MAb Targeting a Link Between ExoN and MTase of TGEV NSP14

Preparation of MAb against TGEV NSP14

The preparation of His-tagged TGEV NSP14 and its corresponding murine MAbs were described previously.^9–11 In brief, 6-week-old female BALB/c mice were immunized subcutaneously with 100 μg of the TGEV NSP14 at 2-week intervals. Mice splenocytes were harvested and fused with SP2/0 using 50% polyethyleneglycol (Sigma-Aldrich, MO). Antibodies in culture supernatants were screened using enzyme-linked immunosorbent assay (ELISA).

The positive hybridoma cells were repeatedly cloned by a limited dilution, and the stable hybridoma clones were injected into abdominal cavities of BALB/c mice to yield sufficient antibody. Subsequently, the MAbs were harvested from the seroperitoneum and purified with an antibody purification kit according to the manufacturer's specifications (NAb™ Protein A/G Spin Kit, Thermo Scientific). The animal study was approved by the ethics committee of the Wuhan University of Bioengineering, China (permit no. WUB20220517).

Immunofluorescence assay

PK-15 cells were seeded into 24-well tissue culture plates (Costar Corning, Inc., NY) at a density of 1 × 10⁵ cells per well. When the cells reached ∼70% confluence, culture medium was removed and then cells were washed three times with phosphate buffer saline (pH7.4) and incubated with TGEV (WH-01 strain) at a multiplicity of infection (MOI) of 0.01 for 1 hour at 37°C.

After removal of supernatant, fresh medium was added and cultures were incubated at 37°C. At 10 hours of postinfection, the infected cells were fixed with absolute methanol and incubated with MAbs for 2 hours, followed by application of fluorescein isocyanate-conjugated goat anti-mouse IgG. Fluorescent images were examined under a fluorescent microscope.

Immunoblotting

The TGEV-infected PK-15 cells were separated by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to nitrocellulose transfer membrane (GE Healthcare). The membrane was blocked for 1 hour at room temperature with blocking solutions containing 1% bovine serum albumin in tris buffered saline (TBS) (20 mM Tris-HCl [pH7.5], 150 mM NaCl), and then incubated overnight with MAb at concentration of 1 μg/mL at 4°C.

After washing three times with T-TBS (20 mM Tris-HCl [pH7.5], 150 mM NaCl, 0.05% Tween 20), the membrane was incubated for 30 minutes with horseradish peroxidase-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). After washing three times with T-TBS, the membrane was developed by treatment with ECL Western Blotting Detection Reagents (GE Healthcare, Darmstadt, Germany).

Enzyme-linked immunosorbent assay

The peptides derived from the 19 amino acids of Hinge I was chemically synthesized (Moonbiochem, Wuhan, China). The epitope on NSP14 recognized by 12F1 was determined by peptide-based ELISA. Generally, 96-well ELISA plates were coated with 100 μL of the indicated antigen (2 μg/mL of peptides) in coating buffer at 4°C overnight.

MAb 12F1 and irrelative MAb 5G10 (a salmonella bacteria flagellin-specific MAb) were detected by using alkaline phosphatase-labeled goat anti-mouse IgG (SouthernBiotech, AL), followed by substrate p-nitrophenyl phosphate (Sigma-Aldrich, MO). The reciprocal values of the last dilution giving an OD_405nm reading that was twice the background of the empty wells were determined as positive.

Preparation of TGEV NSP14

To obtain murine MAb specific for NSP14, we sought to construct and express full-length TGEV NSP14 in pET28a prokaryotic expression system (Novagen), but failed. Fortunately, a truncated construct harboring entire Exon and partial MTase (NSP14 201-460 amino acid, namely NSP14a) in BL21 was successfully expressed upon the induction of 0.5 mM isopropyl β-d-thiogalactoside (IPTG). As shown in Figure 1A, an individual protein band appeared with molecule mess about 27 kDa (Lane 2) that was consistent with the putative molecule weight of NSP14a (260 amino acids).

