Molecular Virology Laboratory, Hellenic Pasteur Institute, 127 Vas. Sofias Avenue, Athens 115 21, Greece
Correspondence
Penelope Mavromara
penelopm{at}hol.gr
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ABSTRACT |
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Published online ahead of print on 17 January 2005 as DOI 10.1099/vir.0.80728-0
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INTRODUCTION |
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HCV is classified into the genus Hepacivirus of the family Flaviviridae (Reed & Rice, 2000). Like all the members of the family, HCV is an enveloped, single-stranded, positive-sense RNA virus. Its genome (about 9600 nt) is flanked at both termini by conserved, highly structured non-translated regions (NTRs) and encodes a polyprotein precursor (about 3000 aa), which is proteolytically processed by host and viral proteases to produce the structural (core, E1, E2 and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins of the virus. Recently, an additional protein has been identified. This protein is encoded by an alternative open reading frame within the core coding region and its function remains unknown (Walewski et al., 2001
; Xu et al., 2001
; Varaklioti et al., 2002
).
Translation initiation of the HCV genome is controlled by an IRES (Hellen & Pestova, 1999; Rijnbrand & Lemon, 2000
). This mechanism first identified for the members of the family Picornaviridae, is also used by the members of Hepacivirus and Pestivirus genera of Flaviviridae. The HCV IRES is located mainly within the 5' NTR of the viral RNA and directs the binding of ribosomes in close proximity to the start codon of the viral open reading frame (Reynolds et al., 1995
; Rijnbrand et al., 1995
; Honda et al., 1996
). Interestingly, the HCV IRES does not have a strict requirement for canonical translation initiation factors (eIFs) other than eIF2 and eIF3, and its activity varies with the cell cycle (Pestova et al., 1998
; Honda et al., 2000
). On the other hand, a number of transacting cellular factors have been shown to interact with the HCV IRES. These include the polypyrimidine-tract-binding protein (Ali & Siddiqui, 1995
), the human La antigen (Pudi et al., 2003
; Izumi et al., 2004
), the poly(rC)-binding protein 2 (Fukushi et al., 2001a
), the heterogeneous nuclear ribonucleoprotein L (Hahm et al., 1998
) and ribosomal protein factors S9 (Fukushi et al., 1999
) and S5 (Fukushi et al., 2001b
). Furthermore, viral sequences located at distal regions from the HCV IRES (Ito et al., 1998
; Ito & Lai, 1999
; Wang et al., 2000
; Murakami et al., 2001
; Imbert et al., 2003
; Kim et al., 2003
) as well as selected viral proteins (Shimoike et al., 1999
; Kato et al., 2002
; Zhang et al., 2002
; He et al., 2003
; Li et al., 2003
) appear to modulate the efficiency of the HCV IRES activity. However, regulation of IRES-mediated translation initiation is poorly understood and most of the results concerning the effects of viral proteins on IRES function remain controversial.
Among the HCV viral proteins, NS5A is a multifunctional serine phosphoprotein of 5658 kDa (Tanji et al., 1995; Reed et al., 1997
; Hirota et al., 1999
) that is implicated in viral pathogenesis (Bartenschlager & Lohmann, 2000
; Blight et al., 2000
; Krieger et al., 2001
; Tan & Katze, 2001
; Tellinghuisen & Rice, 2002
). Although a functional nuclear localization signal (NLS) exists in its carboxy-terminal part, the NS5A protein is found anchored to the cytoplasmic side of the endoplasmic reticulum membranes through an amino-terminal amphipathic
-helix, and modulates virus replication by its direct association with the virus replication complex (Ide et al., 1996
; Elazar et al., 2003
). Interestingly, cell culture-adaptive mutations in the NS5A amino acid sequence significantly enhance the efficiency of HCV replicons (Krieger et al., 2001
; Lohmann et al., 2001
; Blight et al., 2003
). Moreover, NS5A interacts with a number of cellular proteins, thereby affecting numerous cellular pathways (Tan & Katze, 2001
; Macdonald & Harris, 2004
), and plays a major role in controlling host antiviral mechanisms. Notably, recent studies suggest that NS5A may be cleaved by calpains and caspases to produce stable carboxy-terminal truncated forms of the protein (Satoh et al., 2000
; Goh et al., 2001
; Kalamvoki & Mavromara, 2004
).
