key: cord-0713116-qu7umhg5
authors: Cheng, Ya-Wen; Chao, Tai-Ling; Li, Chiao-Ling; Chiu, Mu-Fan; Kao, Han-Chieh; Wang, Sheng-Han; Pang, Yu-Hao; Lin, Chih-Hui; Tsai, Ya-Min; Lee, Wen-Hau; Tao, Mi-Hua; Ho, Tung-Ching; Wu, Ping-Yi; Jang, Li-Ting; Chen, Pei-Jer; Chang, Sui-Yuan; Yeh, Shiou-Hwei
title: Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects
date: 2020-09-23
journal: Cell Rep
DOI: 10.1016/j.celrep.2020.108254
sha: 841ad57e40a82a117511d8af5c5367b923e68db5
doc_id: 713116
cord_uid: qu7umhg5

Development of specific antivirals is an urgent unmet need for SARS-coronavirus 2 (SARS-CoV-2) infections. This study focuses on host proteases that proteolytically activate the SARS-CoV-2 spike protein, critical for its fusion after binding to angiotensin-converting enzyme 2 (ACE2), as antiviral targets. We first validated cleavage at a putative furin substrate motif at SARS-CoV-2 spike by expressing it in VeroE6 cells and found prominent syncytium formation. Both cleavage and syncytium were abolished by treatment with furin inhibitors decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein but not by transmembrane protease serine 2 (TMPRSS2) inhibitor camostat. CMK and naphthofluorescein showed antiviral effects in SARS-CoV-2-infected cells by decreasing viral production and cytopathic effects. Further analysis revealed that, similar to camostat, CMK blocks virus entry, but it further suppresses the cleavage of spike and syncytium. Naphthofluorescein instead acts primarily by suppressing viral RNA transcription. Therefore, furin inhibitors may become promising antivirals for prevention and treatment of SARS-CoV-2 infections.

SARS-coronavirus 2 (SARS-CoV-2) has caused more than 24,000,000 infections and 800,000 deaths after spreading into 184 countries (WHO, 2020) . The pandemic is very difficult to contain at present and SARS-CoV-2 will very likely become a CoV with a sustained ability to infect humans, similar to its predecessor CoVs causing the common cold, such as NL63, OC43, and HKU-1 (Gaunt et al., 2010; Zeng et al., 2018) .

The medical demands for SARS-CoV-2 control are similar to those for seasonal influenza control. In addition to developing vaccines for actively protecting naive people, specific antiviral prophylactics or therapeutics are needed for people already infected, especially those in the high-risk group for serious illness. However, unfortunately, no drug or vaccine has yet been approved to treat human CoVs.

Currently, active trials on repurposing approved or in-development (IND) drugs, including remdesivir, favipiravir, hydroxychloroquine (HCQ) and azithromycin, lopinavir-ritonavir, and convalescent plasma, for the treatment of coronavirus disease 2019 (COVID-19) patients (Sanders et al., 2020) . Preliminary results from limited cases were promising, and more rigorous randomized clinical studies are warranted.

Among the tested drugs, remdesivir was granted Emergency Use Authorization (EUA) by the US Food and Drug Administration (FDA) for patients hospitalized with severe COVID-19, which however appeared to be only mild beneficial for disease recovery in randomized clinical trials (Goldman et al., 2020; Wang et al., 2020b) . Chloroquine (CQ) and hydroxychloroquine were also approved for the EUA by FDA for hospitalized COVID-19 patients, which however was revoked by FDA due to the ineffectiveness and potential serious side effects . Moreover, these J o u r n a l P r e -p r o o f 5 regimens may not be specific enough for SARS-CoV-2, as the action mechanisms of this virus are neither clear nor confirmed.

The molecules targeted by specific antivirals should be essential for the viral life cycle or immune clearance. Such molecules can be divided into two categories: viral molecules and host molecules required for completion of viral replication and infection (Tu et al., 2020) . Among the numerous structural and nonstructural proteins encoded by SARS-CoV-2, viral enzymes are attractive targets for the development of antiviral agents (Li and De Clercq, 2020; Zumla et al., 2016) . Approaches for repurposing existing SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) inhibitors for SARS-CoV-2 were successful and identified molecules potently inhibiting the SARS-CoV-2 M Pro protease (Ton et al., 2020; Zhang et al., 2020) ; numerous such studies are ongoing worldwide. In addition, the replication of SARS-CoV-2 requires several host molecules, which have become candidate targets for drug development. Our previous study identified that the cellular kinase glycogen synthase kinase 3 beta (GSK3β) is important for viral nucleoprotein phosphorylation and for nested viral RNA transcription in the mouse hepatitis virus (MHV) model (Wu et al., 2014; Wu et al., 2009) . Imatinib also showed an antiviral effect on SARS-CoV (Coleman et al., 2016) .

