Abstract
We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors. Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371–1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.
The hepatitis C virus (HCV) nonstructural 3 protein (NS3) performs vital enzymatic functions in the HCV life cycle. The N-terminal one-third serine-like protease domain of the NS3 protein, together with the cofactor NS4A, is required for cleavage of the junction between NS2 and NS3 and the downstream NS3/NS4A, NS4A/NS4B, NS4B/NSA5, and NS5A/NS5B junctions (1, 4). The C-terminal two-thirds of the NS3 protein has NTPase and helicase functions (6, 16). Importantly, the NS3 protein is also one of most conserved proteins of HCV. Collectively, these properties suggest that the NS3 protein may be a suitable target for antiviral therapy.
The variable regions of the heavy and light chains (VH and VL, respectively) of an antibody are essential for the recognition of antigens. Single-chain variable-region fragments (ScFvs) of antibodies, containing VH and VL DNA assembled by a flexible linker sequence, allow for intracellular expression of antibody fragments. Several reports have demonstrated that intracellularly expressed ScFvs may reduce the susceptibility of transfected cells to viral infections or disturb viral enzymes within the life cycles of human immunodeficiency virus type (9, 20), tick-borne encephalitis virus (5), and murine coronavirus (8). The production of ScFvs to the HCV core and envelope proteins was recently described (2). This approach therefore offers an alternative antiviral therapy by blocking vital steps in the viral life cycle inside infected cells. We have developed a murine anti-NS3 monoclonal antibody (MAb) which recognizes the NS3 ATPase/helicase domain. From the NS3-specific MAb we designed and produced a functional NS3-specific ScFv by use of procaryotic and eucaryotic expression systems.
MATERIALS AND METHODS
Cells.
The SP2/0 cell line was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, (FCS), 2 mM l-glutamine, 100 U of penicilin per ml, and 100 μg of streptomycin per ml (GIBCO-BRL, Gaithersburg, Md.). The BHK cell line was maintained in Dulbecco modified Eagle medium (GIBCO-BRL) with 10% fetal calf serum and antibiotics. All cells were incubated at 37°C with 7% CO2.
rNS3 and synthetic peptides.
The expression, purification, and enzymatic characterization of a recombinant HCV NS3 (rNS3), genotype 1a, covering residues 1007 to 1612 has previously been described (6). Synthetic peptides covering the ATPase/helicase domain of HCV NS3 have been described previously (15, 18). Additional peptides covering the identified epitope region were synthesized by using an automated synthesizer (Syro; Syntex, Tubingen, Germany) as described previously (19).
MAb production and characterization.
BALB/c mice were immunized and boosted with rNS3 (10 μg) three times every 2 weeks. Three days after the last injection, spleen cells were harvested and fused with the SP2/0 myeloma cells by standard procedures. Following three cycles of cloning and screening by enzyme immunoassay (EIA), a stable hybridoma, ZX10, was selected for antibody analysis and extraction of mRNA. Purification of the ZX10 antibody was performed by using immobilized protein A/G (Pierce, Rockford, Il.). Analysis of the binding characteristics and the fine specificity of the ZX10 antibody was done by using EIAs as previously described (14, 15).
Construction and expression of ScFv- and HCV NS3/NS4A-encoding plasmids.
Total cellular mRNA was extracted from the ZX10 hybridoma by using magnetic beads coated with oligo(dT)25 (Dynal A.S., Oslo, Norway). The VH and VL of NS3 MAb ZX10 were amplified from cDNA by PCR with a recombinant phase antibody system (Pharmacia Biotech, Uppsala, Sweden). The assembled NS3-ScFv cDNA fragment was directly ligated to the TA cloning vector pCR 2.1 (Invitrogen, San Diego, Calif.). The DNA sequence was determined with an automated sequencer (ALF express; Pharmacia). The NS3-ScFv DNA was reamplified to introduce the cloning sites and to omit the leader sequence.
