(2022) 19:213
Machitani et al. Virology Journal
https://doi.org/10.1186/s12985-022-01948-2

Open Access

BRIEF REPORT

Development of novel monoclonal
antibodies against nsp12 of SARS‑CoV‑2
Mitsuhiro Machitani1, Junko Takei2, Mika K. Kaneko2, Saori Ueki1, Hirofumi Ohashi3,4, Koichi Watashi3,4,
Yukinari Kato2,5 and Kenkichi Masutomi1*

Abstract
A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemicof coronavirus disease 19. Coronaviruses, including SARS-CoV-2, use RNA-dependent RNA polymerase (RdRP) for viral
replication and transcription. Since RdRP is a promising therapeutic target for infection of SARS-CoV-2, it would be
beneficial to develop new experimental tools for analysis of the RdRP reaction of SARS-CoV-2. Here, we succeeded todevelop novel mouse monoclonal antibodies (mAbs) that recognize SARS-CoV-2 nsp12, catalytic subunit of the RdRP.
These anti-nsp12 mAbs, RdMab-2, -13, and -20, specifically recognize SARS-CoV-2 nsp12 by western blotting analysis, while they exhibit less or no cross-reactivity to SARS-CoV nsp12. In addition, SARS-CoV-2 nsp12 was successfully
immunoprecipitated using RdMab-2 from lysates of cells overexpressing SARS-CoV-2 nsp12. RdMab-2 was able todetect SARS-CoV-2 nsp12 transiently expressed in established culture cells such as HEK293T cells by indirect immunofluorescence technique. These novel mAbs against SARS-CoV-2 nsp12 are useful to elucidate the RdRP reaction of
SARS-CoV-2 and biological cell response against it.
Keywords: SARS-CoV-2, RNA-dependent RNA polymerase, nsp12, Monoclonal antibody
Introduction
While human coronaviruses (CoV) are widespread inhumankind as a pathogen for the common cold, severe
acute respiratory syndrome CoV (SARS-CoV), Middle
East respiratory syndrome CoV (MERS-CoV), and SARSCoV-2 cause severe acute respiratory syndrome andhave extremely high mortality rates [1–4]. Among them,
SARS-CoV-2 emerged in Wuhan city, China at the end of2019 and spread worldwide, causing a global pandemic ofcoronavirus disease 19 (COVID-19) [2, 3]. Emerging evidences have clarified the virological properties of SARSCoV-2 and the biological mechanism by which leads tosymptoms of COVID-19 [5].

*Correspondence: kmasutom@ncc.go.jp
1
Division of Cancer Stem Cell, National Cancer Center Research Institute,5‑1‑1 Tsukiji, Chuo‑Ku, Tokyo 104‑0045, Japan
Full list of author information is available at the end of the article

SARS-CoV-2 is a positive-sense single-stranded RNA
virus that has a large RNA genome (~ 30 kb) and shareshigh sequence homology to SARS-CoV (~ 80%) and
MERS-CoV (~ 50%) [6]. A series of non-structural proteins (nsps), which is produced by cleaving two polyproteins, polyprotein1a (pp1a) and polyprotein1ab (pp1ab)translated from the genomic RNA, assemble into viral
replication and transcription complexes [7]. Amongthese nsps, nsp12, which is the catalytic subunit of the
RdRP complex containing nsp7 and nsp8 as cofactors,
catalyzes the synthesis of viral RNA and thus plays a pivotal role in the viral replication and transcription [8–11],
although the detailed transcription mechanism of CoVs
remains unclear. Therefore, nsp12 is considered a promising therapeutic target for antiviral inhibitors such asremdesivir, which is practically utilized to treat COVID19 [12–14]. Recent structural analysis of SARS-CoV-2
nsp12 by electron cryo-microscopy has revealed its righthand shaped structure like RdRPs of other RNA viruses© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
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Machitani et al. Virology Journal(2022) 19:213[8–11]. Intriguingly, the structures of nsp12 encodedby SARS-CoV-2 has a characteristic β-hairpin motif in
the nidovirus RdRP-associated nucleotidyltransferase(NiRAN) domain, which is structurally different fromthat of SARS-CoV [9]. However, difference between the
RdRP reactions of SARS-CoV and SARS-CoV-2 remains
unclarified.
Although a few antibodies (Abs) against nsp12 of
SARS-CoV-2 are commercially available, these Abs are
all polyclonal and not highly specific. In addition, thesepolyclonal Abs cannot discriminate between nsp12 of
SARS-CoV and SARS-CoV-2. To study the molecularmechanism underlying the RdRP reaction of SARSCoV-2, monoclonal antibody (mAb) against nsp12 of
SARS-CoV-2, which has not been developed so far, could
be powerful tools. Furthermore, the mAb that could
specifically detect SARS-CoV-2 nsp12, but not that of
SARS-CoV, would be highly potential to examine difference between the RdRP reactions of SARS-CoV and
SARS-CoV-2. In this study, we have established novel
mouse mAbs against SARS-CoV-2 nsp12 (RdMabs),
and evaluated whether they could be applied for western blotting, immunoprecipitation, and immunostaining
analyses. In addition, we examined the specificity of thesemAbs, especially focusing on whether they discriminate
between nsp12 of SARS-CoV and SARS-CoV-2.

