International Journal of

Molecular Sciences
ArticleSARS-CoV-2 Spike Protein Mutation at Cysteine-488 Impairs Its
Golgi Localization and Intracellular S1/S2 Processing
Yuichiro Yamamoto 1 , Tetsuya Inoue 1 , Miyu Inoue 1 , Mana Murae 1,2 , Masayoshi Fukasawa 1,2 ,Mika K. Kaneko 3 , Yukinari Kato 3,4 and Kohji Noguchi 1,2, *
1

2

3

4

*

Citation: Yamamoto, Y.; Inoue, T.;
Inoue, M.; Murae, M.; Fukasawa, M.;
Kaneko, M.K.; Kato, Y.; Noguchi, K.
SARS-CoV-2 Spike Protein Mutation
at Cysteine-488 Impairs Its Golgi
Localization and Intracellular S1/S2
Processing. Int. J. Mol. Sci. 2022, 23,
15834. https://doi.org/10.3390/

Laboratory of Molecular Targeted Therapy, Faculty of Pharmaceutical Sciences, Tokyo University of Science,
2641 Yamazaki, Noda 278-8510, Chiba, Japan
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1 Toyama,
Shinjuku-ku, Tokyo 162-8640, Japan
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Sendai 980-8575, Miyagi, Japan
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Sendai 980-8575, Miyagi, Japan
Correspondence: noguchi-kj@rs.tus.ac.jp

Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds tothe cellular receptor—angiotensin-converting enzyme-2 (ACE2) as the first step in viral cell entry.
SARS-CoV-2 spike protein expression in the ACE2-expressing cell surface induces cell–cell membranefusion, thus forming syncytia. To exert its fusogenic activity, the spike protein is typically processed
at a specific site (the S1/S2 site) by cellular proteases such as furin. The C488 residue, located at the
spike–ACE2 interacting surface, is critical for the fusogenic and infectious roles of the SARS-CoV-2
spike protein. We have demonstrated that the C488 residue of the spike protein is involved insubcellular targeting and S1/S2 processing. C488 mutant spike localization to the Golgi apparatus
and cell surface were impaired. Consequently, the S1/S2 processing of the spike protein, probed byanti-Ser-686-cleaved spike antibody, markedly decreased in C488 mutant spike proteins. Moreover,brefeldin-A-mediated endoplasmic-reticulum-to-Golgi traffic suppression also suppressed spike
protein S1/S2 processing. As brefeldin A treatment and C488 mutation inhibited S1/S2 processing
and syncytia formation, the C488 residue of spike protein is required for functional spike protein
processing.

ijms232415834
Academic Editor: Masahiro

Keywords: SARS-CoV-2; spike; cysteine; proteolysis; brefeldin A

Fujimuro
Received: 8 October 2022
Accepted: 12 December 2022
Published: 13 December 2022
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Copyright: © 2022 by the authors.
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distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) structural proteins,
such as spike, envelope, and membrane proteins, are transmembrane proteins synthesized in the perinuclear endoplasmic reticulum (ER), carried to the ER–Golgi intermediate
compartment (ERGIC) between the ER and the Golgi apparatus, and mostly assembled
into virions in the early secretory pathway, while some spike proteins are trafficked to theplasma membrane [1]. The spike protein is glycosylated in the ER and Golgi network, andsome spike proteins are further cleaved at a multibasic site into S1 and S2 subunits by the
Golgi enzyme or proprotease furin in the trans-Golgi network (TGN) [2]. The S1 domain
contains a receptor-binding domain (RBD) that binds to the host cell-surface-expressed
receptor protein, angiotensin-converting enzyme-2 (ACE2), while the S2 domain is responsible for viral entry and cell membrane fusion [3]. The second cleavage at the S2’ site in
the SARS-CoV-2 spike, typically mediated by the cell-surface-expressed transmembraneprotease TMPRSS2, followed the S1/S2 cleavage, influences viral infection and virus–cellmembrane fusion. The S1/S2 cleavage causes a conformational change in the spike protein,
and the second cleavage generates a fusion peptide at the S2’ site, which is critical forInt. J. Mol. Sci. 2022, 23, 15834. https://doi.org/10.3390/ijms232415834

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triggering membrane fusion [4]. Therefore, the SARS-CoV-2 spike protein has fusongenic
activity. Since syncytia is formed in SARS-CoV-2-infected cells, intracellular traffic andspike expression in the cell surface are responsible for SARS-CoV-2 cytopathogenicity andcoronavirus disease 2019 (COVID-19) pathogenesis [5,6].
The intracellular trafficking of transmembrane proteins synthesized in the ER throughthe secretory pathway is typically mediated by interactions between their cytoplasmic tails
and the coat proteins COPI and COPII in the ER-to-Golgi apparatus [7]. COPI-mediatedrecycling within the Golgi apparatus causes interactions between the coronavirus spikeand membrane proteins [8]. The SARS-CoV-2 spike protein has a dibasic KxHxx motif at
the cytosolic tail, and mutating this motif reduces SARS-CoV-2 spike expression at the cellsurface and syncytia formation; thus, it is critical for SARS-CoV-2 spike localization to the
ERGIC/Golgi region, glycan processing, protease cleavage at S1/S2 site, and trafficking
to the plasma membrane [1,9]. We have previously reported that mutating the C488
residue of the RBD abrogates the infectious role of the spike protein [10]. In this study, wedemonstrated impaired plasma membrane and Golgi-apparatus-targeting C488 mutant
spikes, which have intact cytoplasmic tails for intracellular trafficking. Consistently, theintracellular S1/S2 processing of C488 mutant spike proteins was suppressed. Overall, thisstudy highlights the importance of C488 residue in the functional subcellular trafficking of
spike proteins.
2. Results
2.1. C488 Is Important for the Fusogenic Activity and Cell-Surface Expression of the Spike Protein
Vero cells were transfected with a spike-expressing plasmid to compare the cell fusion
activity of C488A mutant and wild-type (WT) spike proteins. WT spike protein induced
multinuclear syncytia formation in Vero cells, but the C488A mutant spike protein did not(Figure 1A,B). Moreover, cell-surface C488A mutant spike in non-permeabilized cells wasbarely detected, while WT spike protein was easily detected (Figure 1A, upper images).
However, both WT and C488A mutant spike proteins were clearly detected in Triton-X100permeabilized Vero cells (Figure 1A, lower images). These observations indicate that most
C488A mutant spike proteins were not localized to the plasma membrane.
To confirm the impaired plasma membrane localization of the spike protein, C488R
and C488F mutant spike proteins were examined on the Vero cell surface. Cell-surface
C488 mutant spikes were not observed (Figure 1C, upper images), but whole-cellular WT
and C488 mutant spike proteins were equally visualized in permeabilized cells (Figure 1C,
lower images). Western blot analysis showed that the WT and C488 mutant spike proteinexpression levels were equal (Figure 1D). Confocal microscopy detected WT (Figure 1E,
white arrowheads), but not C488A mutant, spike protein in the plasma membrane. The
cell-surface expression levels were confirmed by flow cytometric analysis (Supplemental
Figure S1). Collectively, these data indicate that C488 mutation compromised the plasma
membrane targeting by the spike protein.