In this study, we investigated the effect of the HCV NS5A protein on HCV IRES-dependent translation by using a transient cell-based expression system. We found that the HCV NS5A protein inhibited HCV IRES-dependent translation in a specific and dose-dependent manner. Moreover, we found that a region of about 120 aa located at the carboxy-terminal part of the protein was critical for this suppression. These findings might help elucidate the predicted temporal regulation of viral RNA translation in the context of a switch from the translation mode to the replication mode of the virus life cycle.
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METHODS |
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For the expression of the HCV-1a NS5A protein and its deleted forms the following plasmids were constructed. Plasmid pHPI1419 expresses the entire NS5A protein (aa 1447) (HCV-1a) and was constructed by insertion of the HindIII blunt-ended fragment from pHPI611 (Kalamvoki et al., 2002), which contains the coding sequence of NS5A, into the XbaI blunt-ended site of pA-EUA2. This plasmid will be referred to as the F-NS5A vector. Plasmid pHPI1433 expresses the amino-terminal half of NS5A protein (aa 1230) and it was constructed by a two-step procedure. Firstly, the BamHIPvuII blunt-ended fragment from pHPI611 (Kalamvoki et al., 2002
), which encodes the amino-terminal half of the NS5A protein (HCV-1a), was ligated into the XbaI blunt-ended site of pCI (Promega) giving rise to pHPI1405. Secondly, the NheINheI blunt-ended fragment of pHPI1405, which encodes the amino-terminal half of the NS5A protein, was ligated into the XbaI blunt-ended site of pA-EUA2. This plasmid will be referred to as the N1-NS5A vector. Plasmid pHPI1435 expresses the carboxy-terminal half of NS5A protein (aa 236447) and it was constructed by a two-step procedure. Firstly, the nucleotide sequence encoding the carboxy-terminal half of NS5A (aa 236447) was amplified by PCR from pHPI611 (Kalamvoki et al., 2002
). The primers used were: sense, 5'-CCAAGCTTGCCATGGCTCCATCTCTC-3' and antisense, 5'-CTCGAGAAGCTTAGCAGCACACGA-3' where the HindIII restriction sites are underlined and the translation initiation and stop codons, respectively, are shown in bold. The PCR conditions were as follows: 95 °C for 60 s followed by 35 cycles of 95 °C for 30 s, 60 °C for 60 s and 75 °C for 60 s and a final extension at 75 °C for 10 min. The amplified fragment was digested with HindIII, blunt-ended and inserted into the XbaI blunt-ended site of pCI, giving rise to pHPI1407. The coding sequence of the carboxy-terminal part of the NS5A protein was verified by dideoxy-sequence analysis. Secondly, the EcoRINotI blunt-ended fragment of pHPI1407, which encodes the carboxy-terminal half of the NS5A protein, was ligated into the XbaI blunt-ended site of pA-EUA2. This plasmid will be referred to as the C-NS5A vector. Plasmid pHPI1436 expresses the amino-terminal part of NS5A protein up to the NLS (aa 1354), and it was constructed by a two-step procedure. Firstly, the HindIIIEcoRV blunt-ended PCR product of NS5A from pHPI611 (Kalamvoki et al., 2002
) was ligated into the XbaI blunt-ended site of pCI giving rise to pHPI1409. The coding sequence of the protein was verified by dideoxy-sequence analysis. Secondly, the EcoRINotI blunt-ended fragment of pHPI1409 that encodes the above amino-terminal part of NS5A was ligated into the XbaI blunt-ended site of pA-EUA2. This plasmid will be referred to as the N2-NS5A vector. All of the above expression cassettes are shown in Fig. 1
.
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Cells and transfection experiments.