Materials and methods
Cells and antibodies

P3X63Ag8U.1 (P3U1) and SV40-transformed humanembryonic kidney cell line HEK-293T (293T) cells wereobtained 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) weredeveloped in this study (see below).
Hybridoma production of mAbs against nsp12of 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.

Page 2 of 7

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 theNiRAN 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 ofeach 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 ScientificInc.). Cultured supernatants were finally screened using
enzyme-linked immunosorbent assay (ELISA) for thedetection 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 theplasmid vectors were codon optimized for expression inhuman 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-FLAGCoV-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

Machitani et al. Virology Journal(2022) 19:213

Page 3 of 7

Fig. 1 Development of mouse monoclonal antibodies against SARS-CoV-2 nsp12. A, B Structure of SARS-CoV-2 nsp12. The RdRP nsp12 comprises
the NiRAN, interface, and the RdRP domains, which has the right-handed structure composed of fingers, palm, and thumb domains. The ribbon
diagrams were cited from Protein Data Bank (PDB) (accession numbers 7BTF). NiRAN, the nidovirus RdRP-associated nucleotidyltransferase domain.
The peptides (#1–3) used to immunize mice are shown. C Western blotting analysis using mouse monoclonal antibodies (mAb) against SARS-CoV-2
nsp12 (RdMab-2, -13, and -20). A FLAG-tagged nsp12 of SARS-CoV-2 transiently expressed in 293T cells was detected using an anti-FLAG antibody
and RdMabs

onto 4–20% gradient sodium dodecyl sulfate (SDS)polyacrylamide gels. After electrophoresis under reducing conditions, bands of protein were transferred tonitrocellulose membranes (Amersham; Cytiva, Marlborough, MA). After blocking with 5% skim milk prepared in
TBS-T (Tween-20, 0.1%), the membrane was incubatedMachitani et al. Virology Journal(2022) 19:213

with 5 μg/mL of RdMab-2, -13, and -20, 1:5000 dilutionof anti-FLAG (clone M2; Merck), or 1:10,000 dilution ofanti-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 transfectedwith a FLAG-tagged nsp12-expressing plasmid (pCMVFLAG-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). Aftersonication, lysates were cleared of insoluble material bycentrifugation at 21,000×g at 4 °C for 15 min. FLAGnsp12 proteins were co-immunoprecipitated using Pierce
Protein A Plus Agarose (Thermo Fisher Scientific) and20 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 aspreviously 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 antimouse 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).

