoto, A. (1997). Genetic analysis of internal ribosomal entry site on hepatitis C virus RNA: implication for involvement of the highly ordered structure and cell type-specific transacting factors. Virology 233, 9–18.[CrossRef][Medline]

Kato, J., Kato, N., Yoshida, H., Ono-Nita, S. K., Shiratori, Y. & Omata, M. (2002). Hepatitis C virus NS4A and NS4B proteins suppress translation in vivo. J Med Virol 66, 187–199.[CrossRef][Medline]

Kien, F., Abraham, J. D., Schuster, C. & Kieny, M. P. (2003). Analysis of the subcellular localization of hepatitis C virus E2 glycoprotein in live cells using EGFP fusion proteins. J Gen Virol 84, 561–566.[Abstract/Free Full Text]

Kolykhalov, A. A., Feinstone, S. M. & Rice, C. M. (1996). Identification of a highly conserved sequence element at the 3' terminus of hepatitis C virus genome RNA. J Virol 70, 3363–3371.[Abstract]

Kolykhalov, A. A., Agapov, E. V., Blight, K. J., Mihalik, K., Feinstone, S. M. & Rice, C. M. (1997). Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science 277, 570–574.[Abstract/Free Full Text]

Kolykhalov, A. A., Mihalik, K., Feinstone, S. M. & Rice, C. M. (2000). Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo. J Virol 74, 2046–2051.[Abstract/Free Full Text]

Kong, L. K. & Sarnow, P. (2002). Cytoplasmic expression of mRNAs containing the internal ribosome entry site and 3' noncoding region of hepatitis C virus: effects of the 3' leader on mRNA translation and mRNA stability. J Virol 76, 12457–12462.[Abstract/Free Full Text]

Laporte, J., Malet, I., Andrieu, T. & 7 other authors (2000). Comparative analysis of translation efficiencies of hepatitis C virus 5' untranslated regions among intraindividual quasispecies present in chronic infection: opposite behaviors depending on cell type. J Virol 74, 10827–10833.[Abstract/Free Full Text]

Lerat, H., Shimizu, Y. K. & Lemon, S. M. (2000). Cell type-specific enhancement of hepatitis C virus internal ribosome entry site-directed translation due to 5' nontranslated region substitutions selected during passage of virus in lymphoblastoid cells. J Virol 74, 7024–7031.[Abstract/Free Full Text]

Li, W. & Brinton, M. A. (2001). The 3' stem loop of the West Nile virus genomic RNA can suppress translation of chimeric mRNAs. Virology 287, 49–61.[CrossRef][Medline]

Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L. & Bartenschlager, R. (1999). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113.[Abstract/Free Full Text]

Lohmann, V., Korner, F., Dobierzewska, A. & Bartenschlager, R. (2001). Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol 75, 1437–1449.[Abstract/Free Full Text]

Lusky, M., Christ, M., Rittner, K. & 7 other authors (1998). In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2A, or E1/E4 deleted. J Virol 72, 2022–2032.[Abstract/Free Full Text]

Michel, Y. M., Borman, A. M., Paulous, S. & Kean, K. M. (2001). Eukaryotic initiation factor 4G-poly(A) binding protein interaction is required for poly(A) tail-mediated stimulation of picornavirus internal ribosome entry segment-driven translation but not for X-mediated stimulation of hepatitis C virus translation. Mol Cell Biol 21, 4097–4109.[Abstract/Free Full Text]

Munroe, D. & Jacobson, A. (1990). mRNA poly(A) tail, a 3' enhancer of translational initiation. Mol Cell Biol 10, 3441–3455.[Medline]

Murakami, K., Abe, M., Kageyama, T., Kamoshita, N. & Nomoto, A. (2001). Down-regulation of translation driven by hepatitis C virus internal ribosomal entry site by the 3' untranslated region of RNA. Arch Virol 146, 729–741.[CrossRef][Medline]

Oh, J. W., Ito, T. & Lai, M. M. (1999). A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA. J Virol 73, 7694–7702.[Abstract/Free Full Text]

Pestova, T. V., Shatsky, I. N., Fletcher, S. P., Jackson, R. J. & Hellen, C. U. (1998). A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes Dev 12, 67–83.[Abstract/Free Full Text]

Reed, K. E. & Rice, C. M. (2000). Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol Immunol 242, 55–84.[Medline]

Reich, N. C., Sarnow, P., Duprey, E. & Levine, A. J. (1983). Monoclonal antibodies which recognize native and denatured forms of the adenovirus DNA-binding protein. Virology 128, 480–484.[CrossRef][Medline]

Shimoike, T., Mimori, S., Tani, H., Matsuura, Y. & Miyamura, T. (1999). Interaction of hepatitis C virus core protein with viral sense RNA and suppression of its translation. J Virol 73, 9718–9725.[Abstract/Free Full Text]

Shine, J. & Dalgarno, L. (1974). The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A 71, 1342–1346.[Abstract]

Tanaka, T., Kato, N., Cho, M. J. & Shimotohno, K. (1995). A novel sequence found at the 3' terminus of hepatitis C virus genome. Biochem Biophys Res Commun 215, 744–749.[CrossRef][Medline]

Tarun, S. Z., Jr, & Sachs, A. B. (1996). Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G. EMBO J 15, 7168–7177.[Abstract]

Tsukiyama-Kohara, K., Iizuka, N., Kohara, M. & Nomoto, A. (1992). Internal ribosome entry site within hepatitis C virus RNA. J Virol 66, 1476–1483.[Abstract]

Vende, P., Piron, M., Castagne, N. & Poncet, D. (2000). Efficient translation of rotavirus mRNA requires simultaneous interaction of NSP3 with the eukaryotic translation initiation factor eIF4G and the mRNA 3' end. J Virol 74, 7064–7071.[Abstract/Free Full Text]

Wang, T. H., Rijnbrand, R. C. & Lemon, S. M. (2000). Core protein-coding sequence, but not core protein, modulates the efficiency of cap-independent translation directed by the internal ribosome entry site of hepatitis C virus. J Virol 74, 11347–11358.[Abstract/Free Full Text]

Yanagi, M., St Claire, M., Emerson, S. U., Purcell, R. H. & Bukh, J. (1999). In vivo analysis of the 3' untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone. Proc Natl Acad Sci U S A 96, 2291–2295.[Abstract/Free Full Text]

Zhang, J., Yamada, O., Yoshida, H., Iwai, T. & Araki, H. (2002). Autogenous translational inhibition of core protein: implication for switch from translation to RNA replication in hepatitis C virus. Virology 293, 141–150.[CrossRef][Medline]

Received 17 October 2002; accepted 10 February 2003.