1 Division of Microbiology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
2 Division of Diabetes, Digestive and Kidney Diseases, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
Correspondence
Hak Hotta
hotta{at}kobe-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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The non-structural protein 5A (NS5A) of HCV has been reported to be multifunctional. NS5A is localized in the cytoplasmic perinuclear region, despite the presence of a functional nuclear localization signal (Ide et al., 1996; Song et al., 1999
, 2000
), and represents two forms with different degrees of phosphorylation, p56 and p58 (Kaneko et al., 1994
; Tanji et al., 1995
; Song et al., 1999
; Reed & Rice, 2000
). Mutation in the central region of NS5A (aa 237276) has been demonstrated to be correlated with responsiveness to IFN treatment in patients chronically infected with HCV subtype 1b (HCV-1b) and, therefore, this region has been designated the IFN sensitivity-determining region or ISDR (Enomoto et al., 1996
). We have also reported that mutations in the ISDR inversely correlated with HCV RNA titres in patients infected with HCV-1b, HCV-1c and HCV-2a (Lusida et al., 2001
). In experimental settings, NS5A has been demonstrated to rescue encephalomyocarditis virus (EMCV) replication in IFN-treated cell cultures (Polyak et al., 1999
; Song et al., 1999
). It was also reported that NS5A inhibited antiviral activity of IFN by binding to the double-stranded RNA-dependent protein kinase (PKR) through the ISDR and its adjacent region, called the PKR-binding region (aa 237302) (Gale et al., 1997
, 1998
). However, apparently controversial observations were reported in that the ISDR sequence variation did not account for different IFN resistance in patients (Duverlie et al., 1998
) and also in an HCV subgenomic RNA replicon system (Guo et al., 2001
). Moreover, expression of NS5A or the entire HCV polyprotein was reported to counteract the antiviral effect of IFN in a PKR-independent, ISDR-independent manner (Francois et al., 2000
; Podevin et al., 2001
). The possibility therefore still remains that a molecule(s) other than PKR is involved in the NS5A-mediated inhibition of IFN.
In addition to PKR, the antiviral effects of IFN are executed through the functions of various proteins including 2',5'-oligoadenylate synthetase (2-5AS), RNase L and Mx (Staeheli & Pavlovic, 1991; Hassel et al., 1993
; Sen & Ransohoff, 1993
; Li et al., 1998
). The role of the human MxA protein, in regulating HCV infection might be marginal (Frese et al., 2001
). In the present study we investigated a possible interaction between NS5A and 2-5AS. We report here that the N-terminal one-third of NS5A [NS5A(1148)], which does not contain the ISDR or PKR-binding domain, physically interacted with 2-5AS and counteracted the antiviral activity of IFN. Introduction of a point mutation (Phe to Asn) to residue 37 of NS5A(1148) significantly reduced 2-5AS-binding activity and negated the otherwise more significant IFN-inhibitory activity of NS5A(1148). The same mutation introduced to an HCV subgenomic RNA replicon abolished its replication competence. These results collectively suggest that NS5A interacts with 2-5AS and inhibits the antiviral activity of IFN in an ISDR-independent manner.
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METHODS |
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The coding sequence for glutathione S-transferase (GST) in pGEX-4T-1 (Pharmacia Biotech) was cloned into the unique SmaI site of pBlueScript II SK- (Stratagene). This vector plasmid was designated pBS-GSTBam. pBS-GSTBam was digested with BamHI, the ends filled in by treatment with the Klenow fragment of Escherichia coli DNA polymerase I and then self-ligated to create an in-frame stop codon after the GST sequence. This plasmid, designated pBS-GST, was used to express control GST. The full-length coding sequences for NS5A and 2-5AS were each subcloned in frame to the GST sequence into the unique BamHI site of pBS-GSTBam to generate pBS-GST-NS5A-F and pBS-GST-2-5AS-F, respectively. Plasmids for various deletion mutants of GST-tagged 2-5AS (see Fig. 1B) were constructed by cloning the PCR products, which had been amplified using appropriate sets of primers (Table 1
), into the unique BamHI site of pBS-GSTBam.
We also used the entire coding sequence for human 2-5AS isoform 1 (DDBJ/EMBL/GenBank accession no. D00068) in pHE25AS (a kind gift from Y. Sokawa). The human 2-5AS sequence was fused in frame to GST in pBS-GSTBam and pcDNA vectors to generate pBS-GST-Hu-2-5AS-F and pcDNA-GST-Hu-2-5AS-F, respectively.
The plasmid pFK5B2884Gly containing an HCV subgenomic RNA replicon with a cell culture-adaptive Arg-to-Gly mutation at residue 2884 (Lohmann et al., 2001) was a kind gift from R. Bartenschlager, University of Heidelberg, Heidelberg, Germany. The F37L and F37N mutations were introduced to pFK5B2884Gly by site-directed mutagenesis, as described above.
