Cells and antibodies
P3X63Ag8U.1 (P3U1) and SV40-transformed human embryonic kidney cell line HEK-293T (293T) cells were obtained from the American Type Culture Collection (Manassas, VA). P3U1 cells were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) that was supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.). 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, streptomycin (100 μg/ml), and penicillin (100 U/ml).
Anti-β-actin (AC-15) mouse mAb (Merck, Darmstadt, Germany) and anti-FLAG mouse mAb (M2) (Merck) were used for western blotting analysis. mAbs against nsp12 of SARS-CoV-2 (RdMab-2, -13, and -20) were developed in this study (see below).
Hybridoma production of mAbs against nsp12 of SARS-CoV-2
Three female BALB/c mice (6-weeks-old) were purchased from CLEA Japan (Tokyo, Japan). The animals were housed under specific pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved all animal experiments (Permit number: 2019NiA-001). We designed three peptides (#1–3) of nsp12 as immunogens to immunize mice around the NiRAN domain (Fig. 1A, B). To develop mAbs against nsp12 of SARS-CoV-2, three synthesized peptides, such as 34AFDIYNDKVAGFAKFLKTNC53, 74RHTFSNYQHEETIYNLLKDC83, and 248TRALTAESHVDTDLTKPYIC266, which were keyhole limpet hemocyanin (KLH)-conjugated (Eurofins Genomics, Tokyo, Japan) were immunized intraperitoneally (i.p.) with Imject Alum (Thermo Fisher Scientific Inc.) into BALB/c mice (100 μg of each peptide/one mouse). The procedure included three additional immunization procedures (100 μg of each peptide), followed by a final booster intraperitoneal injection (100 μg of each peptide) 2 days before its spleen cells were harvested. The harvested spleen cells were subsequently fused with P3U1 cells, using polyethylene glycol 1500 (PEG1500; Roche Diagnostics, Indianapolis, IN). Then, hybridomas were grown in an RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher Scientific Inc.). Cultured supernatants were finally screened using enzyme-linked immunosorbent assay (ELISA) for the detection of nsp12 peptides [15]. Of 24 clones (RdMab-1 to -24), three clones (RdMab-2, 13, 20) were cultured using Hybridoma-SFM medium (Thermo Fisher Scientific Inc.), and were purified with Ab-Capcher (ProteNova, Kagawa, Japan).
Plasmids
The plasmid vectors, pCMV-FLAG-CoV-nsp12 and -CoV-2-nsp12, containing a FLAG-tagged nsp12 expression cassette driven by a CMV promoter, was designed and constructed by VectorBuilder Inc. (Chicago, IL). The SARS-CoV and -CoV-2 nsp12 genes (GenBank: NC_004718 and MN908947, respectively) cloned into the plasmid vectors were codon optimized for expression in human cells.
Transfection of plasmids
To overexpress SARS-CoV and -CoV-2 nsp12 transiently, 293T cells were transfected with plasmid vectors carrying a FLAG-tagged nsp12 expression cassette, pCMV-FLAG-CoV-nsp12 and -CoV-2-nsp12 using Lipofectamine 2000 (Thermo Fisher Scientific). After 72 h of incubation, cells were lysed in RIPA buffer (Nacalai Tesque) for western blotting analysis.
Western blotting analysis
Western blotting assay was performed as previously described [16]. Briefly, whole-cell extracts were prepared and 30 μg of total protein per lane was loaded onto 4–20% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gels. After electrophoresis under reducing conditions, bands of protein were transferred to nitrocellulose membranes (Amersham; Cytiva, Marlborough, MA). After blocking with 5% skim milk prepared in TBS-T (Tween-20, 0.1%), the membrane was incubated with 5 μg/mL of RdMab-2, -13, and -20, 1:5000 dilution of anti-FLAG (clone M2; Merck), or 1:10,000 dilution of anti-β-actin (clone AC-15; Merck), followed by incubation in the presence of horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody (1:5000; Cell Signaling Technology, Danvers, MA).
Immunoprecipitation assay
Immunoprecipitation assays were performed as previously described [15]. Briefly, 293T cells were transfected with a FLAG-tagged nsp12-expressing plasmid (pCMV-FLAG-CoV-2-nsp12). After 72 h of incubation, 1 × 107 cells were lysed in 1 ml of Lysis buffer A (0.5% NP-40, 20 mM Tris–HCl (pH 7.4), and 150 mM NaCl). After sonication, lysates were cleared of insoluble material by centrifugation at 21,000×g at 4 °C for 15 min. FLAG-nsp12 proteins were co-immunoprecipitated using Pierce Protein A Plus Agarose (Thermo Fisher Scientific) and 20 µg of anti-nsp12 mAb (RdMab-2) from the lysate, and then precipitated immune complexes were eluted in 2 × SDS loading buffer (2% β-mercaptoethanol, 20% glycerol, 4% SDS, and 100 mM Tris–HCl (pH 6.8)). The eluted proteins were detected by western blotting analysis. To detect the precipitated FLAG-nsp12 proteins, anti-FLAG mouse mAb (1:5000; M2) and Mouse TrueBlot ULTRA Anti-Mouse Ig HRP (1:4000; Rockland, Gilbertsville, PA) were used for western blotting analysis.
Immunofluorescence cell staining
Immunofluorescence cell staining was performed as previously described [16]. Briefly, cells were fixed with 4% formaldehyde in PBS, permeabilized with 0.2% TritonX-100 in PBS, and blocked with 2% bovine serum albumin in PBS. The cells were incubated with 10 μg/mL the primary antibody RdMab-2, followed by incubation in the presence of Alexa488-labeled goat anti-mouse IgG (1:1000; Thermo Fisher Scientific). The cells were mounted in ProLong Glass Antifade Mountant with NucBlue Stain (Thermo Fisher Scientific) and imaged under a fluorescent microscope (IX81, Olympus, Tokyo, Japan).