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Figure 1. Loss of cell−surface expression of C488 mutant spike protein: (A) Wild−type (WT) and
Figure1. Loss of cell-surface expression of C488 mutant spike protein: (A) Wild-type (WT) andC488A mutant spike−expressing plasmids were transfected in Vero cells for 2 days. Cell surface
C488A mutant spike-expressing plasmids were transfected in Vero cells for 2 days. Cell surface spike
spike protein was detected by anti−spike (1A9) antibodies and Alexa 488−conjugated secondary anprotein
wasdetected by anti-spike
antibodies
and Alexa 488-conjugatedsecondarytibodies
in non−permeabilized
cells(1A9)(upperpanels). Intercellularspike protein was
detectedantibodies
in Triinton−X100
non-permeabilized
cells(upperpanels).
spikefromprotein
was detectedin Triton-X100
permeabilized
cells(lowerpanels).Intercellular(B) Green signals
spike−probed
antibodyin (A)
were measuredcells
by ImageJ
At least
imageswerefrom
analyzedin each setting.
The spike−expermeabilized(lower1.44o.
panels).
(B)fiveGreen
signals
spike-probed
antibodyin (A) werepressing byarea
in C488A−expressing
permeabilized
cells was insignificantly
than that inmeasured
ImageJ
1.44o. At least fiveimages were analyzed
each setting.larger
The spike-expressing
non−permeabilized cells. The Student’s t−test was performed to assess statistical significance. ** inarea in C488A-expressing permeabilized cells was significantly larger than that in non-permeabilized
dicates p < 0.05. (C) Cell−surface expression of different C488 mutant spike proteins. WT and C488
cells. The Student’s t-test was performed to assess statistical significance. ** indicates p < 0.05. (C) Cellmutant spike proteins were expressed in Vero cells for 2 days. Cell surface (upper panels) and wholesurfaceexpression
different
C488
mutantproteins.
WTTheandnumbers
C488 mutant
proteins
intercellular(lowerof
panels)spike
proteins
werespikedetected,
as in (A).
#1 andspike#2 indicate
wereexpressed
in
Vero
cells
for
2
days.
Cell
surface(upperpanels)andwholeintercellular(lowertwo independent images. (D) Mutant spike protein expression was confirmed by Western blot analysis.
GAPDH
is
shown
as
a
loading
control.
(E)
Subcellular
localization
of
WT
and
C488A−mutant
panels) spike proteins were detected, as in (A). The numbers #1 and #2 indicate two independent
spike proteinsin permeabilizedVerocells wereanalyzedby confocal
microscopy.
The green
and isimages.
(D) Mutant
spike protein
expression
wasconfirmed
by Western
blot analysis.
GAPDH
blue signals represent spike proteins and DAPI−stained nuclei, respectively. The white arrowheads
shown as a loading control. (E) Subcellular localization of WT and C488A-mutant spike proteins inshow spike proteins detected at plasma membrane.
permeabilized Vero cells were analyzed by confocal microscopy. The green and blue signals represent
spike proteins and DAPI-stained nuclei, respectively. The white arrowheads show spike proteinsdetected at plasma membrane.

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2.2. C488 Is Involved in Golgi−Targeting by the Spike Protein

2.2. C488 Is Involved in Golgi-Targeting by the Spike Protein

Intracellular spike localization in Vero cells was examined by confocal microscopy.

IntracellularGreen
spike signals
localizationin Verocells wasexaminedbyanti−spike
confocal microscopy.
are spike
proteins
probedwith themonoclonal Green
antibody 1A9.
signals are spikeThe
proteins
probedwiththeanti-spike
monoclonal
antibody
1A9. Theco-calnexinco−localization at the ER and ERGIC were visualized by co−staining
withlocalization at theand
ERERGICand ERGIC
were visualized
by co-stainingwithcalnexin and
ERGIC
53, signals
53, respectively(red signal;
Figure 2A,B,
respectively).
Mergedyellow
respectively (redsuggest
signal; that
Figure2A,B,
respectively).
Mergedspikeyellow
signalssuggestthatdetectedboth in theboththe WT
and C488A mutant
proteins
weresimilarly
the WT and C488A
spikeThe
proteins
were similarly
detectedin the ER
ERGIC.
ER mutantand ERGIC.
co−localization
at the Golgi
apparatus
wasandobservedbyTheco−staining
GM130apparatus(Figure 2C).
described
WT
spike (Figure
expression
co-localization atwith
the Golgi
wasAs
observed
bypreviously
co-staining[11],
with
GM130
2C).induced
Golgi fragmentation(Figure2C, left panels);
the C488A mutant
spike
As described previously[11], WT spike
expression
inducedhowever,
Golgi fragmentation(Figure2C,was notco−localized
with
GM130
and
did
not
induceGolgi
fragmentation(Figure2C,
right panleft panels); however, the C488A mutant spike was not co-localized with GM130 and did
els).
These
data
suggestthat
C488A
mutationsuppressed
spike
proteintrafficking
not induce Golgi fragmentation (Figure 2C, right panels). These data suggest that C488A to theGolgi apparatus.
mutation suppressed
spike protein trafficking to the Golgi apparatus.