Results
Establishment of anti‑nsp12 mAbs

Structural analyses using electron cryo-microscopy haveshown that SARS-CoV-2 nsp12 protein has a closed,right-handed structure, which consists of palm, finger,
and thumb domains (Fig. 1A, B) [8, 9]. Comparative
analysis of structures of nsp12 encoded by SARS-CoVand SARS-CoV-2 suggested that SARS-CoV-2 nsp12 hasa characteristic β-hairpin motif in the NiRAN domain,which is structurally different from that of SARS-CoV[9]. We hypothesized that antibodies recognizing thePage 4 of 7

NiRAN domain in SARS-CoV-2 nsp12 could discriminate between nsp12 of SARS-CoV and SARS-CoV-2.
Therefore, we designed and utilized three peptides (#1–
3) of nsp12 around the NiRAN domain, but not recombinant nsp12 protein, as immunogens to immunize mice
(Fig. 1A, B) and subsequently established hybridomas
producing anti-nsp12 mAbs from the mouse spleen.
We first screened the culture supernatants by ELISA forbinding to nsp12 peptides, which selected 24 mAb clones(RdMab-1-24) (Table 1). To examine whether these putative clones binds to the nsp12 protein, we transiently
expressed FLAG-tagged nsp12 of SARS-CoV-2 in 293T
cells and then performed western blotting analysis using
the culture supernatants of hybridomas producing theseclones. We confirmed that anti-FLAG mAb (M2) recognized the FLAG-tagged nsp12 of SARS-CoV-2 in theposition of the predicted size (~ 100 kDa) (Additional File
1: Fig. S1). The screening of 24 mAb clones by western
blotting analysis demonstrated that 6 clones (RdMab-1,-2, -13, -15, -16, and -20) could detect SARS-CoV-2 nsp12(Additional File 1: Fig. S1). To further evaluate whether
they discriminate between nsp12 of SARS-CoV and
SARS-CoV-2, we performed western blotting analysis
Table 1 Subclass of RdMabs
Clone