HepG2 cells (epithelium from human hepatocellular carcinoma) obtained from the ATCC, WRL-68 cells (human liver embryonic hepatoma) kindly provided by A. Budkowska (Institute Pasteur, Paris) and BHK-21 cells (baby hamster kidney) obtained from the ATCC were maintained in minimal essential medium (Gibco-BRL) (HepG2) and Dulbecco's modified eagle medium (Biochrom KG) (WRL-68 and BHK-21), supplemented with 10 % fetal bovine serum (Gibco-BRL), penicillin/streptomycin (5 IU ml1/50 mg ml1) and 2 mM L-glutamine. Cells, seeded in 12-well plates at confluence of about 30 % for HepG2 and 50 % for WRL-68 and BHK-21, were transfected using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's protocol. In the transfection experiments that were performed with only one plasmid vector, 0·3 µg DNA per well was used. In the co-transfection experiments either 0·8 or 0·6 µg total plasmid DNA per well was used. At least three independent experiments were carried out for each set of transfections. The medium was replaced 24 h post-transfection. At 48 h post-transfection the cells were washed twice with ice-cold 1x PBS and lysed in 160 µl 1x Luciferase lysis buffer (Promega) according to the manufacturer's instructions.
Quantification of LUC and CAT.
Quantification of LUC protein was performed by mixing 20 µl (out of the 160 µl) cell extracts with 100 µl Luciferase assay reagent (Promega) and the luminescence was measured directly by a Turner TD-20/20 luminometer. Quantification of CAT protein was performed with the CAT-ELISA kit (Boehringer Mannheim) according to manufacturer's instructions.
Immunoblotting.
Samples (40 of 160 µl) of cell extracts were used in Western blot analysis. SDS-PAGE loading buffer was added to each sample, the samples were boiled for 3 min, analysed in 12 % denaturing polyacrylamide gels and transferred onto nitrocellulose membranes.
After blocking for 1·5 h [in 1x PBS, 0·02 % (v/v) Tween 20, 10 % (w/v) dried milk], the membranes were incubated with the primary antibodies overnight at 4 °C. A rabbit polyclonal anti-NS5A antibody [diluted 1 : 100 in 1x PBS, 0·04 % (v/v) Tween 20, 2 % (w/v) dried milk] was used for the detection of NS5A protein and deletion mutants of this protein (Kalamvoki et al., 2002; Kalamvoki & Mavromara, 2004
), whereas a mouse monoclonal anti-
-galactosidase antibody (Gibco-BRL) (diluted 1 : 500 in the same buffer as anti-NS5A antibody) was used for the detection of
-galactosidase protein. The membranes then were washed three times (10 min each) with a solution containing 1x PBS, 0·04 % (v/v) Tween 20, 2 % (w/v) dried milk, and incubated at room temperature with the secondary antibodies diluted 1 : 1000 in the same solution. The secondary antibodies, anti-rabbit for NS5A protein (Dako) and anti-mouse for
-galactosidase protein (Dako), are conjugated with horseradish peroxidase. After washing, the membranes were soaked in enhanced chemiluminescence reagent (Pierce) and exposed to film (Kodak).
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RESULTS |
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DISCUSSION |
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Notably, the inhibitory effect on HCV IRES was NS5A dose-dependent as it was clearly proportional to the protein amount, ranging from almost no inhibition (20 %) to almost total inhibition (90 %) (Fig. 3). Therefore, the inhibitory effect of the NS5A protein on the HCV IRES activity depends either on the absolute levels of the NS5A protein or on the stoichiometry of the HCV IRES and NS5A molecules. Moreover, the ability of NS5A to repress HCV IRES-mediated activity appeared to be cell-type independent, even though in BHK-21 cells LUC production was not as significantly inhibited as in cells of hepatic origin. This inhibition was not related to the recently described HCV 5' NTR-related promoter activity (Dumas et al., 2003
), because the production of the LUC protein from the vector that carries the HCV IRES bicistronic cassette, but lacks the HCMV promoter sequence that controls the transcription of the bicistronic unit, was negligible in the cell lines of hepatic origin (Fig. 4
).