For Escherichia coli expression, the forward primer 5′-CCG CTC GAG CAG GTG AAG CTG CAG-3′ and reverse primer 5′-CGG ATC CTA CCG TTT GAT TTC CAG-3′ were used to amplify the NS3-ScFv to introduce 5′ BamHI and 3′ XhoI restriction sites (underlined). The digested fragment was inserted into the bacterial expression vector pET-15b (Novagen, Madison, Wis.) to give NS3-ScFv-pET.
For eucaryotic expression, the primer pair 5′-CCG GGT ACC ACA GGT GAA GCT GCA G-3′ and 5′-GCC TCT AGA CTA CCG TTT GAT TTC CAG-3′ was used to introduce KpnI and XbaI restriction sites (underlined). After PCR amplification, the reamplified NS3-ScFv was digested with KpnI and XbaI and then inserted in the pcDNA3.1/His A vector (Invitrogen). This produces an NS3-ScFv (NS3-ScFv-pcDNA) which should be retained intracellularly.
A 2.1-kb DNA fragment of HCV NS3/NS4A, encoding a region covering the amino acid sequence from HCV position 1007 to 1711, was amplified with high-fidelity polymerase (Expand High Fidelity PCR; Boehringer, Mannheim, Germany) from a patient infected with HCV genotype 1a and was inserted into a BamHI- and XbaI-digested pcDNA3 vector (Invitrogen). All expression constructs were sequenced once again to ensure that the reading frame was correct. The cloning and characterization of this gene will be described in detail elsewhere (unpublished data).
Characterization of the NS3-ScFv.
The expression of NS3-ScFv-pET was induced with IPTG (isopropyl-β-d-thiogalactopyranoside) in E. coli BL21 transformants. The expressed protein was purified by using an Ni-nitrilotriacetic acid (Ni-NTA) chelating resin under denaturating conditions according to the recommendation of the manufacturer (Qiagen Gmbh, Hilden, Germany). The cells were pelleted and lysed in 8 M urea in Tris-HCl buffer, pH 8. The clarified supernatant was loaded on the Ni-NTA resin column. The bound protein was eluted with Tris buffer (pH 5.9, 5.0, and 4.0) containing 8 M urea or containing 0.1 M EDTA. Protein-containing fractions were analyzed by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Expression of NS3-ScFv-pET was confirmed by Western blot analysis. The samples containing NS3-ScFv-pET were applied on an SDS-PAGE minigel (PhastSystem; Pharmacia) and transferred electrophoretically to a nitrocellular membrane (Pharmacia). The gel was developed by silver staining. The membrane was blocked with a 2% bovine serum albumin–phosphate-buffered saline solution for 1 h at room temperature or overnight at 4°C prior to probing with Ni-NTA conjugate (Qiagen) or a goat anti-mouse Fab conjugate (A-2179; Sigma, St. Louis, Mo.). The bands were visualized with an insoluble substrate, BCIP-NBT (B-5655; Sigma).
The specificity of the purified NS3-ScFv-pET was determined by both direct and competitive EIAs. The NS3-ScFv-pET, alone or in mixture with MAb ZX10, was added to plates coated with 1 μg of rNS3 per ml. In the direct EIA, anti-Xpress antibody (Invitrogen) directed against an epitope carried by the NS3-ScFv-pET fusion protein was added prior to addition of a goat anti-mouse antibody conjugate (A-1047; Sigma). In the competitive EIA, the amount of remaining ZX10 MAb was indicated by the goat anti-mouse antibody conjugate (A-1047; Sigma). The enzyme reaction was developed by addition of 100 μl of p-nitrophenyl phosphate (Sigma), and optical densities were read at 405 nm.
Coexpression of NS3-ScFv and HCV NS3/NS4A.