Immunogen

Animal

Subclass
HC

LC
Kappa

RdMab-1#2, RHTFSNYQHEETIYNLLKDC

Mouse

IgG1

RdMab-2#3, TRALTAESHVDTDLTKPYIC

Mouse

IgG2a

Kappa

RdMab-3#1, AFDIYNDKVAGFAKFLKTNC

Mouse

IgM

Kappa

RdMab-4#2, RHTFSNYQHEETIYNLLKDC

Mouse

IgG1

Kappa

RdMab-5#2, RHTFSNYQHEETIYNLLKDC

Mouse

IgG1

Kappa

RdMab-6#3, TRALTAESHVDTDLTKPYIC

Mouse

IgG1

Kappa

RdMab-7#1, AFDIYNDKVAGFAKFLKTNC

Mouse

IgG1

LambdaRdMab-8#2, RHTFSNYQHEETIYNLLKDC

Mouse

IgG1

Kappa

RdMab-9#2, RHTFSNYQHEETIYNLLKDC

Mouse

IgG1

Kappa

RdMab-10#2, RHTFSNYQHEETIYNLLKDC

Mouse

IgG1

Kappa

RdMab-11#3, TRALTAESHVDTDLTKPYIC

Mouse

IgM

Kappa

RdMab-12#3, TRALTAESHVDTDLTKPYIC

Mouse

IgG1

Kappa

RdMab-13#3, TRALTAESHVDTDLTKPYIC

Mouse

IgM

Kappa

RdMab-14#2, RHTFSNYQHEETIYNLLKDC

Mouse

IgG1

Kappa

RdMab-15#2, RHTFSNYQHEETIYNLLKDC

Mouse

IgG1

Kappa

RdMab-16#2, RHTFSNYQHEETIYNLLKDC

Mouse

IgG1

Kappa

RdMab-17#3, TRALTAESHVDTDLTKPYIC

Mouse

IgG1

Kappa

RdMab-18#1, AFDIYNDKVAGFAKFLKTNC

Mouse

IgG1

LambdaRdMab-19#1, AFDIYNDKVAGFAKFLKTNC

Mouse

IgG1

LambdaRdMab-20#3, TRALTAESHVDTDLTKPYIC

Mouse

IgG2b

Kappa

RdMab-21#3, TRALTAESHVDTDLTKPYIC

Mouse

IgM

Kappa

RdMab-22#3, TRALTAESHVDTDLTKPYIC

Mouse

IgG1

Kappa

RdMab-23#1, AFDIYNDKVAGFAKFLKTNC

Mouse

IgM

Kappa

RdMab-24#1, AFDIYNDKVAGFAKFLKTNC

Mouse

IgG1

LambdaMachitani et al. Virology Journal(2022) 19:213

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using 293T cells transiently expressing the FLAG-tagged
nsp12 of SARS-CoV or SARS-CoV-2, suggesting thatthe culture supernatants of three clones (RdMab-2, -13,and -20) could specifically detect SARS-CoV-2 nsp12(Additional File 1: Fig. S2). Next, we purified these clones(RdMab-2, -13, and -20) from the hybridoma supernatants, which recognized SARS-CoV-2 nsp12 but not thatof SARS-CoV (Fig. 1C). Together, we established threeclones (RdMab-2, -13, and -20) which could specificallydetect SARS-CoV-2 nsp12.
Application to immunoprecipitation assay
and immunofluorescence cell staining

Fig. 2 Immunoprecipitation of SARS-CoV-2 nsp12 using RdMab-2.
SARS-CoV-2 nsp12 proteins were immunoprecipitated by RdMab-2
from 293T cells transiently expressing a FLAG-tagged nsp12 of
SARS-CoV-2, and then detected by an anti-FLAG antibody. Mouse­IgG2a was used as an isotype control for the immunoprecipitation

We next performed immunoprecipitation assay using
lysates of 293T cells transiently expressing the FLAGtagged nsp12 of SARS-CoV-2. Immunoprecipitation with
RdMab-2 successfully isolated the FLAG-tagged nsp12 of
SARS-CoV-2 from the lysates (Fig. 2), whereas RdMab-13
and -20 failed to isolate it by immunoprecipitation (datanot shown). We confirmed using an isotype matchedirrelevant control that the immune-complex precipitated by the RdMab-2 is specific signal (Fig. 2). Finally,
we applied RdMab-2 to immunostaining analysis. No signals were detected in non-transfected 293T cells (Fig. 3,
upper). When we transiently overexpressed the nsp12
in 293T cells, we found discrete signal of nsp12, which
was spreading throughout the cytoplasm (Fig. 3, lower).

Fig. 3 Immunofluorescence imaging of SARS-CoV-2 nsp12 using RdMab-2. 293T cells transiently expressing a FLAG-tagged nsp12 protein of
SARS-CoV-2 were immunostained with RdMab-2. Scale bar: 10 μm

Machitani et al. Virology Journal(2022) 19:213

This results are consistent with previous reports that the
RdRP reaction of CoVs, including SARS-CoV, is conducted in the cytoplasm [17–19]. Together, these findings
indicated the utility of RdMab-2 for immunoprecipitation and immunofluorescence cell staining assays.

Discussion
The recent global pandemic of SARS-CoV-2 has caused
catastrophic consequences [2, 3]. The research about
infection with SARS-CoV-2 and the development of therapeutic agents for COVID-19 is ongoing. Among several
SARS-CoV-2 proteins, its RdRP protein, nsp12, is one of
the crucial therapeutic targets, because it plays essential
roles in viral RNA replication and transcription [7, 14]. In
this study, we have established three novel mouse mAbs
against SARS-CoV-2 nsp12 (RdMab-2, -13, and -20).
They all detected SARS-CoV-2 nsp12 by western blotting
analysis more specifically than that of SARS-CoV (Fig. 1).
In addition, RdMab-2 were also suitable for immunoprecipitation (Fig. 2) and immunostaining analyses (Fig. 3).
Both SARS-CoV and SARS-CoV-2 use RdRP for viral
replication and transcription, although the detailedtranscription mechanism of CoVs remains unclear. It is
assumed that the RdRP complex of SARS-CoV-2 executes
two types of transcription; continuous transcription of
the genomic RNA (gRNA) and discontinuous transcription of various sizes of subgenomic RNAs (sgRNAs)[20, 21]. Regarding the discontinuous transcription, it ishypothesized that the RdRP can skip to the 3’-end leader
sequence of the template gRNA from transcription-regulatory sequences (TRSs) located in the middle of gRNA
[6, 20, 22, 23], although there has been no experimental evidence for the transcription mechanism of CoVs,
including SARS-CoV-2. In addition, difference between
the RdRP reactions of SARS-CoV and SARS-CoV-2
remains unclarified. Therefore, the mAb that could distinguish nsp12 from SARS-CoV and SARS-CoV-2 would
be potentially valuable to investigate difference between
the RdRP reactions of SARS-CoV and SARS-CoV-2.
RdMab-2 developed in the present study may contribute to the elucidation of this mechanism, because it has
the potential for various biochemical and cell biologicalexperiments.
While several rabbit polyclonal antibodies against
nsp12 of SARS-CoV-2 are now commercially available
from Cell Signaling Technology, Proteintech, GeneTex,
and so on, no mAb against nsp12 of SARS-CoV-2 havebeen reported. Notably, Yamada et al. [24] reported thatthe mAb against nsp12 of SARS-CoV (clone 4E6; Novus
Biologicals, Centennial, CO) can recognize nsp12 of
SARS-CoV-2, although the signals from western blotting analysis are faint. Since nsp12 of SARS-CoV and
SARS-CoV-2 shares high amino acid sequence homology