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FIG. 1.

Expression of prokaryotic TGEV-NSP14a. (A) BL21 containing recombinant plasmid pET28a-NSP14a was induced by IPTG, and then the whole bacteria BL21 lysates were subject to SDS-PAGE analysis. Lane 1, without IPTG inducing. Lane 2, IPTG inducing. Arrowhead indicates prokaryotic NSP14a protein about 27 kDa in molecule mass. (B) The cell lysates of whole bacteria were subject to WB. Lane 1, without IPTG inducing. Lane 2, IPTG inducing. The anti-Histag recognizes the TGEV-NSP14a that expresses in BL21. Arrowhead indicates prokaryotic TGEV-NSP14a. IPTG, isopropyl β-d-thiogalactoside; SDS-PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis; TGEV, transmissible gastroenteritis virus; WB, western blotting.

To further confirm whether the protein about 27 kDa mentioned earlier was NSP14a, we utilized anti-Histag (Proteintech, CN) to immunoblot with induced BL21 upon IPTG by western blotting (WB). As seen in Figure 1B, the induced BL21 containing pET28a-NSP14a plasmid generated a single band (∼27 kDa) (Lane 2), which was in agreement with the result of SDS-PAGE.

Subsequently, the induced target protein (NSP14a) was then purified using affinity chromatography Ni resin (GE) after mass culture of BL21 containing pET28a-NSP14a. Collectively, a truncated NSP14 (NSP14a) was produced with high purity. In turn, the NSP14a was used to immune five BALB/c mice.

Characterization of the MAb toward TGEV NSP14

The five BALB/c mice were immunized with purified NSP14a as described previously.^9–11 The fusion hybridoma cell secreting specific antibody against NSP14a were selected by using ELISA and limited dilution approach. Finally, a stable positive hybridoma cell line, designated MAb 12F1, was established. The characteristics of 12F1 were determined by indirect fluorescent assay (IFA) and WB. PK-15 was infected by TGEV (WH-01 strain) at MOI 0.01 for 10 hours. The TGEV-infected cells were subject to IFA. As shown in Figure 2A, fluorescent signal can be seen when 12F1, but not 5G10 (a MAb against bacterial flagellin), ⁹ was applied as detection antibody.

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FIG. 2.

MAb 12F1 recognizes eukaryotic TGEV NSP14. (A) PK-15 cell was infected with TGEV (WH-01 strain) at MOI 0.01 for 10 hours. Immunofluorescence assay was performed to evaluate whether MAb 12F1 could react with TGEV-NSP14. TGEV-NSP14-specific green fluorescent signal was able to be detected in PK-15 that was infected with TGEV. An irrelative MAb 5G10 served as a control. Scale bar, 100 μm. (B) The above cell lysates of TGEV-infected PK-15 cells were subject to WB, showing that a ∼52 kDa band (arrowhead indicated) can be detected when 12F1 was acted as detection antibody. (Lane 2) Uninfected PK-15 served as negative control. (Lane 1) (C) The purified MAb 12F1 was subject to SDS-PAGE, the result show 12F1 have both typical heavy chain (about 50 kDa) and light chain (about 20 kDa). MAb, monoclonal antibody; MOI, multiplicity of infection.

TGEV NSP14 is dominantly located in cytoplasm of PK-15 at 10 hours of postinfection. Moreover, the binding activity of 12F1 to NSP14 was evaluated by immunoblotting. As seen in Figure 2B, 12F1 can specifically interact with viral NSP14 (about 52 kDa). The purified MAb 12F1 was subject to SDS-PAGE, the result showed 12F1 have both typical heavy chain and light chain (Fig. 2C). ELISA result revealed that MAb 12F1 belongs to IgG1 subtype. (Data not shown) Therefore, the MAb 12F1 we developed here can specifically bind to viral NSP14.