The sequences of all the plasmids constructed were verified by sequence analysis.
Transient and stable expression.
Transient expression was performed as described previously (Muramatsu et al., 1997). In brief, HeLa cells, maintained in Dulbecco's modified Eagle's medium supplemented with 10 % fetal bovine serum, were infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3) for 1 h and then transfected with the expression plasmids using Lipofectin reagents (Life Technologies). After cultivation for 1216 h, the cells were analysed for the expression of the respective proteins and their possible interaction, as described below.
To establish cell clones stably expressing NS5A, mouse fibroblast L929 and human hepatoma Huh-7 cell lines were used. The cells were co-transfected with a selection plasmid, pSV2neo, and either pCAGGS-FLAG-NS5A-F, pCAGGS-FLAG-NS5A(1-148), pCAGGS-FLAG-NS5A(1-148)F37N or the pCAGGS vector using FuGene 6 transfection reagents (Roche). After cultivation in the presence of G418 (1 mg ml-1) for 23 weeks, resultant colonies were cloned using cloning cylinders.
Huh-7 human hepatoma cells harbouring the HCV subgenomic RNA replicon were also generated by transfecting RNA that had been transcribed in vitro from pFK5B2884Gly (Lohmann et al., 2001), followed by G418 selection.
GST pull-down assay.
HeLa or Huh-7 cells were transiently transfected with the expression plasmids for GST-tagged NS5A and HA-tagged 2-5AS or GST-tagged 2-5AS and FLAG-tagged NS5A, as described above. After 1216 h, the cells were washed once with PBS and lysed in NETN buffer consisting of 150 mM NaCl, 1 mM EDTA, 10 mM Tris/HCl (pH 7·4) and 0·5 % NP40. The lysates were centrifuged at 14 000 r.p.m. for 5 min and the supernatants were mixed with 20 µl glutathione-conjugated Sepharose beads at 4 °C for 90 min. The beads were washed five times with NETN buffer, and possible association between NS5A and 2-5AS was analysed by immunoblotting using appropriate antibodies, as described below. To verify that comparable amounts of the proteins were being analysed, the cell lysates were directly (without pull-down) subjected to immunoblotting.
Immunoblot analysis.
Samples dissolved in a solution consisting of 50 mM Tris/HCl (pH 6·8), 100 mM dithiothreitol, 2 % SDS, 0·1 % bromophenol blue and 10 % glycerol were resolved by SDS-PAGE and electrophoretically blotted on to a PVDF filter (Bio-Rad). After blocking in PBS containing 5 % non-fat dried milk, the filters were incubated with mouse monoclonal antibodies against NS5A (a kind gift from I. Fuke, Research Institute for Microbial Diseases, Kan-Onji Branch, Osaka University, Kan-Onji, Kagawa, Japan), the FLAG (F-3165; Sigma) or HA peptide (16B12; BabCO). After five washes with PBS containing 0·5 % Tween 20, the filters were incubated with peroxidase-labelled goat anti-mouse IgG (MBL). After a further five washes, the protein bands were visualized by an enhanced chemiluminescence method (Amersham Pharmacia). The intensity of the signals was quantified by using NIH image 1.62 software.
Co-immunoprecipitation analysis.
HeLa cells transiently transfected with the expression plasmids were lysed with NETN buffer and the lysates were clarified by centrifugation. The resultant supernatants were incubated at 4 °C for 3 h with 0·5 µg anti-HA rabbit polyclonal antibody (Y-11; Santa Cruz Biotechnology) to immunoprecipitate HA-tagged 2-5AS. Normal rabbit IgG served as a control. The mixtures were then incubated with 10 µl of protein G-coupled Sepharose (Amersham Pharmacia). After six washes with NETN buffer, the immunoprecipitates were subjected to immunoblot analysis using anti-FLAG antibody to detect NS5A.
Immunofluorescence analysis.
HeLa cells transiently transfected with the expression plasmids were fixed with 95 % ethanol and double-stained with anti-FLAG mouse monoclonal antibody and anti-HA rabbit polyclonal antibody. After five washes with PBS, the cells were incubated with Texas-Red-conjugated anti-mouse IgG (Amersham Life Science) and FITC-conjugated anti-rabbit IgG (MBL). After a further five washes with PBS, the cells were analysed by confocal laser-scanning microscopy (MRC-1024; Bio-Rad).
IFN antiviral activity assay.