Figure2. Subcellular
localizationof spike
C488 mutant
proteinin Verocells. Wild−type
Figure 2. Subcellular
localizationof C488
mutantproteinspikein Verocells.
Wild-type(WT) and(WT) andC488A mutant spike−expressing
plasmids wereexpressed
2 days,and their subcelC488A mutant spike-expressing
plasmids were expressed
in Verocells forin2Verodays,cells
andfortheirsubcellular
lular co−localization with (A) calnexin, (B) ERGIC53, and (C) GM130 were analyzed by confocal
co-localization with (A) calnexin, (B) ERGIC53, and (C) GM130 were analyzed by confocal microscopy.
microscopy. WT and C488A mutant spikes (green signals) were probed with anti−spike (1A9) antiWT and C488A mutant
spikes(green markers
signals)(red
wereprobedwithanti-spike(1A9) antibodies
andbodies andsubcellularsignals)used
were:calnexin, ERGIC53,
and GM130
as ER, ERsubcellular markers(redsignals)
used were:calnexin, A
ERGIC53,
andyellow
GM130
as ER,ERGIC, and
Golgi
GIC,
and
Golgi markers,
respectively.
co−localized
signalis visualized
in themerged images A(lowerpanels). yellow signal is visualized in the merged images (lower panels).
markers, respectively.
co-localized

2.3. C488 Mutation
S1/S2
Processingof theSpike Protein
2.3.Suppresses
C488 Mutation
Suppresses
S1/S2
Processingof the Spike Protein
The
SARS−CoV−2
spike
can beby
processedby the proprotein
convertase
The SARS-CoV-2
spike
protein canbeproteinprocessed
the proprotein
convertase
furin furin
at
the
S1/S2
site.
Furin
is
ubiquitously
expressed
in
the
Golgi
apparatus
andtrans−Golgi
at the S1/S2 site. Furin is ubiquitously expressed in the Golgi apparatus and trans-Golgi
network[12,13].
As
thecell−surface
expression
of
the
C488A
mutantwas
disturbed,
we
network [12,13]. As the cell-surface expression of the C488A mutant was disturbed, we next
next
compared
S1/S2
processing
of
WT
and
C488A
mutantspike
proteins.
The
S1/S2
cleavcompared S1/S2 processing of WT and C488A mutant spike proteins. The S1/S2 cleavage
age of the WT, but not the C488A mutant, spike protein was detected by an anti−Ser−686
of the WT, but not the C488A mutant, spike protein was detected by an anti-Ser-686 cleaved
cleaved spike monoclonal antibody (Figure 3A, bottom panel). As the C488A mutation
spike monoclonal antibody (Figure 3A, bottom panel). As the C488A mutation abrogatedabrogated disulfide−bond formation between C480 and C488, we tested the effect of an
disulfide-bond formation
C480 andC488, wetestedtheof foran protein
inhibitorof in theinhibitor of between
protein disulfide
isomerases(PDI),
whichis effect
requiredfolding
protein disulfideER,
isomerases(PDI),
whichisrequiredfor
proteinfolding
in
the
ER,
on
on the spike protein S1/S2 processing. After treatment with the PDI inhibitor CCF642,
the spike protein
the PDI
CCF642,the cleaved
theS1/S2
wholeprocessing.
spike proteinAfterlevel treatment
was slightlywith
decreased,
andinhibitor
subsequently,
Ser−686
whole spike protein level was slightly decreased, and subsequently, Ser-686 cleaved spike
protein level was reduced. (Figure 3B). Thus, non-specific inhibition of disulfide-bondformation in the spike protein was not a major cause of the C488A mutation-mediated
S1/S2 processing defect. Interestingly, a recent study showed that other cysteine mutants,
such as C301F and C379F, reduced the S2 fragment spike protein, although the reason was
not discussed [14]. Overall, these data suggested that the C488A mutation significantly

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Int. J. Mol. Sci. 2022, 23, 15834