Epitope determination of the MAb 12F1

The entire TGEV NSP14 consists of an Exon domain, two hinge regions, and two N7-MTase domains. (Fig. 3A) Next we further clarify the precise epitope recognized by 12F1. Three constructs derived from NSP14a with various truncations as indicated in Figure 3A. The BL21s containing various recombinant plasmids were induced by IPTG, subsequently the cell samples were subject to immunoblotting analysis by the aforementioned method.

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FIG. 3.

12F1 recognizes a hydrophilic epitope of TGEV-NSP14. (A) Diagram of truncated constructs of pET28a-NSP14. NSP14-FL, NSP14 full length (1-520 aa, amino acid); NSP14a, 201-460 aa; NSP14b, 282-460 aa; NSP14c, 201-300 aa; NSP14d, 201-410 aa. (B) After inducing by IPTG, BL21s containing various plasmids (pET28a-NSP14a, pET28a-NSP14b, pET28a-NSP14c, pET28a-NSP14d) were subject to WB with or without induction of IPTG. The arrowhead indicated each expressed truncated protein, NSP14a, NSP14b, NSP14c, and NSP14d, respectively. (C) Four synthetic partially overlapping peptides based on TGEV-NSP14a were used to identify the precise domain recognized by MAb 12F1. (D) The synthetic peptides and the TGEV-NSP14a were coated on 96-well plate and detected by MAb 12F1 using enzyme-linked immunosorbent assay. Irrelative MAb 5G10 acted as a control. The synthetic peptide P3 (WSIVYPFID) and the entire P0, as well as the intact TGEV-NSP14a can be specifically recognized by MAb 12F1. (E) The epitope that was recognized by MAb 12F1 was localized on hinge region between N7-methyltransferase and ExoN of the crystal structure model NSP14 (PDB: 7r2v).

As shown in Figure 3B, three truncates, along with NSP14a, show obvious signal, demonstrating the binding capacity of MAb 12F1 with all four various constructs derived from NSP14. Thus, the four NSP14 truncates shared the epitope of 12F1. Based on these results, the epitope recognized by MAb 12F1, therefore, should be set in the first hinge region (hinge I, ²⁸²IHDCFVKRVDWSIVYPFID³⁰⁰) of NSP14.

To accurately identify the epitope of 12F1, the four overlapping peptides derived from hinge I region were synthesized as indicated in Figure 3C. Each peptide was coated on the 96-well ELISA plate, respectively, to assess the binding activity of 12F1 by ELISA. The irrelative MAb 5G10 could not associate with any indicated peptide. As observed in Figure 3D, 12F1 had the strongest binding capacity with peptide 3 (P3, ²⁹²WSIVYPFID³⁰⁰), followed by P3 (²⁸⁶FVKRVDWSI²⁹⁴). Therefore, the P3 (²⁹²WSIVYPFID³⁰⁰) was the core epitope recognized by 12F1.

To further determine the spatial position of the epitope of 12F1 on NSP14, the peptide was localized on the model using PyMOL. Specifically, the peptide (²⁹²WSIVYPFID³⁰⁰) was fixed in the PyMOL molecule model (PDB: 7R2V). ¹² As shown in Figure 3E, the epitope of 12F1 was a hinge region between N7-MTase and ExoN, and exposed on the surface of the molecule model, suggesting its hydrophilic property.

A proper interface between MTase and polymerase of flavivirus plays important role in RNA initiation and elongation. ¹³ The MAb 12F1 developed in this study exclusively targets a hinge region between N7-MTase and ExoN, which orchestrate the TGEV genome RNA replication. Therefore, the application of 12F1 may perturb in the interface of N7-MTase and ExoN and disturb the TGEV replication. In addition, 12F1 associating with NSP14 probably interfere the interaction of NSP10 and NSP14 through steric hindrance effect, thereby inhibiting virus RNA replication. Therefore, a genetically modified 12F1 warrants further investigation for its potential application.