To further characterize the specificity of the NS3-ScFv and the ability to recognize coexpressed HCV NS3 in a eucaryotic system, the T7 coupled reticulocyte lysate system (Promega, Madison, Wis.) was used. In vitro translation of the NS3-ScFv-pcDNA and control pcDNA vectors was carried out at 30°C with [35S]methionine (Amersham International plc, Buckinghamshire, United Kingdom). The labeled proteins were separated on an SDS–12% polyacrylamide gel and visualized by exposure to X-ray film (Hyper film-MP, Amersham) for 6 to 18 h. NS3-ScFv and NS3 complex formation was analyzed by immunoprecipitation. Protein A/G-Sepharose beads (Pierce) were incubated with the anti-Xpress antibody (Invitrogen) or anti-NS4A mouse serum (19) at 4°C for 1 h. The gel was then washed with 0.05 M Tris-HCl (pH 8.0) containing 1 mM EDTA, 0.15 M NaCl, 0.25% gelatin, and 0.02% NaN3 (NET buffer). The antibody-loaded protein A/G-Sepharose was then incubated for 2 h at 4°C under agitation together with a mixture of separately translated NS3/4A and NS3-ScFv or with these two proteins cotranslated. The immunoprecipitates were washed five times with NET buffer supplemented with 0.5 M NaCl and 0.3% SDS. The pellet was resuspended in 20 μl of 2× SDS sample buffer and heated at 100°C for 3 min. The proteins were then analyzed by SDS-PAGE.
RESULTS
Production of a MAb (ZX10) against HCV NS3.
The specificity of the NS3 MAb ZX10 was characterized by using rNS3 and 16-amino-acid synthetic peptides covering the ATPase/helicase domain of NS3 (Fig. 1). The epitope of the ZX10 MAb could be finely mapped to the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 (Fig. 2). A much lower reactivity to the linear synthetic peptide corresponding to NS3 residues 1367 to 1382 than to rNS3 was observed (Fig. 3). This region is similar to the one identified by a human MAb (12). Thus, the ZX10 MAb most likely recognizes a conformationally dependent epitope of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 may be a part in composing the complete epitope. Overall, the reactivity of the ZX10 MAb is similar to those previously observed in immunized mice and HCV-infected humans (3, 7, 15, 18).
FIG. 1.
Characterization of the specificity of binding of MAb ZX10 to rNS3 and 16-amino-acid synthetic peptides with a 6-amino-acid overlap by solid-phase EIA. OD, optical density.
FIG. 2.
Fine mapping of the peptide-specific reactivity by using deletion analogues of the NS3 peptide covering residues 1367 to 1382 by solid-phase EIA. OD, optical density.
FIG. 3.
Characterization of the preferred binding of MAb ZX10 by testing serial dilutions of MAb ZX10 with rNS3 (□) and the peptide p1367 to 1382 (▴) by solid-phase EIA. OD, optical density.
Construction of ZX10-ScFv expression vectors.
Sequence analysis using the V BASE database with the DNAPLOT software (http://www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO .html) showed that the rearranged VH and VL domains of the ZX10 MAb used sequences from the VH1 and VκIII gene families (Fig. 4). The NS3-ScFv-pET and NS3-ScFv-pcDNA vectors were constructed as outlined in Fig. 4. Sequence analysis did not show any sequence alterations during the cloning steps (data not shown). The DNA corresponding to complete NS3-ScFv together with the (Gly4Ser)3 linker comprises about 780 bp and encodes a protein of 32 kDa (Fig. 2).
FIG. 4.
Deduced amino acid sequence of NS3-ScFv and outline for amplification and cloning of NS3-ScFv in the pET and pcDNA3.1/His vectors. The thin solid line indicates the VH sequence, the thick solid line indicates the linker sequence, and the dotted line indicates the VL sequence of the NS3-ScFv. Primers containing the indicated restriction site are indicated by horizontal half arrows.
E. coli-expressed NS3-ScFv contains histidine residues as an amino-terminal leader sequence, which facilitates purification and detection. Both soluble and insoluble fractions were isolated, and the NS3-ScFv protein was located in the insoluble fraction. The NS3-ScFv was solubilized in Tris buffer (pH 8.0) containing 8 M urea and was purified by use of Ni-NTA chelating resin. The eluted fractions were analyzed by Western blotting. A detectable 32-kDa NS3-ScFv band, corresponding to the expected molecular mass, was identified by using Ni-NTA conjugate (Fig. 5).