Page 6 of 7(~ 96%) (GenBank: NC_004718 and MN908947), it seems
that the mAb against nsp12 of SARS-CoV (clone 4E6;
Novus Biologicals, Centennial) can recognize nsp12 ofboth SARS-CoV and SARS-CoV-2. We now found that
RdMab-2, -13, and -20 could specifically detect the nsp12of SARS-CoV-2 by western blotting analysis (Figs. 1, 2
and 3). These novel anti-nsp12 mAbs would be the firstmAb specialized in detection of SARS-CoV-2 nsp12.
In this study, we designed three peptides (#1–3) for
producing mAbs, focusing on the structure around theNiRAN domain in the nsp12, because SARS-CoV-2
nsp12 has a characteristic β-hairpin motif in the NiRAN
domain, which was not found by structure analysis of
SARS-CoV nsp12 [9]. Intriguingly, the established mAbs
(RdMab-2, -13, and -20) were all obtained from mice
immunized with the same peptide #3 (Table 1). The
amino acid sequence of the peptide #3 derived of SARSCoV-2 nsp12 had lower homology (5 mutations/19 aminoacids) to the corresponding sequence of SARS-CoV
nsp12 than that of peptides #1 and #2 (1 mutation and
2 mutations/19 amino acids). In addition, RdMab-2, -13,and -20 preferentially recognized nsp12 of SARS-CoV-2
rather than that of SARS-CoV (Fig. 1C). These results
suggests that the structure surrounding peptide #3 in the
NiRAN domain might be unique to SARS-CoV-2 nsp12.
Taken together, we have successfully developed threenovel mouse mAbs against SARS-CoV-2 nsp12 (RdMab2, -13, and -20), which are suitable for western blotting,immunoprecipitation, and immunostaining analyses.
These novel anti-nsp12 mAbs were able to discriminate
between nsp12 of SARS-CoV and SARS-CoV-2. Generally, elucidation of specific protein functions criticallydepends on the ability to recognize the target protein
specifically, and we obtained mAbs that could specificallyrecognize SARS-CoV-2 nsp12. These novel anti-nsp12
mAbs could be utilized for investigation of the RdRP
reaction of SARS-CoV-2 and responses against it in hostcells. In this study, we could develop mAbs against SARSCoV-2 nsp12 as an initial step. We hope that further studies using these anti-nsp12 mAbs will lead to elucidationof the RdRP reactions of SARS-CoV-2 and developmentof more effective anti-viral strategies.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12985-​022-​01948-2.
Additional file1. Figure S1: Screening of anti-nsp12 mouse monoclonal
antibodies using the culture supernatant of hybridomas. Figure S2: Selection of anti-nsp12 mouse monoclonal antibodies to discriminate between
nsp12 of SARS-CoV and SARS-CoV-2.
Acknowledgements
We would like to thank the members of our laboratory for their help and many
useful suggestions.