5 of 14spike protein level was reduced. (Figure 3B). Thus, non−specific inhibition of disulfide−bond formation in the spike protein was not a major cause of the C488A muta5 of 13tion−mediated S1/S2 processing defect. Interestingly, a recent study showed that othercysteine mutants, such as C301F and C379F, reduced the S2 fragment spike protein, although the reason was not discussed [14]. Overall, these data suggested that the C488A
mutationsignificantly
attenuates
ER–Golgi
trafficking
and S1/S2
the spikeattenuates
ER–Golgi
trafficking
and S1/S2
processing
ofprocessing
the spike ofprotein.
The mutantprotein.
The mutant−mediatedprocessing
defect was
analyzedin othervariant
mediated
S1/S2 processingS1/S2
defectwas analyzed
in othervariant
spike
proteins using
spike
proteins
using
other 3C).
cell lines(FigureWhen
C488
mutantandproteinsvariant spikeothercell lines(FigureWhen
C488 3C).
mutantandvariant
spike
were expressed
proteins
were
expressed
in
HEK293Tcells
and
ACE2−transfected
human
lung
adenocarin HEK293T cells and ACE2-transfected human lung adenocarcinoma
NCI-H522 clone
cinoma NCI−H522 clone cells (NCI−H522/ACE2), Ser−686 cleavage in the C488A, C488R,
cells (NCI-H522/ACE2), Ser-686 cleavage in the C488A, C488R, and C488F spikes were
and C488F spikes were reduced in both cell lines. The C488R and C488F mutations are
reduced in both cell lines. The C488R and C488F mutations are naturally recorded in thenaturally
recordedinthe
SARS−CoV−2
database
COV−GLUE
SARS-CoV-2 database COV-GLUE (https://cov-glue.cvr.gla.ac.uk/index.php, accessed on(https://cov−glue.cvr.gla.ac.uk/index.php, accessed on 30 September 2022) with frequen30ofSeptember
2022)
withrespectively.
frequenciesTheof Ser−686
0.000002
and 0.000003,
respectively.
The Ser-686
cies
0.000002 and
0.000003,
cleavagein Wuhan−Hu−1,
D614G,
cleavagein
Wuhan-Hu-1,
D614G,
and
Delta
spike
proteins
werecomparable,
and Delta spike proteins were comparable, while those in Alpha and Omicron BA.1 werewhile thosein Alpha
andOmicronsyncytium−inducing
BA.1 were relatively
low. of
Functional
syncytium-inducing
activity
relatively
low.
Functional
activity
Alpha variant
spikes was obof Alphavariant spikescells
was(Supplemental
observed in NCI-H522/ACE2
cells(Supplemental
servedin NCI−H522/ACE2
Figure S2); therefore,
S1/S2
processing in Figure S2);
cells
may haveS1/S2
a low impact
on Alpha
spike
activity.
relatively
lowtherefore,
processing
in cells
may
haveThea lowimpactonsyncytium−inducAlpha spike activity. The
ing
activity oflow
Omicron−typespikes is consistent
the results of aspikes
recentisstudy[15]. with therelatively
syncytium-inducing
activity withof Omicron-type
consistent
These
resultssuggestthat
C488
mutationcausesunique
S1/S2
processing
defects
inresults of a recent study [15]. These results suggest that C488 mutation causes theunique S1/S2
spike
protein. defects in the spike protein.
processing

Figure 3. Defect of S1/S2 processing in C488 mutant spike proteins. (A) Spike-expressing plasmid
was transfected into VeroE6/TMPRSS2 cells, and the cell lysate was prepared 20 h post-transfection.
Full-length and Ser-686 cleaved spike proteins were probed with anti-spike (1A9) and cleaved SARSCoV-2 spike (Ser686) antibodies, respectively. GAPDH is shown as a loading control. (B) Wild-type
spike-expressing plasmid was transfected in HEK293 cells, which were treated with CCF642 for 13 h
at 5 h post-transfection, followed by cell lysate preparation. Full-length and Ser-686 cleaved spike
proteins were detected with anti-spike (1A9) and cleaved SARS-CoV-2 spike (Ser686) antibodies,
respectively. (C) Each mutant spike-expressing plasmid was transfected in NCI-H522/hACE2 (leftpanels) and HEK293T (right panels) cells. After 20 h, cell lysates were prepared, and full-length andSer-686 cleaved spike proteins were detected with anti-spike (1A9) and cleaved SARS-CoV-2 spike(Ser686) antibodies, respectively.

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6 of 132.4. SARS-CoV-2 Membrane Protein Does Not Compensate C488 Mutation of Spike Protein
Previous reports have shown that co-expression of the SARS-CoV-2 membrane (M) protein induces spike and nucleocapsid (N) protein accumulation in the Golgi apparatus [16,17].
WT spike-induced syncytium formation in VeroE6/TMPRSS2 cells was apparently suppressed by co-expression of M, but not by N, which confirmed functional interplay between
the spike and M protein (Figure 4A). Therefore, we examined the effect of M protein coexpression on C488A localization in the Golgi apparatus. Immunofluorescence microscopic
analysis showed that green signals of WT spike protein in most cells were co-detected with
GM130 red signals, and the merged yellow signals were clear, showing Golgi-targeting of
WT spike proteins in Vero cells 20 h post-transfection (Figure 4B (left images),C (quantitative results)). In contrast, co-localization of the C488A mutant spike protein with GM130
did not increase, even in the presence of M protein co-expression. Precise analysis indicatedthat GM130 red signals co-localized with the WT, but not the C488A mutant, spike signals(Supplemental Figure S3). However, co-expression of N and M combination slightly, but
statistically significantly, enhanced co-localization of C488A mutant spike with GM130(Figure 4B (right images),C (quantitative results)). Since Golgi localization affects S1/S2
processing, we examined the effect of M and N protein co-expression on spike protein
S1/S2 processing (Figure 4D). The co-expression of M protein decreased S1/S2 processing(cleavage at Ser-686) of the C488A mutant spike, and co-expression of N protein appeared
to show little impact on S1/S2 processing (Figure 4D). Quantitative, triplicated experiments indicated that M protein might increase S1/S2 processing of WT spike protein, butconversely decrease that of C488A mutant spike protein (Figure 4E). Full-length spike
protein levels were comparable in the presence of M protein. Although previous studies
revealed a critical role of the C-terminal cytoplasmic tail of the spike protein in ER–Golgi
retention [9,16], our data suggest that the C488 mutation may affect spike protein targeting
to the Golgi apparatus and functional interplay with M protein.
2.5. Brefeldin A Inhibits SARS-CoV-2 Spike Protein Processing
Brefeldin A (BFA) is a fungal metabolite that disrupts intracellular membrane traffic
at the ER–Golgi complex junction [18]. Hackstadt et al. reported that BFA-mediatedGolgi apparatus disruption inhibited viral replication [11]. To confirm the involvementof ER–Golgi trafficking in S1/S2 processing of spike proteins, we tested the effect of BFA
and observed that BFA treatment disrupted WT spike plasma membrane localization(Figure 5A), but not furin localization (red signal). Further, Western blot analysis using
anti-Ser-686 antibodies indicated that BFA treatment suppressed S1/S2 processing of every
spike protein variant (Figure 5B). BFA treatment also affected the electrophoretic mobilityof Ser-686 cleaved S2 protein on Western blot analysis, suggesting that BFA-mediatedGolgi-targeting inhibition might suppress glycosylation maturation of the spike protein.
The function of the spike protein after BFA treatment was examined using a cell–cellfusion assay. BFA treatment inhibited WT, Delta, and Omicron spike-mediated syncytium
formation in VeroE6/TMPRSS2 cells (Figure 5C). The spike-mediated syncytium was notinhibited by CID-1067700, a Rab7 GTPase competitive inhibitor that blocks lysosomal
trafficking during beta-coronavirus egress [19]. Thus, ER-to-Golgi trafficking and S1/S2
processing of spike proteins are critical for the functional targeting of spike proteins to theplasma membrane.