FIG. 5.
Analysis of NS3-ScFv-pET expressed in E. coli by use of a silver-stained SDS minigel (right) and by Western blotting (left). Total E. coli lysate of the pET-15b vector without insert (lanes 1 and 7) and lysate of the E. coli pellet containing NS3-ScFv-pET (lanes 2 and 8) or containing the NS3-ScFv-pET eluted from the Ni-NTA column by Tris buffer with 8 M urea at pH 5.0 (lanes 3 and 9) or pH 4.0 (lanes 5 and 11) or with 0.1 M EDTA (lanes 6 and 12) are shown. Lanes 4 and 10, molecular size markers. The Western blot was developed with an Ni-NTA conjugate and the insoluble substrate BCIP-NBT.
Binding activity of ZX10-ScFv.
E. coli-expressed NS3-ScFv-pET was characterized by EIA. First, a γ chain-specific antibody (A-1047; Sigma) did not recognize the NS3-ScFv (Fig. 6a), whereas a Fab-specific antibody (A-2197; Sigma) bound the immobilized NS3-ScFv (Fig. 6a). Second, serial dilutions of NS3-ScFv in renaturating buffer (20% glycerol, 0.5 M NaCl, 2 mM β-mercaptoethanol, 0.05 M Tris-HCl, pH 7.4) were mixed with a constant concentration of MAb ZX10. The NS3-ScFv inhibited the binding of the ZX10 MAb to rNS3, confirming the specificity of the NS3-ScFv. As a negative control, pET-15b without any insert but expressed, purified, and diluted exactly like the NS3-ScFv was used and had no effect on the binding of ZX10 to NS3 (Fig. 6). Moreover, despite repeated experiments, we could not observe that the NS3-ScFv bound to the epitope peptide of the ZX10 MAb (data not shown). Overall, these experiments show that the recognition of rNS3 was retained but that the fine specificity, or the avidity, of the NS3-ScFv was reduced.
FIG. 6.
(a) E. coli-expressed ScFv in renaturating buffer applied directly to microplates was indicated by γ chain- and Fab-specific antibodies. (b) Analysis of the inhibition of MAb ZX10 binding to rNS3 by NS3-ScFv-pET or pET-15b vector without insert, both expressed and purified from E. coli and diluted in renaturating buffer. Residual binding of ZX10 was indicated by goat anti-mouse immunoglobulin G. OD, optical density.
The interaction between the NS3-ScFv and HCV NS3 was further evaluated by using eucaryotic expression systems. An immunoprecipitation assay was used for analysis of the interaction between NS3-ScFv-pcDNA and NS3/4A expressed in a rabbit reticulocyte lysate in vitro translation system. As positive controls, anti-NS4A immunoprecipitated in vitro-translated NS3/4A (Fig. 7, lane 1), whereas anti-Xpress immunoprecipitated in vitro-translated NS3-ScFv (Fig. 7, lane 4). Coexpressed NS3-ScFv and NS3/4A could be immunoprecipitated by either anti-NS4A (Fig. 7, lane 3) or anti-Xpress (Fig. 7, lanes 2 and 7). Moreover, the mixture of separately translated NS3/4A and ZX10-ScFv also formed a complex that was immunoprecipitated by anti-NS4A (Fig. 7, lane 5) or by anti-Xpress (Fig. 7, lane 6). Thus, the NS3-ScFv-pcDNA plasmid expresses a functional single-chain antibody fragment that recognizes and binds coexpressed HCV NS3.
FIG. 7.
Analysis of protein-protein interaction of the NS3-ScFv-pcDNA and NS3/4A proteins translated in vitro by a rabbit reticulocyte translation assay followed by immunoprecipitation. The proteins were labeled with [35S]methionine during translation and were then immunoprecipitated as described in Materials and Methods. Proteins were separated by SDS–8% PAGE. The NS3-ScFv-pcDNA and NS3/4A proteins and the sizes of the molecular mass markers are indicated. Positive controls are NS3/4A immunoprecipitated by anti-NS4A (lane 1) and NS3-ScFv-pcDNA immunoprecipitated by anti-Xpress (lane 4). Coexpressed NS3/4A and NS3-ScFv-pcDNA were immunoprecipitated by anti-NS4A serum (lane 3) and antibody against Xpress (lanes 2 and 7). Mixtures of separately translated NS3/4A and NS3-ScFv-pcDNA were also immunoprecipitated by anti-NS4A (lane 5) and anti-Xpress (lane 6) antibodies.