Cells were seeded in six-well tissue culture plates at a density of 2x105 cells per well and cultivated for 24 h. The cells were treated with recombinant mouse IFN-A (PBL Biomedical Laboratory) or human IFN-
2a (Roche) at concentrations of 5 and 25 U ml-1 or left untreated for another 24 h, then inoculated with 5060 p.f.u. EMCV (strain DK-27) per well (Dan et al., 1995
; Song et al., 1999
). After 1 h with intermittent rocking, fresh medium containing 1 % methylcellulose was added to each well of the plates. After cultivation for 2 days, the plates were stained with crystal violet and the number of plaques in each well was counted. The percentage of plaques on IFN-treated cells compared with untreated cells was calculated.
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RESULTS |
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The N-terminal 148 residues of NS5A are involved in the interaction with 2-5AS
Various deletion mutants of FLAG-tagged NS5A (see Fig. 1A) were co-expressed with GST-tagged 2-5AS-F to determine the region responsible for the interaction with 2-5AS. All the NS5A deletion mutants tested were localized in the cytoplasm (data not shown). Immunoblot analysis using anti-FLAG antibody revealed that FLAG-tagged NS5A(1232) and NS5A(1148), but not NS5A(1109) or NS5A(27148), were efficiently pulled down by GST-tagged 2-5AS-F (Fig. 3
A, upper panel). It should be noted that NS5A(1232) and NS5A(1148) bound to 2-5AS-F more efficiently than did NS5A-F. Comparable degrees of expression of NS5A-F and the deletion mutants (Fig. 3A
, lower panel) and GST-tagged 2-5AS-F (data not shown) in each transfected cell culture were verified. Neither NS5A(167) nor NS5A(187) were pulled down by GST-tagged 2-5AS-F (data not shown).
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Two separate portions of 2-5AS are independently involved in the interaction with NS5A
To determine the region(s) of 2-5AS responsible for the interaction with NS5A, various deletion mutants of GST-tagged 2-5AS (see Fig. 1B) were co-expressed with FLAG-tagged NS5A(1148), pulled down by glutathione-conjugated Sepharose beads and subjected to immunoblot analysis using an anti-FLAG antibody. GST-tagged 2-5AS(1104), 2-5AS(52144) and 2-5AS(184275), but not 2-5AS(160), 2-5AS(184235) or the control GST, pulled down NS5A(1148) (Fig. 3C
, upper panel). Comparable degrees of expression of FLAG-tagged NS5A(1148) (Fig. 3C
, lower panel), GST-tagged 2-5AS-F and the deletion mutants (data not shown) in each transfected cell culture were verified. Collectively, these results suggested that two separate regions of 2-5AS (aa 52104 and aa 184275) are independently involved in physical interaction with NS5A(1148).
Mutation of residue 37 of NS5A affects its interaction with 2-5AS
We previously noticed that a single point mutation of NS5A at residue 37 might be correlated with serum HCV RNA titres (data not shown). Therefore, we were interested in testing the possible effects of NS5A mutations at residue 37 (F37L, F37N, F37S and F37Y) on its interaction with 2-5AS. FLAG-tagged mutants of NS5A(1148) were co-expressed with GST-tagged 2-5AS-F or the control GST in HeLa cells. The cell lysates were subjected to a GST pull-down assay using glutathione-conjugated Sepharose beads, followed by immunoblot analysis using an anti-FLAG antibody. The F37L mutation of NS5A significantly augmented complex formation with 2-5AS, whereas the F37N mutation significantly decreased it (Fig. 4A, upper panel). F37S or F37Y mutations did not significantly affect the complex formation. A comparable degree of expression of NS5A(1148) and the single point mutants (Fig. 4A
, lower panel) in each transfected cell culture was verified. Mean values of relative degrees of complex formation between each NS5A mutant and 2-5AS obtained from four independent experiments are shown in Fig. 4(B)
.
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The possible inhibitory effects of NS5A on antiviral activity of IFN were also assessed using Huh-7 human hepatoma cells. We first confirmed the physical interaction between NS5A and human 2-5AS in this cell type. As shown in Fig. 6(A), NS5A(1148), NS5A(1148)F37L and NS5A(1148)F37N physically interacted with GST-tagged human 2-5AS. NS5A-F was also shown to interact with human 2-AS (data not shown). We then tested the antiviral activity of IFN in Huh-7 cells stably expressing NS5A-F or NS5A(1148) or the non-expressing control. The result obtained showed that the antiviral activity of IFN was significantly counteracted by NS5A-F and NS5A(1148) (Fig. 6B
), the result being consistent with that obtained with the L929 mouse cell system.