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Figure 4. M protein did not affect C488A-mutant spike. (A) VeroE6/TMPRSS2 cells were coFigure 4. M protein did
not affect C488A−mutant spike. (A) VeroE6/TMPRSS2 cells were co−transtransfected with spike-, M-, N-expressing plasmids with pEGFP-C1. The cells were fixed at 20 h
fected with spike−, M−,
N−expressing plasmids with pEGFP−C1. The cells were fixed at 20 h
post-transfection, and the green fluorescence protein (GFP) signals were photographed with convenpost−transfection, andtional
the fluorescencegreen fluorescence
protein(GFP) signals
were
withbyconmicroscopy. Areasof GFP-positive
green cells
were photographedmeasured in each picture
software, andaverageofarea
size of GFP-positive
cells incellsa picture
was measured
plotted in the rightgraph.picventional fluorescenceImageJ
microscopy.
Areas
GFP−positive
green
were
in eachOne-way
ANOVA
withTukey-HSD
wasperformed
toassessstatisticalsignificance.
n.s.
means
ture by ImageJ software, and average area size of GFP−positive cells in a picture was plotted in the“not significant”, and * indicates p < 0.01. (B) Spike-expressing plasmids were co-transfected withright graph. One−way ANOVA with Tukey−HSD was performed to assess statistical significance.
SARS-CoV-2 M or N protein-expressing plasmid in Vero cells, which were fixed and permeabilized.
n.s. means “not significant”,
and *ofindicatesp <with0.01.
(B) (red)
Spike−expressing
plasmids
were co−transCo-localization
spikes (green)
GM130
were analyzed by confocal
microscopy.
Whitefected with SARS−CoV−2
or Ncomplete
protein−expressing
plasmid
inwithVerocells,
wereoffixed
arrowsM
indicateco-localization (yellow)of spikes
GM130.
(C)whichCo-localization
spikesandand GM130 wasin the transfected
cells (n ≥ 156),
andwerethe ratioof co-localizationis shown. mipermeabilized. Co−localizationof examined
spikes (green)with GM130(red)analyzedby confocal
One-way
ANOVA
withTukey-HSD
wasperformed
toassessstatisticalsignificance.
n.s.
means
croscopy. White arrows indicate complete co−localization (yellow) of spikes with GM130. (C)“notCo−losignificant”, and * indicates p < 0.01. (D,E) SARS-CoV-2 spike-expressing plasmid was co-transfected
calization of spikes andGM130 was examined in the transfected cells (n ≥ 156), and the ratio ofwith M- or N-expressing plasmid in HEK293T cells. The cell lysate was prepared after 20 h, andco−localization is shown.
One−way
ANOVA
withTukey−HSD
performed
to assessstatisticalfull-lengthand Ser-686
cleaved spike
proteins
were detectedwas
by Western
blotting with
anti-spike(1A9)
significance. n.s. means“notsignificant”,
and(Ser686)* indicatesp < respectively.
0.01. (D,E)Triplicated
SARS−CoV−2
andcleavedSARS-CoV-2 spike
antibodies,transfectedspike−expresssamples wereanalyzed (Exp.with
1–3), M−and signal
intensity was quantified
by ImageJ(right graphin E). The cell lysate
ing plasmid was co−transfectedor N−expressing
plasmidin HEK293T
cells.
was prepared after 20 h, and full−length and Ser−686 cleaved spike proteins were detected by Western blotting with anti−spike (1A9) and cleaved SARS−CoV−2 spike (Ser686) antibodies, respectively.
Triplicated transfected samples were analyzed (Exp. 1–3), and signal intensity was quantified by
ImageJ (right graph in E).

2.5. Brefeldin A Inhibits SARS−CoV−2 Spike Protein Processing
Brefeldin A (BFA) is a fungal metabolite that disrupts intracellular membrane traffic
at the ER–Golgi complex junction [18]. Hackstadt et al. reported that BFA−mediated Golgi
apparatus disruption inhibited viral replication [11]. To confirm the involvement of ER–

Int. J. Mol. Sci. 2022, 23, 15834

lysosomal trafficking during beta−coronavirus egress [19]. Thus, ER−toand S1/S2 processing of spike proteins are critical for the functional
8 of 13proteins to the plasma membrane.