DISCUSSION
The HCV NS3 protein, which is most likely not present within HCV virions, can be detected inside the infected host cell. During HCV infection, the NS3 protein participates in the processing of the HCV polyprotein precursor and unwinding of viral RNA helices. Therefore, it may be possible to interfere with the enzymatic functions of NS3, which may disturb the viral life cycle. Intracellular therapies against HCV infection have been tested by using antisense oligonucleotides and ribozymes (10, 17). In addition, recombinant antibody fragments, or ScFv, expressed intracellularly have been demonstrated to inhibit numerous viruses (5, 8, 9, 11, 20).
We first produced MAb ZX10 against the NTPase/helicase domain of HCV NS3. The epitope of the ZX10 MAb could be finely mapped to the linear sequence GEIPFYGKAIPL at residues 1371 to 1382. However, the ZX10 MAb recognized rNS3 around 36 times more efficiently than the linear synthetic peptide epitope, which suggests that the complete epitope for the ZX10 MAb is conformationally dependent. This is not a unique phenomenon. We have previously characterized a MAb to the hepatitis B core antigen (HBcAg) that preferentially binds native HBcAg (13, 14). Within HBcAg one linear sequence could be precisely mapped, which most likely constitutes a part of the complete discontinuous epitope (13, 14). However, the peptide binding was of very low affinity, since in solution the peptide was unable to block the binding of the MAb to recombinant HBcAg (13). Due to the significant similarities, we propose the same type of binding characteristics for the HCV NS3-specific MAb ZX10. Importantly, the ZX10 MAb seems to recognize HCV NS3 in a similar way as do humans (3). We therefore designed an intracellular ScFV based on the ZX10 MAb.
The obtained sequences from the VH and VL domains of the ZX10 hybridoma showed that the rearranged sequences used the gene families VH1 and VκIII. The VH and VL cDNA fragments from the ZX10 hybridoma were assembled by a linker sequence to the NS3-ScFv, which was cloned into both procaryotic and eucaryotic expression vectors. E. coli-expressed NS3-ScFv retained most of the binding characteristics of the original MAb despite the recombination of the VH, VL, and linker DNA fragments. However, we were unable to repeat the peptide recognition exerted by the ZX10 MAb with the NS3-ScFv. This indicates that the NS3-ScFv may have a slightly altered fine specificity or that the affinity had been reduced. This is not surprising, since the peptide recognition of the ZX10 MAb was rather weak and presumably of low affinity.
Studies were performed to mimic the intracellular eucaryotic expression of HCV NS3 and the NS3-ScFv. By in vitro translation assays we found that the NS3-ScFv recognized and bound cotranslated NS3/NS4A, since the complex could be immunoprecipitated by antibodies to NS4A. This verifies that most of the binding activity of the ZX10 MAb was also retained by the recombined VH and VL domains expressed by the NS3-ScFv-pcDNA. We also noted that the NS3-ScFv could be expressed transiently and stably by several cell lines, indicating that the NS3-ScFv expression was nontoxic (data not shown). Collectively, this suggests that we have designed a eucaryotic vector coding for a single-chain antibody specific for the NTPase/helicase domain of HCV NS3 which can be stably expressed by mammalian cells. Hypothetically, intracellularly expressed NS3-ScFv may recognize endogenously expressed NS3 protein and may thereby possibly interfere with its intracellular functions.
ACKNOWLEDGMENTS
Financial support was obtained from the Swedish Medical Research Council (grant K98-06X-12617-01A) and funds provided by the School of Dentistry at the Karolinska Institutet.
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