Currently, attention has been focused specifically on the spike (S) protein, the key determinant for viral entry. The spike protein contains an N-terminal S1 domain for binding with the host ACE2 receptor and a C-terminal S2 domain for membrane fusion (Li et al., 2003; Tortorici and Veesler, 2019) . Small molecules targeting ACE2

were thus developed to reduce viral infection, pending the results from clinical trials (Gurwitz, 2020) . Whereas binding to ACE2 is a key step in establishing infection, proteolytic activation of the spike protein by host proteases at the putative cleavage J o u r n a l P r e -p r o o f 6 site located at the S1/S2 boundary (S1/S2 cleavage site) and within the S2 domain (S2' cleavage site) has been documented critical for its fusion activity in CoVs (Belouzard et al., 2009) . Several host proteases, including endosomal cathepsins, cell surface TMPRSS2 proteases, furin, and trypsin, were identified to be responsible for spike protein cleavage during viral entry or viral protein biogenesis in CoVs, depending on their distribution in cells. In addition to regulating viral entry, spike protein cleavage might also regulate the host tropism and pathogenesis of CoV infections (Coutard et al., 2020; Hoffmann et al., 2020b; Park et al., 2016; Wang et al., 2020a) .

The SARS-CoV-2 genomic sequences indicate that the viral spike protein contains conserved putative motifs for several cellular proteases. Via a pseudovirus approach, Markus Hoffmann et al. recently reported that camostat, an inhibitor of the TMPRSS2 protease, can inhibit the viral entry (Hoffmann et al., 2020b) . In addition to TMPRSS2, which targets the S2' cleavage site, furin has been proposed as another host protease mediating cleavage of the SARS-CoV-2 spike protein at the S1/S2 cleavage site (Hoffmann et al., 2020a) . The S1/S2 boundary features a polybasic stretch of an RRAR motif, matching the consensus sequence of the substrate for furin and related proprotein convertase (PC) family members (Seidah and Prat, 2012) . This site was identified in SARS-CoV-2 but not in SARS-CoV or other lineage of β-CoVs, although it is preserved in some other human CoVs, including HCoV-OC43, MERS-CoV, and HKU1 (Coutard et al., 2020) . A recent study showed that abolishing this site in pseudotyped SARS-CoV-2 S viral particles did not affect infectivity (Walls et al., 2020) . However, the function of this furin cleavage site in viral pathogenesis, especially for spreading and cytopathic effects (CPE), needs to be more thoroughly addressed in a virus infection system. J o u r n a l P r e -p r o o f 7 Cleavage by furin and related PCs has been documented as a key event in activating the envelope glycoprotein for its fusion activity, critical for viral entry and pathogenesis, in numerous pathogenic virus families, including Herpes-, Corona-, Flavi-, Toga-, Borna-, Bunya-, Filo-, Orthomyxo-, Paramyxo-, Pneumo-and Retroviridaeviruses (Braun and Sauter, 2019; Izaguirre, 2019) . Furin/PCs thus become attractive therapeutic targets for various infectious diseases. In recent years, several peptide-based and small-molecule inhibitors targeting furin/PCs have been developed as putative antivirals, many of which can block the maturation of viral envelop proteins and thus its fusion activity in various viruses (Braun and Sauter, 2019; Izaguirre, 2019) . We thus tested four furin/PC inhibitors in SARS-CoV-2-infected VeroE6 cells. In addition to confirming cleavage at the putative furin site at the S1/S2 boundary, we evaluated the inhibitory effects on the viral replication cycle and CPE such as syncytium formation. The results supported the essential role of host furin/PC proteases in the viral replication and pathogenesis of SARS-CoV-2 infections. Thus, furin/PC inhibitors may become specific antiviral leads warranting further development.

J o u r n a l P r e -p r o o f 8

We first tried to validate the function of the furin cleavage site at R685/S686 in the RRAR↓ ↓ ↓ ↓S motif within the SARS-CoV-2 spike protein. To this end, two expression plasmids for spike proteins that were codon optimized for human expression-one for the wild-type (WT) protein and the other for the R682A mutant (in which the furin cleavage site was mutated from RRAR↓ ↓ ↓ ↓S to ARARS)-with an HA tag at the C-terminus were constructed ( Figure 1A ). After transfection into VeroE6 cells, SARS-CoV-2 spike protein cleavage was examined in cell lysates by western blotting using either anti-spike or anti-HA tag antibodies (Abs). The wild-type full-length spike protein was readily processed into smaller ones, indicating efficient cleavage.