Machitani et al. Virology Journal(2022) 19:213

Page 7 of 7

Author contributions
MM, SU and HO performed biochemical and cellular experiments. JT, MKK and
YK generated monoclonal antibodies against nsp12 of SARS-CoV-2. MM, KW,
and KM designed the experiments and discussed the interpretation of theresults. MM and KM wrote the manuscript. All authors read and approved thefinal manuscript.

9.

Funding
This work was supported in part by the Mitsubishi Foundation to M.M. andseveral grants from the Japan Agency for Medical Research and Development (AMED): the Emerging/Re-emerging Infectious Diseases Project Grant(No. 20fk0108274h0001 to K.M. and 21fk0108589j0001 to K.W.), Basis for
Supporting Innovative Drug Discovery and Life Science Research (BINDS) (Nos.
JP21am0401013 and JP22ama121008) to Y.K., and Science and Technology
Platform for Advanced Biological Medicine (No. JP21am0101078) to Y.K.

11.

Availability of data and materials
All data generated or analyzed during this study are included in this publishedarticle.

10.

12.

13.
14.

Declarations

15.

Ethics approval and consent to participate
The Animal Care and Use Committee of Tohoku University approved all animal
experiments (Permit Number: 2019NiA-001).

16.

Consent for publication
Not applicable.

17.

Competing interests
The authors declare no potential conflicts of interest.

18.

Author details
1
Division of Cancer Stem Cell, National Cancer Center Research Institute,5‑1‑1 Tsukiji, Chuo‑Ku, Tokyo 104‑0045, Japan. 2 Department of Antibody
Drug Development, Tohoku University Graduate School of Medicine, 2‑1
Seiryo‑Machi, Aoba‑Ku, Sendai, Miyagi 980‑8575, Japan. 3 Research Center
for Drug and Vaccine Development, National Institute of Infectious Diseases,
1‑23‑1 Toyama, Shinjuku, Tokyo 162‑8640, Japan. 4 Department of Virology II, National Institute of Infectious Diseases, 1‑23‑1 Toyama, Shinjuku,
Tokyo 162‑8640, Japan. 5 Department of Molecular Pharmacology, Tohoku
University Graduate School of Medicine, 2‑1 Seiryo‑Machi, Aoba‑Ku, Sendai,
Miyagi 980‑8575, Japan.
Received: 25 March 2022 Accepted: 6 December 2022

References
1. Fung TS, Liu DX. Human coronavirus: host-pathogen interaction. Annu
Rev Microbiol. 2019;73:529–57.
2. Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, Hu Y, Tao ZW, Tian JH, Pei
YY, et al. A new coronavirus associated with human respiratory disease in
China. Nature. 2020;579:265–9.
3. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Zhang L, Fan G, Xu J, Gu X,
et al. Clinical features of patients infected with 2019 novel coronavirus in
Wuhan, China. Lancet. 2020;395:497–506.
4. Zumla A, Chan JF, Azhar EI, Hui DS, Yuen KY. Coronaviruses - drug discovery and therapeutic options. Nat Rev Drug Discov. 2016;15:327–47.
5. Wiersinga WJ, Rhodes A, Cheng AC, Peacock SJ, Prescott HC. Pathophysiology, Transmission, diagnosis, and treatment of coronavirus disease 2019
(COVID-19): a review. JAMA. 2020;324:782–93.
6. Kim D, Lee JY, Yang JS, Kim JW, Kim VN, Chang H. The Architecture of
SARS-CoV-2 transcriptome. Cell. 2020;181(914–921): e910.
7. Snijder EJ, Decroly E, Ziebuhr J. The nonstructural proteins directing
coronavirus RNA synthesis and processing. Adv Virus Res. 2016;96:59–126.
8. Hillen HS, Kokic G, Farnung L, Dienemann C, Tegunov D, Cramer P. Structure of replicating SARS-CoV-2 polymerase. Nature. 2020;584:154.

19.
20.
21.
22.
23.
24.

