Lancemaside A inhibits SARS-CoV-2 entry by blocking S
protein-mediated viral membrane fusion
Having observed that LA inhibits two main SARS-CoV-2 entry routes to
similar extent, we reasoned that a possible target for LA could be a
common step of both viral entry pathways. Therefore, we first examined
the effect of LA on the interaction between SARS-CoV-2 spike (S) protein
and ACE2. To do so, a recombinant protein containing receptor-binding
domain (RBD) of the SARS-CoV-2 S protein fused with GFP (S-RBD-GFP) was
produced and added into ACE2+ H1299 cells (Figure 3a).
Flow cytometry showed that more than 97% of the cells were bound with
S-RBD-GFP (Figure 3b), reflecting SARS-CoV-2 S attachment to cellular
receptor for viral cell entry. Importantly, pretreatment of
ACE2+ H1299 cells with CL root extracts or LA did not
affect the binding of S-RBD-GFP to ACE2 on the cells within the
effective concentration range we obtained in the anti-SARS-CoV-2 assays
(Figure 1a and 2), despite somewhat inhibitory effects were seen at
higher concentrations (Figure. 3b). Moreover, LB, LD, and triterpenoid
aglycone such as echinocystic acid (EA) did not display any inhibitory
effects until their concentration reached 20 µM (Figure S1). Overall,
these results suggest that S-ACE2 protein-protein interaction is not a
main target for LA to inhibit SARS-CoV-2 infection. Thus, as a next
step, we tested another possibility that LA may prevent fusion between
the viral and host membranes, a process that commonly occurs in both
entry pathways. For the assay, we established two stable cell lines: one
that overexpresses S protein with EGFP from a single bicistronic mRNA in
HEK293 cells (Spike-HEK293) and the other overexpressing mRuby in
ACE2/TMPRSS2+ H1299 cells. Then, dual imaging
time-lapse microscopy was performed to monitor cell fusion between these
two cells as indicative of the S-mediated membrane fusion during viral
entry (Figure S2). Addition of Spike-HEK293 cells to a monolayer of
ACE2/TMPRSS2+ H1299 rapidly induced the cell-to-cell
fusion, which allows them to fuse continuously with neighboring
ACE2/TMPRSS2+ H1299 due to the S protein displayed on
the surface of fused hybrid cells, eventually giving rise to enlarged
multinucleated cells (Figure 3c). The heterologous cell fusion is
mediated by the interaction between S protein and ACE2, because this
process was not observed in co-culture of HEK293 expressing GFP only
with ACE2/TMPRSS2+ H1299 cells (Figure 3c). We next
examined the effects of LA on the cell-to-cell fusion event. Strikingly,
LA treatment almost completely blocked the fusion of Spike-HEK293 cells
with ACE2/TRMPRSS2+ H1299 cells, implicating
impairment of SARS-CoV-2 entry by LA at the membrane fusion step (Figure
3c). Furthermore, we quantified the cell-to-cell fusion by flow
cytometry analysis after co-culturing the two cell types in a ratio 1 to
3. The results showed that approximately 30% of total cells were
double-positive for mRuby and GFP, indicating that most of Spike-HEK293
were participated in the fusion event (Figure 3d). Meanwhile, the
double-positive cells were barely detected when control GFP-HEK293 cells
(no S protein) were co-cultured with ACE2/TMPRSS2+cells, again demonstrating the requirement of S protein during the
cell-to-cell fusion (Figure 3d). Notably, LA pretreatment strongly
blocked the fusion event, with less than 4 % of cells generating cell
fusion hybrids (Figure 3d). On the other hand, LB and LD showed limited
or no anti-fusion activity (Figure 3d), further supporting our results
obtained in pSARS-CoV-2 entry assay (Figure 1d). Next, we investigated
how LA may affect membrane fusion. We previously reported that PD, a
triterpenoid saponin derived from PG, redistributes membrane cholesterol
and inhibits SARS-CoV-2 infection (Kim et
al., 2021). Membrane cholesterol is essential component for SARS-CoV-2
to gain entry into the host cells through membrane fusion