Figure 5. Brefeldin A (BFA) interferes with SARS-CoV-2 spike protein function. (A) Vero cells were

Figure5. Brefeldin
A (BFA)
interferes
with
spike
proteinfunctiontransfectedwith spike-expressing
plasmid.
The cells
wereSARS−CoV−2
treated with or without
1 µM
BFA for an
additional 15 h
at 9 hspike−expressing
post-transfection. The spike
protein The
localization
permeabilizedwastransfectedwithplasmid.
cells inweretreated cells
withor witho
examined using confocal microscopy. The numbers #1 and #2 indicate two independent images.
additional 15 h at 9 h post−transfection. The spike protein localization in perm
White arrowheads indicate plasma membrane localization of spike protein. (B) HEK293T cells were
examinedusing confocal microscopy. The numbers #1 and #2 indicate two intransfected with spike-expressing plasmid. The cells were treated with 1 µM BFA and 1 µM CIDWhite
indicate
membranelocalizationof spike
protein.
(B)
1067700arrowheads(CID) for additional
19 h atplasma
9 h post-transfection.
Full-lengthand Ser-686
cleavedspike
proteins
weredetectedbyWesternblottingwith
anti-spike
(1A9)
and
cleavedSARS-CoV-2
spike
transfected with spike−expressing plasmid. The cells were treated with 1 µ(Ser686) antibodies, respectively. (C) VeroE6/TMPRSS2 cells were transfected with spike-expressing
CID−1067700(CID) for additional 19 h at 9 h post−transfection. Full−length aplasmid with pEGFP-C1. The cells were treated with BFA or CID-1067700 for an additional 15 h at 5 h
spike
proteinsThe
were
Westernblottingwithanti−spike(1A9) and cl
post-transfection.
greendetected
fluorescenceby
protein(GFP) signals
and phasecontrast
cell morphologies
spike(Ser686) antibodies,
respectively.
(C) VeroE6/TMPRSS2 cells were transfe
were photographedwith conventional
fluorescence microscopy.
pressing
plasmid with pEGFP−C1. The cells were treated with BFA or CID−10677
3. Discussion
15 h at
5 h post−transfection.
The greenprotein(GFP)
signalsand
SARS-CoV-2
infection causes cell–cellfusionfluorescenceto form cytopathic
syncytia,
whichremorphologies
werephotographed
withprotein
conventional
fluorescencemicroscopy.
quires cell-surface
expressionof the viral spikeand its binding
to ACE2 expressedon the neighboring cell surface [6]. The disulfide bond between C480 and C488 in the spike
protein stabilizes the conformation of the extended loop structure at the RBD, which is
3.required
Discussion
for ACE2-binding [20,21]. Previously, we have reported SARS-CoV-2 spike proteinfunction loss by mutation at C488 residue [10] and demonstrated the novel effects of C488
SARS−CoV−2 infection causes cell–cell fusion to form cytopathic s
residue on spike protein subcellular targeting and its S1/S2 processing. As BFA inhibited
quirescell−surface
expressionof the viral
spike
proteinits bindingtboth ER-to-Golgi
traffic and
the S1/S2 processingof spike
protein,our dataand
indicatethatthetheC488
residue of spike cell
proteinis required
forThethe functional
processing
spike proteinsonneighboring
surface[6].
disulfidebondofbetween
C480 andin cells.
proteinstabilizesspike
theprotein
conformationof the extended
looptranslocatedstructure at t
The SARS-CoV-2
is a type I transmembraneprotein initiallyinto the ER,for
glycosylated
in the ER and
Golgi apparatus,and further
into S1/S2SARS−
required
ACE2−binding[20,21].
Previously,
weprocessed
have reported
subunits by furin in the trans-Golgi network [2]. The spike protein’s C-terminal cytoplasmic
teinfunction loss by mutation at C488 residue [10] and demonstrated ttail, rather than the RBD, regulates intracellular trafficking and S1/S2 processing of the spike
protein[1,9]. The on
non-canonical
COPI-binding
motif in thetargeting
SARS-CoV-2andspikeits
C-terminal
C488
residue
spike protein
subcellular
S1/S2 proce
tail is speculated as allowing more spikes to reach the surface [8], and the sub-optimal

hibited both ER−to−Golgi traffic and the S1/S2 processing of spike protcate that the C488 residue of spike protein is required for the functio
spike proteins in cells.
The SARS−CoV−2 spike protein is a type I transmembrane protei