Gao Y, Yan L, Huang Y, Liu F, Zhao Y, Cao L, Wang T, Sun Q, Ming Z, Zhang
L, et al. Structure of the RNA-dependent RNA polymerase from COVID-19
virus. Science. 2020;368:779–82.
Chen J, Malone B, Llewellyn E, Grasso M, Shelton PMM, Olinares PDB,
Maruthi K, Eng ET, Vatandaslar H, Chait BT, et al. Structural basis forhelicase-polymerase coupling in the SARS-CoV-2 replication-transcription
complex. Cell. 2020;182(1560–1573):e1513.
Yan L, Ge J, Zheng L, Zhang Y, Gao Y, Wang T, Huang Y, Yang Y, Gao S, Li
M, et al. Cryo-EM structure of an extended SARS-CoV-2 replication andtranscription complex reveals an intermediate state in cap synthesis. Cell.
2021;184(184–193):e110.
Sheahan TP, Sims AC, Graham RL, Menachery VD, Gralinski LE, Case JB,
Leist SR, Pyrc K, Feng JY, Trantcheva I, et al. Broad-spectrum antiviral
GS-5734 inhibits both epidemic and zoonotic coronaviruses. Sci Transl
Med. 2017;9:eaal3653.
Beigel JH, Tomashek KM, Dodd LE, Mehta AK, Zingman BS, Kalil AC,
Hohmann E, Chu HY, Luetkemeyer A, Kline S, et al. Remdesivir for thetreatment of Covid-19: preliminary report. N Engl J Med. 2020;383:1813.
Machitani M, Yasukawa M, Nakashima J, Furuichi Y, Masutomi K. RNAdependent RNA polymerase, RdRP, a promising therapeutic target forcancer and potentially COVID-19. Cancer Sci. 2020;111:3976–84.
Yasukawa M, Ando Y, Yamashita T, Matsuda Y, Shoji S, Morioka MS, Kawaji
H, Shiozawa K, Machitani M, Abe T, et al. CDK1 dependent phosphorylation of hTERT contributes to cancer progression. Nat Commun.
2020;11:1557.
Machitani M, Taniguchi I, Ohno M. ARS2 regulates nuclear Paraspeckle
formation through 3’-end processing and stability of NEAT1 long noncoding RNA. Mol Cell Biol. 2020. https://​doi.​org/​10.​1128/​MCB.​00269-​19.
V’Kovski P, Kratzel A, Steiner S, Stalder H, Thiel V. Coronavirus biology and replication: implications for SARS-CoV-2. Nat Rev Microbiol.
2021;19:155–70.
Gosert R, Kanjanahaluethai A, Egger D, Bienz K, Baker SC. RNA replicationof mouse hepatitis virus takes place at double-membrane vesicles. J Virol.
2002;76:3697–708.
Ivanov KA, Thiel V, Dobbe JC, van der Meer Y, Snijder EJ, Ziebuhr J.
Multiple enzymatic activities associated with severe acute respiratory
syndrome coronavirus helicase. J Virol. 2004;78:5619–32.
Sola I, Almazan F, Zuniga S, Enjuanes L. Continuous and discontinuous
RNA synthesis in coronaviruses. Annu Rev Virol. 2015;2:265–88.
Zhang Y, Huang K, Xie D, Lau JY, Shen W, Li P, Wang D, Zou Z, Shi S, Ren H,
et al. In vivo structure and dynamics of the SARS-CoV-2 RNA genome. Nat
Commun. 2021;12:5695.
Spaan W, Delius H, Skinner M, Armstrong J, Rottier P, Smeekens S, van der
Zeijst BA, Siddell SG. Coronavirus mRNA synthesis involves fusion of noncontiguous sequences. EMBO J. 1983;2:1839–44.
Furuichi Y: Caps on Eukaryotic mRNAs. In eLS. Wiley, Chichester; 2014.
Yamada T, Sato S, Sotoyama Y, Orba Y, Sawa H, Yamauchi H, Sasaki M,
Takaoka A. RIG-I triggers a signaling-abortive anti-SARS-CoV-2 defense in
human lung cells. Nat Immunol. 2021;22:820–8.

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