The use of different NS5A deletion mutants suggested that the functional domain of the NS5A protein that is critical for the inhibitory effect on HCV IRES activity resides at the carboxy-terminal half of the protein and includes a sequence of about 120 aa that contains the interferon sensitivity-determining region and the proline-rich region. Clearly, the amino-terminal part of the protein, based on the results from the N1-NS5A form as well as from shorter amino-terminal forms of the protein (data not shown), had no effect on HCV IRES function (Fig. 5). Although the molecular basis of the inhibitory effect of NS5A on HCV IRES remains to be elucidated, it is of interest to note that the putative inhibitory domain of NS5A is known to represent a hot spot for mutations that accumulate in the replicon system (Krieger et al., 2001
; Lohmann et al., 2001
; Blight et al., 2003
).
While our work was in progress, it was reported that the NS5A protein enhances the HCV IRES-mediated translation while NS3, NS4A, NS4B and NS5B do not exhibit any significant effect on the activity level of the HCV IRES (He et al., 2003). Although the reason for this discrepancy is still not known, we have considered a number of possibilities to explain these data. Firstly, that the study was largely based on the use of NS5A sequences derived from the HCV-1b replicon, which is known to contain adaptive mutations within the carboxy-terminal half of the NS5A protein (Lohmann et al., 1999
; Blight et al., 2000
; Bartenschlager & Lohmann, 2001
). However, the selection of this replicon is based on the use of neomycin antibiotic and the gene that is responsible for the resistance (neomycin phosphotransferase) is placed under the translational control of the HCV IRES. Neomycin affects the translation process of the cell (Eustice & Wilhelm, 1984
). Thus, it is tempting to speculate that the selection for neomycin might result in the selection of mutations in NS5A that will suppress or even reverse the negative effect of NS5A on HCV IRES-dependent translation, allowing the survival of the replicon.
Secondly, according to our data, the inhibitory effect of NS5A on the HCV IRES activity is dose-dependent. Because He et al. (2003) have reported results from only a single plasmid concentration, it is likely that the experimental conditions used in that study may not be appropriate to detect repression of the IRES activity. Interestingly, He et al. (2003)
failed to detect repression of the HCV IRES activity by the HCV NS4A and NS4B proteins. Both proteins were shown to have a dose-dependent inhibitory effect on HCV IRES-driven translation (Kato et al., 2002
). A third explanation for this discrepancy might be the HCV genotype studied. In this study, HCV genotype 1a was used, whereas in the other study (He et al., 2003
) HCV 1b was examined. Although this explanation may be less likely, it is strengthened by the fact that NS5A functions are affected by the sequence variability of the protein (Gale et al., 1997
; Pellerin et al., 2004
).
To summarize our work, we showed that NS5A acts as a negative regulator for HCV IRES-mediated translation. The role of NS5A inhibitory effect on HCV RNA translation in virus life cycle remains currently unknown. However, in the case of other positive-sense RNA viruses such as poliovirus, it has been demonstrated that the replication of viral RNA begins only after translation has been inhibited, because the RNA-dependent RNA polymerase of the virus cannot replicate the viral RNA while it is being translated by ribosomes (Gamarnik & Andino, 1998; Barton et al., 1999
). Poliovirus inhibits the translation of its own RNA by producing a non-structural precursor protein, 3CD. 3CD protein binds to the cloverleaf just before the IRES of the virus, represses translation and facilitates negative-strand synthesis (Gamarnik & Andino, 1998
). Furthermore, the existence of a negative-feedback mechanism for the regulation of the initiation of viral RNA replication has been suggested for EMCV, another member of the family Picornaviridae (Svitkin & Sonenberg, 2003
).
Thus, it is intriguing to speculate that the NS5A protein might be part of the biological switch mechanism that is responsible for the inhibition of HCV IRES-driven translation favouring the initiation of the HCV genome replication. Furthermore, this NS5A inhibitory effect on HCV IRES occurs under conditions that do not disturb the translation of the cellular mRNAs and by this way may allow the establishment of the viral persistence.
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ACKNOWLEDGEMENTS |
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Received 27 October 2004;
accepted 17 December 2004.
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