Int. J. Mol. Sci. 2022, 23, 15834

9 of 13

COPI-binding site of SARS-CoV-2 spike protein C-terminal tail appears to contribute toits efficient plasma membrane localization for syncytia formation [1,9]. The M protein,another SARS-CoV-2 viral protein, induces spike protein retention in the ERGIC and Golgi
apparatus [1,16]. Another viral structural, protein N, is known to be responsible for COVID19 virulence as it affects the rate of replication of the virus [22]. Interaction among thecoronaviral structural proteins (N, E, S, and M), as well as a host membrane envelope
obtained from the site of budding, is required for viral assembly [23]. A recent study hassuggested a predominant role for M protein in controlling the spatial organization of thetransmembrane proteins and initiating the assembly of SARS-CoV-2, and that the M protein
is responsible for the recruitment of the N protein to the Golgi/ERGIC membranes [17].
Our data consistently showed that the M + N combination enhanced C488A mutant spiketargeting to the Golgi, suggesting a possible role of interaction between M and N proteinsin the spike protein traffic mechanism. M-protein-mediated spike protein retention at the
ERGIC and Golgi is also dependent on the intact spike protein C-terminal cytoplasmic
tail, and M protein cannot reverse C488 mutation-mediated ER-to-Golgi traffic defects.
In contrast, M protein decreased C488A mutant spike S1/S2 processing although theprecise mechanism is unknown. As the C-terminal tail, which is required for intracellulartrafficking to both the Golgi and plasma membranes, is intact in the C488 mutant spike
protein, C488 mutation might affect mechanisms other than the C-terminal-tail-involved
ER–Golgi–plasma membrane traffic. Tien et al. showed that LYQD mutations at 611–614
residues alter intracellular spike protein trafficking and reduce cell-surface expression
levels [24]. While the way in which the LYQD motif might coordinate spike protein
maturation with ER export and ER retrieval signals in the C-terminus is unclear, theseobservations raise the possibility that mechanisms other than the spike C-terminal tail may
be involved in the intracellular SARS-CoV-2 spike protein maturation process.
S1/S2 processing in Alpha- and Omicron (BA.1)-type spike proteins detected by Ser686 cleaved spike antibodies was relatively decreased (Figure 3C). Recent studies suggestthat P681H mutation acquisition may inhibit cleavage by furin at low pH levels [25].
Therefore, the decrease in Ser-686 cleaved S2 spike protein levels in Alpha and BA.1 spike
proteins may be due to the 681H mutation although the C488 mutants in this study do nothave the 681H mutation. Consistent with this, the RBD is a determinant of reduced S1/S2
proteolysis in the Omicron BA.1 spike [26], and the N-terminal domain of the Delta spikeallosterically modulates S1/S2 processing [27]. Furthermore, similar to the C488 mutationsin RBD, which disrupt the C480–C488 disulfide bond of the spike protein, Yamaoka et al.
recently showed that disulfide-bond formation is critical for RBD functioning, and C488A
and C379A mutations lost their ACE2-binding ability [28]. In addition, Zhang et al. showedthat the C301F and C379F mutations eliminate the C291–C301 and C379–C432 disulfide
bonds located in the S1 N-terminal domain and RBD, respectively, thus causing the S1/S2
processing loss of the spike protein [14]. As S1/S2 processing was still detected during
CCF642 treatment, a PDI inhibitor that blocks disulfide-bond-mediated proper protein
folding in the ER (Figure 3B), the non-specific inhibition of disulfide bond formation would
not be a major cause of the S1/S2 processing loss. These findings, along with our data here,
might imply an important role for the RBD in the S1/S2 processing of spike protein in cells,
and further studies will be needed to elucidate this mechanism.
4. Materials and Methods
4.1. Cells, Plasmids, and Reagents
HEK293T, Vero (ATCC, Cat#CCL-81), and VeroE6/TMPRSS2 (JCRB, Cat#JCRB1819)
cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with
7.5% (v/v) fetal bovine serum (FBS) and 25 µg/mL kanamycin. NCI-H522 (ATCC, Cat#CRL5810) cells were cultured in RPMI1640 medium supplemented with 7.5% (v/v) FBS and 25µg/mL kanamycin. NCI-H522/ACE2 cells were generated by isolating human ACE2-DYKexpressing plasmid (synthesized by GenScript Japan Inc., Tokyo, Japan)-transfected stable
cells. The plasmids used are listed in Table 1. BFA was purchased from Fujifilm Wako PureInt. J. Mol. Sci. 2022, 23, 15834

10 of 13

Chemical Corp. (Osaka, Japan). CCF642 (Cat#S8281) and CID-1067700 (Cat#E0135) werepurchased from Selleck Biotech Corp. (Tokyo, Japan).
Table 1. Plasmids used in this study.
Plasmid Names

Remarks, Protein Sequence and Mutation

pEGFP-C1

BD Biotech Clontech

pcDNA3.1–spike/WT

Wuhan Hu-1 spike, YP_009724390.1

pcDNA3.1–M

Wuhan Hu M, YP_009724393

pcDNA3.1 SARS-CoV-2 N

pcDNA3.1 SARS-CoV-2 N was a gift from Jeremy Luban(Addgene plasmid # 158079;
http://n2t.net/addgene:158079, accessed on 15 November
2022; RRID:Addgene_158079) [29]pcDNA3.1–spike/D614G

D614G

pcDNA3.1–spike/C488A

C488A

pcDNA3.1–spike/C488R

C488R

pcDNA3.1-spike/C488F

C488F

pcDNA3.1–spike/Alpha

H69/V70-del, Y144-del, N501Y, A570D, D614G, P681H,
T716I, S982A, D1118H

pcDNA3.1–spike/Delta

L5F, T19R, E156G, F157/R158-del, L452R, T478K, D614G,
P681R, D950N

pcDNA3.1–spike/Omicron (BA.1)

A67V, H69/V70-del, T95I, G142D, del143–145, ins214EPE,
NL211–212I, G339D, S371L, S373P, S375F, K417N, N440K,
G446S, S477N, T478K, E484A, Q493R, G496S, Q498R,
N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H,
N764K, D796Y, N856K, Q954H, N969K, L981F