However, the R682A mutant spike protein remained largely intact, with minimal cleavage ( Figure 1B ). These results thus indicate that the furin cleavage motif at the S1/S2 boundary is functional.

Furthermore, expression of the wild-type spike protein alone induced an extensive syncytial phenotype, with fused cells containing multiple nuclei visible under light and fluorescence microscopy ( Figure 1C -D). However, this syncytial phenotype did not occur in cells expressing the R682A mutant spike protein ( Figure   1C -D), despite similar protein expression levels. Therefore, cleavage of the SARS-CoV-2 spike protein at the S1/S2 furin substrate site occurs and possibly contributes to syncytium formation.

protein cleavage and development of the syncytial phenotype in VeroE6 cells.

J o u r n a l P r e -p r o o f 9 To further explore this possibility, we evaluated the effects of several furin/PC inhibitors on SARS-CoV-2 spike protein cleavage and syncytium formation. Four potent furin/PC inhibitors two peptide inhibitors (decanoyl-RVKR-chloromethylketone (CMK) and hexa-D-arginine amide (D6R)) and two small molecule inhibitors (SSM 3 trifluoroacetate (SSM3) and naphthofluorescein)-were evaluated (Coppola et al., 2008; Henrich et al., 2003; Remacle et al., 2010; Sarac et al., 2002) . Camostat, an inhibitor targeting TMPRSS2, another spike cleavage protease, was used as a control. VeroE6 cells expressing the wild-type spike protein were treated with the inhibitors for 24 hr at an effective dose as suggested in previous studies (Coppola et al., 2008; Croissandeau et al., 2002; Hoffmann et al., 2020b; Sarac et al., 2002) .

After treatment with CMK or naphthofluorescein, immunoblot analysis of cell lysates showed a dramatic decrease in the levels of processed spike protein fragments in VeroE6 cells. The full-length but not the cleaved spike protein was detected after treatment with either of these two inhibitors. In contrast, no suppression of spike protein cleavage was detected in cells treated with the other inhibitors ( Figure 2A ).

Notably, cleavage of cellular integrin αV, a furin substrate, into heavy and light chain fragments was decreased in cells treated with CMK and naphthofluorescein but not the other inhibitors (Figure 2A ), suggesting that CMK and naphthofluorescein but not the other inhibitors suppressed furin/PC activity in VeroE6 cells.

Consistent with these observations, microscopic observation revealed significant Microscopic observation showed that prominent CPE were induced by SARS-CoV-2 infection and that these CPE was suppressed by treatment with CMK or naphthofluorescein but not by treatment with D6R or SSM3 ( Figure 3A ). These results were similar to those obtained in spike-expressing VeroE6 cells. The levels of not only the cleaved S1/S2 subunits but also the full-length spike protein and the nucleocapsid (N) protein in cell lysates were diminished by treatment with CMK or naphthofluorescein ( Figure 3B ). Moreover, the virus titer of progeny viruses harvested from the supernatant was significantly decreased by CMK and naphthofluorescein ( Figure 3C ). The inhibitory effect of these two inhibitors has been further validated by multi-step virus growth cycle setup, in which the VeroE6 cells In addition to VeroE6 cells, the similar antiviral effects of these five inhibitors for SARS-CoV-2 infection were validated in another cell line, the MK2 cells, which is also susceptible for SARS-CoV-2 infection (Supporting Figure 2) .

production and CPE in association with reduced viral RNA and protein levels.

Then, we conducted a dose-response experiment with CMK, naphthofluorescein and camostat to assess suppression of CPE and viral production. The results confirmed the suppressive effects of the inhibitors on CPE and viral production (by plaque assay) and showed that the effective dose for CMK was as low as 5 µM ( Figure Figure 5A (preinfection treatment) and Figure 5E (postinfection treatment).

In the preinfection treatment experiments, CPE was abolished by treatment with CMK or camostat but only mild by treatment with naphthofluorescein ( Figure 5B ).

Consistent with this result, the virus titer was strikingly decreased by treatment with CMK or camostat but less significant by naphthofluorescein ( Figure 5C ). The northern blot and western blot analysis results further showed a marked reduction in J o u r n a l P r e -p r o o f 13 viral RNA and protein only by treatment with CMK and camostat but not by naphthofluorescein ( Figure 5D ). Co-administration of CMK, camostat and naphthofluorescein with virus infection all effectively decreased the virus titer in supernatant and viral RNA and protein levels in the infected cells (Supporting Figure   3 ). The results suggested that CMK and camostat but not naphthofluorescein might target the early stage of the viral life cycle before viral RNA synthesis.