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DISCUSSION |
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In the present study we have demonstrated that HCV NS5A physically interacts with 2-5AS, with the N-terminal region of NS5A (aa 1148) and two separate regions of 2-5AS (aa 52104 and aa 184275) being involved in the interaction (Figs 2 and 3). We used two different 2-5AS molecules: the isoform 1a (p42) of mouse 2-5AS and the isoform 1 (p40) of human 2-5AS. While the overall sequence similarity between them is 68 %, the NS5A-binding portions show even higher sequence similarities (83 and 85 %, respectively). Our results also demonstrated that NS5A(1148) inhibited the antiviral activity of IFN against EMCV, as did the full-length NS5A-F, in both mouse and human cell culture systems (Figs 5 and 6
). Since NS5A(1148) does not harbour the ISDR or PKR-binding domain, IFN inhibition by NS5A(1148) is likely independent of ISDR and PKR. The major phosphorylation sites of NS5A (Reed & Rice, 1999
; Katze et al., 2000
) and residues undergoing adaptive mutation in NS5A of HCV replicons (Blight et al., 2000
; Krieger et al., 2001
; Lohmann et al., 2001
), which are located in the central and C-terminal portions of NS5A, are unlikely to be involved in the interaction with 2-5AS or IFN inhibition, since NS5A(1148) does not contain these residues. We could not confirm, however, the inhibition of 2-5AS enzymic activity in NS5A-expressing cells (data not shown). We assume that the possible inhibition might have been masked due to a technical limitation, since 2-5AS in lysates of the cells, either NS5A-expressing cells or the control, is unavoidably activated to some extent during the experimental procedures.
In our previous study on NS5A sequence diversity among HCV isolates in Indonesia (Lusida et al., 2001), we noticed that a mutation at residue 37 of NS5A might be correlated with HCV viraemia titres (data not shown). In an attempt to find a correlation between the mutation and IFN inhibition, we introduced various point mutations: F37L, F37N, F37S and F37Y. Interestingly, our results revealed that NS5A(1148)F37L interacted with 2-5AS twice as strongly as the wild-type NS5A(1148) (Fig. 4
). However, the interaction between another mutant, NS5A(1148)F37N, and 2-5AS was much weaker than that between the wild-type NS5A(1148) and 2-5AS. This weaker interaction may account for the weaker inhibitory effect of NS5A(1148)F37N on the antiviral activity of IFN compared with the wild-type NS5A(1148). Moreover, our result suggests that the F37N mutation is highly disadvantageous for the replication of the HCV subgenomic RNA replicon (Fig. 7B
). It should be noted that, while Phe, Leu and Tyr are hydrophobic and found at this position in clinical isolates of HCV, Asn is hydrophilic and has not been found so far at this position in clinical isolates.
There are a number of functional domains in the N-terminal half of NS5A. The N-terminal 30 residues of NS5A have been reported to include a membrane-anchor domain that determines the cytoplasmic localization of NS5A (Satoh et al., 2000
; Song et al., 2000
; Brass et al., 2002
). It has also been demonstrated that the N-terminal half of NS5A (aa 1224) binds to apolipoprotein A1 and co-localizes with the HCV core protein on lipid droplets (Shi et al., 2002
). Moreover, a region of NS5A spanning from aa 105 to 162 forms a complex with NS5B and modulates its RNA-dependent RNA polymerase activity (Shirota et al., 2002
). The 2-5AS-binding region of NS5A determined in the present study (aa 1148) overlaps those domains. For 2-5AS, we identified two independent regions that are responsible for the interaction with NS5A: one spanning from aa 52 to 104 and the other from aa 184 to 275. The former region contains an ATP-binding motif (P-loop) followed by an Asp76-Ala-Asp78 sequence (D-box), while the latter contains a region with a high Lys and Arg content (KR-rich region). The P-loop, D-box and KR-rich region are important for the enzymic activity of 2-5AS, and mutations in these motifs impair the enzymic activity (Yamamoto et al., 2000
). It is likely, therefore, that NS5A interferes with 2-5AS functions by binding to the active sites.
Whether NS5A(1148) or its equivalent(s) is actually generated in the cell would be an interesting issue to address. We have observed that NS5A was cleaved to generate a cleavage product of 19 kDa in FL cells undergoing apoptosis and that the cleavage was inhibited by the caspase inhibitor Z-VAD (data not shown). An NS5A cleavage product of 19 kDa in apoptotic cells was previously reported by Satoh et al. (2000)
, with the estimated cleavage site being residue 154. Goh et al. (2001)
also reported NS5A cleavage by a caspase-like protease(s) that was activated by co-expressed HCV core protein. Collectively, these results suggest the possibility that NS5A(1148) or its equivalent(s) is generated in the cell under certain conditions and interferes with 2-5AS functions more strongly than does full-length NS5A.
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ACKNOWLEDGEMENTS |
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Received 17 July 2003;
accepted 8 December 2003.