pcDNA3.1-hACE2-DYKGenScript, Cat No:OHu20260D

4.2. Transfection
Synthetic cDNA with human codon optimization to express SARS-CoV-2 spike andmembrane proteins were purchased from GenScript and cloned into the expression plasmid
pcDNA3.1. Plasmid DNA was transfected using PEIpro® transfection reagent (Cat#101000017,
Polyplus Transfection, New York, NY, USA) for Western blot and immunofluorescenceanalyses, as described previously [10].
4.3. Immunofluorescence Microscopic Analysis and Antibodies
Immunofluorescence analysis was performed as described previously [10]. Fluorescenceconjugated secondary antibodies (goat anti-mouse IgG (H + L) cross-adsorbed with Alexa
Fluor® 488, Cat#A-11001, and donkey anti-rabbit IgG (H + L) cross-adsorbed with Alexa
Fluor® Plus 594, Cat#A-32754) were purchased from Thermo Fisher Scientific (Life Technologies Corp.; Carlsbad, CA, USA), and antifade (Vectashield Vibrance Antifade Mounting
Medium with DAPI, Cat#H-1700) was purchased from Vector Laboratories Inc. (Burlingame,
CA, USA). Conventional immunofluorescence images were captured using a BZ-9000 microscope (KEYENCE; Osaka, Japan). The green fluorescence protein (GFP) signal wasmeasured using ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health;Bethesda, MD, USA, http://imagej.nih.gov/ij/, 1997–2012, accessed on 14 September2022). Confocal microscopy was performed using a TCS SP8 confocal laser scanning microscope (Leica Microsystems GmbH; Wetzlar, Germany) and Leica Application Suite X
software (Leica). TIFF images were merged using Adobe Photoshop CS4 Extended software(Adobe Systems Inc., San Jose, CA, USA). Anti-calnexin (Cat#PM060MS) and anti-GM130(Cat#PM061MS) antibodies were purchased from Medical & Biological Laboratories Co.,
Ltd. (Aichi, Japan). Anti-ERGIC53/LMAN1 (Cat#EPR6979) and anti-furin (Cat#EPR14674)
antibodies were purchased from Abcam K.K. (Tokyo, Japan).

Int. J. Mol. Sci. 2022, 23, 15834

11 of 134.4. Hybridoma Production of mAbs against SARS-CoV-2
Two 6-week-old female BALB/c mice were purchased from CLEA Japan (Tokyo,
Japan) and housed under specific pathogen-free conditions. The Animal Care and Use
Committee of Tohoku University approved all of the animal experiments (permit number:
2019NiA-001). Each BALB/c mouse was intraperitoneally (i.p.) immunized with 100 µg
N-terminal His-tagged S1 spike protein of SARS-CoV-2 (Ray Biotech, Cat#230-01102) using
Imject Alum (Thermo Fisher Scientific Inc., Walthan, MA, USA). The procedure included
three additional immunization procedures (100 µg/mouse), followed by a final booster
intraperitoneal injection (100 µg/mouse) 2 days before harvesting the spleen cells, whichwere subsequently fused with P3U1 cells (ATCC, CRL-1597) using polyethylene glycol
1500 (PEG1500; Roche Diagnostics; Indianapolis, IN, USA). The hybridomas were thengrown in RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine
for selection (Thermo Fisher Scientific, Inc.). The culture supernatants were screened using
an enzyme-linked immunosorbent assay (ELISA) for detecting SARS-CoV-2 S1. Clone
CvMab-6 culture supernatant using hybridoma-SFM medium (Thermo Fisher ScientificInc.) was purified using Ab-Capcher (ProteNova, Kagawa, Japan).
4.5. Immunoblotting
Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Cat#16488-34,
Nacalai tesque, Inc., Kyoto, Japan)-containing protease inhibitors. Proteins were resolved
by electrophoresing on a 5–20% gradient sodium dodecyl sulfate polyacrylamide gel andelectrotransferred to an Immobilon-P membrane (Cat#IPVH00010, EMD Millipore; Billerica,
MA, USA). The primary antibodies against SARS-CoV-2 anti-spike (Cat#GTX632604, clone
1A9), anti-nucleocapsid (Cat#GTX635679, HL344), and anti-membrane (Cat#GTX636245,
clone HL1087) proteins were purchased from GeneTex Inc. (Irvine, CA, USA). Cleaved
SARS-CoV-2 spike protein (Ser686) antibodies (Cat#84534) and anti-mouse IgG–HRP(Cat#7076) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).
Anti-rabbit IgG–HRP (Cat#NA934) was purchased from Cytiva™ (Tokyo, Japan). Antiglyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH; Cat#171-3, clone 3H12) was
purchased from Medical & Biological Laboratories Co., Ltd. (Aichi, Japan). Immunoblot
signals were developed using EzWestLumi plus® (Cat#WES-7120, ATTO Corp.; Tokyo,
Japan) and recorded with an ImageQuant LAS4000 mini-image analyzer (GE Healthcare
Japan Corp.; Tokyo, Japan).
4.6. Statistical Analysis
Quantitative results are presented as mean ± standard deviation (SD) from a minimumof three samples (n ≥ 3). The Student’s t-test or one-way ANOVA was used to evaluate
the significance of differences between the experimental and control groups with similarvariances. Differences were considered statistically significant at p < 0.05. Each experiment
was independently repeated at least twice, and similar results were obtained. The statisticalanalysis and graphing were performed using GraphPad Prism 9 software.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms232415834/s1.
Author Contributions: Conceptualization, K.N.; Investigation, Y.Y., T.I., M.I., M.M., M.F. and K.N.;
Resources, M.K.K. and Y.K.; Writing—original draft preparation, K.N.; Writing—review and editing,Y.Y., M.F., Y.K. and K.N.; Funding acquisition, Y.Y., M.F., Y.K. and K.N. All authors have read andagreed to the published version of the manuscript.
Funding: This work was supported by an on-campus grant (K20-077) in TUS, funded by donations
from the “Account for Donations to Develop Vaccine and Medicine to Treat COVID-19” establishedby the Sumitomo Mitsui Trust Bank to K.N.; research grants from the Japan Agency for Medical
Research and Development (AMED), the Japan Program for Infectious Diseases Research and Infrastructure (Interdisciplinary Cutting-edge Research) to Y.Y. and M.F.; and a JSPS KAKENHI Grant(JP22K15284) to Y.Y. This research was partially supported by the Japan Agency for Medical Research

Int. J. Mol. Sci. 2022, 23, 15834

12 of 13and Development (AMED) under Grant Numbers JP22ama121008 (to Y.K.) and JP21am0101078(to Y.K.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We would like to thank the members of our laboratory for their assistance.
Conflicts of Interest: The authors declare no conflict of interest.

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