In the postinfection treatment experiments, the CPE and virus titer were reduced by treatment with naphthofluorescein ( Figure Since CMK and camostat affected the viral entry stage, we examined whether they disrupt the binding of the spike protein to the ACE2 receptor. Via an in vitro binding assay, we determined that the binding of the SARS-CoV-2-spike protein (RBD)-Fc to human ACE2 expressing 293T cells was not affected by treatment with these inhibitors (Supporting Figure 5) , thus eliminating the possibility that binding disruption is a mechanism of these inhibitors.

J o u r n a l P r e -p r o o f 14 Interestingly, although the postinfection treatment with CMK did not inhibit the expression of the spike protein, it did significantly reduce spike protein cleavage into the S1 and S2 subunits (the spike protein appears mainly as the uncleaved form in Figure 5H ). Consistent with these findings, uncleaved spike protein was enriched in virus harvested from the supernatant after treatment with CMK ( Figure 5I ). Moreover, as revealed by the plaque assay, the virus titer and infectivity of these virions from CMK treated cells were not decreased compared with the control virions ( Figure 5G and 5J), indicating that the cleavage of the spike protein by furin/PCs during biogenesis might not be essential for viral assembly and infectivity.

Notably, although the postinfection treatment with CMK did not affect viral replication and assembly, it decreased the CPE induced by SARS-CoV-2 infection.

Such a decrease of CPE instead did not occur in the camostat treated cells ( Figure 5F ).

Therefore, from a therapeutic viewpoint, blockade of furin/PC-mediated spike protein cleavage after viral infection likely ameliorates CPE and thus the virulence and pathogenicity.

Similar to numerous pathogenic viruses, cleavage of the spike protein by host proteases has been documented to be important for its fusion activity after its binding to the host receptor in several CoVs (Heald-Sargent and Gallagher, 2012; Hoffmann et al., 2018) . The putative furin substrate site located at the S1/S2 boundary and the putative TMPRSS2 substrate site located within the S2 domain of the SARS-CoV-2 spike protein thus attract considerable interest. Our study not only confirmed the critical role of the furin cleavage site for the fusion activity of spike protein in contributing to viral production and syncytium, but also identified two related antiviral lead compounds targeting the furin cleavage site in SARS-CoV-2, laying the groundwork for further development of antivirals against SARS-CoV-2.

Furin/PC-mediated processing of the SARS-CoV-2 spike protein may be clinically significant. In autopsies of COVID-19 victims, many multinucleated giant cells resulting from syncytia of pneumocytes were found in the lungs (Xu et al., 2020) .

In infections with other CoVs, syncytium formation confers multiple pathogenic advantages over cell-free spread. Direct cell-to-cell spread of CoV is more efficient than cell-free spread, which requires engagement with cell membrane-specific receptors. In addition, syncytium formation allows the virus to evade innate humoral and cellular defenses (Sattentau, 2008; Sattentau, 2011) . Therefore, in infections with many CoVs, syncytium formation was found to be associated with increased viral pathogenesis (Frana et al., 1985; Nakagaki et al., 2005; Park et al., 2016) , for example, greatly enhancing the infectivity of avian coronavirus infectious bronchitis (Yamada et al., 2009) . Therefore, suppression of syncytium formation in SARS-CoV-2 infection by furin/PC inhibitors could be a strategy to reduce virus spread and ameliorate virulence and disease progression. In support of the role of furin/PC-mediated spike protein cleavage in formation of syncytia from SARS-CoV-2-infected cells, insertion of the furin cleavage site at the S1/S2 boundary of the SARS-CoV genome, which lacks such a site, greatly increased syncytium formation but not viral infectivity (Watanabe et al., 2008) . This result was consistent with our postinfection treatment experiment, showing that syncytium formation was suppressed; however, neither the virus titer nor infectivity was affected by CMK. Furin/PC-mediated cleavage of the spike protein during biogenesis and assembly, which is accomplished mainly by furin/PC proteases localized at the trans-Golgi network, is required for syncytium formation but not essential for efficient virion assembly or infectivity in VeroE6 cells.

However, preinfection treatment with CMK did block the early stage of the viral life cycle before viral RNA synthesis. As noted, release of the fusion peptide from the spike protein via cleavage by host proteases is a prerequisite for virus-cell membrane fusion and thus viral entry (Ou et al., 2016) . As CMK treatment did not affect the binding of spike to ACE2 receptor, it suggests that preinfection treatment with CMK might suppress the cleavage and release of the fusion peptide from spike protein after its binding with receptor. In addition to furin, the putative target protease for CMK at the membrane to help release the fusion peptide could be other PC family members or other off-target proteases, for example TMPRSS2, and these possibilities are worthy of investigation.