(Sanders et al., 2021). As LA has a
similar chemical structure to PD, we reasoned that LA might also alter
membrane cholesterol content and undertook confocal microscopy analysis
of filipin staining to investigate membrane cholesterol distribution
after LA treatment. We found that 1 h treatment of
ACE2+ H1299 cells with 10 μM LA significantly led to
an increase in cholesterol content at the plasma membrane and in
endosomes (Figure 3e and 3f). Importantly, these redistributions of
cholesterol were also observed upon treatment of parental H1299 cells
with LA (Figure 3e and 3f), indicating that LA affects cell membrane
directly, not by affecting ACE2. Moreover, we treated
ACE2+ H1299 cells with LB, LD, and EA, and compared
their effects on membrane cholesterol with that of LA. The cells showed
that LB also increased the amount of cholesterol in plasma membrane by
about 1.7-fold above DMSO-treated control, as revealed by filipin
staining, but it was lower than that of LA (2.3-fold increase) (Figure
S3). In addition, LD and EA did not show any effects on membrane
cholesterol content (Figure S3). Intriguingly, the increased levels of
membrane cholesterol caused by triterpenoid saponins from CL is well
correlated with their inhibitory ability against S-mediated membrane
fusion and viral infection (Figure 3d and Figure 1d), supporting the
idea that membrane cholesterol might be a direct target for LA to exert
its anti-SARS-CoV-2 activity. Taken together, our data suggest that LA
interferes with the S-mediated membrane fusion, possibly by altering
distribution of cholesterol on the host cell membrane, leading to
inhibition of main SARS-CoV-2 infection routes.
Lancemaside A blocks syncytia formation Given that LA has an
inhibitory activity for membrane fusion, we next sought to investigate
the effects of LA on the formation of multinucleated giant cells called
syncytia, which is a result of the continuous fusion of
SARS-CoV-2-infected cells with neighboring cells and often detected in
lung tissue from COVID-19 patients
(Buchrieser et al., 2020;
Bussani et al., 2020). For the assay, we
employed a spit-GFP complementation technology, in which half of GFP is
expressed separately in different cells and a functional GFP protein can
be reconstituted upon cell-to-cell fusion.
ACE2/TMPRSS2+ H1299 cells stably expressing each of
the two fragments of the reporter protein were generated and plated in
equal number, and then S protein was ectopically expressed in these cell
mixtures to induce syncytia formation, which can be detected by GFP
fluorescence (Figure 4a and 4b). In DMSO-treated control cells, multiple
enlarged GFP+ cells with clustered nuclei were
detected, indicating syncytia were formed (Figure 4c, upper panel).
Using automated image analysis, we quantified the efficiency of syncytia
formation by dividing the number of nuclei in GFP+multinucleated cells by the total number of nuclei in the field of view
(Figure 4c, lower panel) and found that approximately 30% of the total
number of cells plated formed GFP+ syncytia (Figure
4d). Importantly, the number of GFP+ cell clusters was
dose-dependently reduced by LA treatment, with IC50values of 3.94 μM, indicating that LA effectively inhibits the
S-mediated syncytia formation (Figure 4c and 4d). As virus-induced
syncytia formation generally promotes programmed cell death
(Hooper, Zaki, Daniels & Middleton,
2001; Nardacci, Perfettini, Grieco,
Thieffry, Kroemer & Piacentini, 2015), we next asked if LA is able to
prevent the viral activation of apoptotic pathways. Consistent with
previous studies, the S-mediated syncytia promoted apoptosis as
evidenced by western blot analysis of the proteolytic cleavage of
caspase 3 and 9 (Figure 4e). Notably, these apoptotic markers were not
detected under no S expression and LA pretreatment conditions (Figure
4e). Together, these results indicate that LA blocks the S-induced
syncytia formation and consequent apoptotic cell death.