As shown in our virus infection system, CMK, naphthofluorescein and camostat, Alternatively it might have off target effects on decreasing viral RNA levels.

Therefore, in addition to study the exact mechanism for naphthofluorescein to decrease viral transcription, if this mechanism depends on furin activity or other new targets warrants to be clarified.

Although the mechanism underlying the distinct effects of these two furin/PC inhibitors in the viral replication cycle still remains to be investigated, our studies proposed that these three inhibitors might be positioned for different antiviral strategies. The results of the preinfection treatment experiments indicated that CMK and camostat might function to prevent viral entry. Therefore, they could be used as See also Figure S1 and S2. 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Shiou-Hwei Yeh (shyeh@ntu.edu.tw)

All materials in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

The original sequencing datasets for hCoV-19/Taiwan/NTU03/2020 can be found on the GISAID under Accession ID: EPI_ISL_413592.

Sputum specimens obtained from SARS-CoV-2-infected patients were maintained in viral transport medium. Virus in the specimens was propagated in VeroE6 cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 µg/mL tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (Sigma-Aldrich).

Culture supernatant was harvested when CPE were seen in more than 70% of cells, and virus titers were determined by a plaque assay. The virus isolate used in the current study is hCoV-19/Taiwan/NTU03/2020 (Accession ID: EPI_ISL_413592)

Plaque assay J o u r n a l P r e -p r o o f 26 The plaque assay was performed as previously described with minor modifications (Su et al., 2008) . In brief, VeroE6 cells (2 x 10 5 cells/well) were seeded in triplicate in 24-well tissue culture plates in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics one day before infection. SARS-CoV-2 was added to the cell monolayer for 1 hr at 37°C. Subsequently, virus was removed, and the cell monolayer was washed once with PBS before being overlaid with medium containing 1% methylcellulose and incubated for 5-7 days. Cells were fixed with 10% formaldehyde overnight. After removal of the overlay medium, cells were stained with 0.7% crystal violet, and plaques were counted. The vial titer presented in a histogram from the mean of three independent experiments. 

Western blotting was performed as previously described (Wu et al., 2009) . In brief, cell lysates were harvested in 1X RIPA buffer (Merck Millipore) containing 1X proteinase inhibitor (Merck Millipore) and 1X phosphatase inhibitor (Calbiochem).

Protein samples were added to 4X SDS loading dye and denatured for 10 min at 95°C.

Proteins were separated on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% milk in 1X TBST prior to incubation with primary antibodies at 4°C overnight. Membranes were then reacted with a secondary antibody. Antigen-antibody complexes were visualized 

VeroE6 cells were fixed with 4% paraformaldehyde for 10 min at room temperature.

After three washes with 1X PBS, cells were permeabilized with 0.1% Triton X-100 at 4°C for 10 min. Next, cells were incubated in blocking buffer (1X PBS containing 5% were applied for 1 hr at room temperature. Images were acquired using a Zeiss AXIO Imager A1 microscope.

RNA was extracted using NucleoSpin RNA Kit (Macherey-Nagel) according to the instruction manual. Northern blotting was performed as previously described (Wu et al., 2014) . In brief, 0.5 µg of RNA was denatured and processed for electrophoresis on an 0.8% agarose/formaldehyde gel at 70 volts for 2 hr and 100 volts for 3 hr.

Before capillary transfer, the agarose gel was submerged in 50 mM NaOH for 50 min, 

The cytotoxicity effect of specific inhibitors was evaluated by a CCK-8 assay (Dojindo Molecular Technologies). In brief, 5x10 3 VeroE6 cells were seeded in 96-well plates for 24 hr before treatment with serial doses of the inhibitors. Ten microliters of CCK-8 reagent was added to each 96-well plate after 24 hr of inhibitor J o u r n a l P r e -p r o o f 30 treatment and further incubated in a 37°C incubator for 2 hr before measurement of the optical density at 450 nm. The data from three independent experiments was used to calculate the CC50 by nonlinear regression using GraphPad Prism V5.0 software. 

In brief, VeroE6 cells (2 x 10 5 cells/well) were seeded in triplicate in 24-well tissue culture plates in DMEM supplemented with 10% fetal bovine serum (FBS) and

antibiotics one day before infection. 

The virus titers quantified by plaque assay in triplicate were shown as the means ± standard deviations. The difference between the control cells (without inhibitor treatment) and the cells treated with specific inhibitors were evaluated by Student's t test. The P values of 0.05 or lower were considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P <0.001).

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