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Mol Cell。2020May21；78（4）：779-784.e5。
Published online2020May1。 doi:10.1016/j.molcel.2020.04.022
PMCID:PMC7194065
PMID:32362314

A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2Is Essential for Infection of Human Lung Cells

Markus Hoffmann单击功能区上，1，∗ Hannah Kleine-Weber单击功能区上，1，2and，andStefan Pöhlmann1，2，3，∗∗
Author information Article notes Copyright and License information PMC Disclaimer

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数据可用性状态

Abstract

The pandemic coronavirus SARS-Cov-2threatens public health worldwide.The viral spike protein mediates SARS-Cov-2entry into host cells and harbors a1/1cleavage site containing multiple arginine residues（multibasic）not found in closely related animal coronaviruses。However，the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown.Here，we report that the cellular protease furin cleaves the spike protein at the S1/S2site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells.Moreover，optiming the S1/sicell， but not virus-cell，fusion，suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence.Our results suggest that acquisition of aS1/S2multibasic cleavage site was essential for SARS-Cov-2infection of humans and identiffurinas intertient on。

Keywords:SARS-COV-2，COVID-19，spike，cleavage，S1/S2，furin，TMPRSS 2，entry，membrane fusion

Graphical Abstract

Coronavirus spike proteins are activated by host cell proteases.Hoffmann and colleagues show that the pandemic SARS-CoV-2harbors a highly cleavable S1/S2 cleavage site not found in closely related coronavruses.Cleavage at this site is mediated by furin and is requireed for viral entry into humang。

基础，基础

It is believed that the severe acute respiratory syndrome coronavirus2（SARS-CoV-2，previously termed nCoV-2019）was introduced into the human population from a poorly characterized animal reservoir in late2019（Geet al。，2013单击功能区上，Wang et al。，2020单击功能区上，Zhou et al。，2020b单击功能区上，Zhu et al。，2020）。The epicenter of the subsequent SARS-CoV-2 spread was Wuhan，Hubei province，China，with more than65000 cases occurring in this area（WHO，2020a）。However，infections have now been detected in more than110 countries and massive outbreaks are currently on going in the United States，Italy，and Spain（WHO，2020a单击功能区上，WHO，2020b）。Understanding which features of SARS-CoV-2are essential for infection of human cells should provide insights into viral transmissibility and pathogenesis and might reveal targets for intervention。

The spike protein of coronaviruses is incorporated into the viral envelope and facilitates viral entry into target cells.For this，the surface unit S1 binds to a cellular receptor while the transmembrane unit S2 facilitates fusion of the viral membrane with a cellular membrane（Hoffmann et al。，2018单击功能区上，Hulswit et al。，2016单击功能区上，Millet and Whittaker，2018）。第一节课，第一节课，第二节课，第二节课，第二节课火焰1A），which results in S protein activation（Hoffmann et al。，2018单击功能区上，Hulswit et al。，2016单击功能区上，Millet and Whittaker，2018）。Cleavage of the S protein can occur in the constitutive secretory pathway of infected cells or during viral entry into target cells and is essential for viral infectivity.Therefore，the responsible enzymes constitute potential targets for antiviral intervention。

An external file that holds a picture，illustration，etc。Object name is gr1_lrg.jpg

The Multibasic Motif at the S1/S2 Cleavage Site of SARS-2-S Is Unique among Related Group 2b Betacoronaviruses

（A）RBD，receptor-binding domain；RBM，receptor-binding motif；TD，transmembrane domain）。

（B）Protein models for SARS-S and SARS-2-S based on the PDB:5X5B structure（元宝。，2017）as a template。Colored in red are the S1/S2and S2'cleavage sites.Further，the S1subunit（blue），including the RBD（purple），and the S2subunit（gray）are depicted。

（C and D）Amino acid sequence alignment of residues around the S1/S2andS2’cleavage sites of group2b betacoronaviruses found in humans，civet cats，raccoon dog，pangolin，and bats（C）or coronaviruses that are able to infect humans（D）。Basic amino acid residues are high lighted in red，while gray boxes mark the presence of multibasic motifs.Numbers refer to amino acid residues（n/a，no information available）.The symbol“∗“refers to amino acid residues that are conserved among all tested sequences，while the symbols”：“and”indicate positions with heterogeneous amino acid residues that share highly similar or similar biochemical properties。

Our previous work revealed that the activity of the cellular serine protease TMPRSS 2，which activates several coronaviruses（Bertram et al。，2013单击功能区上，Gierer et al。，2013单击功能区上，Glowacka et al。，2011单击功能区上，Matsuyama et al。，2010单击功能区上，Shirato et al。，2013单击功能区上，Shirato et al。，2016单击功能区上，Shulla et al。，2011），is also required for robust SARS-CoV-2 infection of human lung cells（Hoffmann et al。，2020）。However，it is conceivable that the activity of other cellular proteases is also necessary.Thus，the Middle East respiratory syndrome coronavirus spike protein（MERS-S）is activated by atwo-step process:MERS-Sis first cleaveed by furin at the S1/S2site in ininininfected cells，subsequent TMPRSS 2-mediated cleavage at the S2’site（火焰1A）during viral entry into lung cells（Kleine-Weber et al。，2018单击功能区上，Park et al。，2016单击功能区上，Millet and Whittaker，2014）。A cathepsin B/L-dependent auxiliary activation pathway is operative in many TMPRSS 2-什么cell lines but seems not to be available in viral target cells in the lung because TMPRSS 2-dependent activation of the S protein is essential for robust MERS-Cov and SARS-Cov spread and pathogenesis in the infected host（Iwata-Yoshikawa et al。，2019单击功能区上，Simmons et al。，2005单击功能区上，Zhou et al。，2015）。

TheS1/S2site in SARS-CoV-2forms an exposed loop（火焰1B）that harbors multiple arginine residues（multibasic）（Walls et al。，2020单击功能区上，Wrapp et al。，2020）that are not found in SARS-Cov-related coronaviruses（SARSr-Cov）but are present in the human coronaviruses OC43，HKU1，and MERS-Cov（火焰1C）.However，the contribution of this multibasic cleavage site to SARS-Cov-2infection of human cells is unknown and was in the focus of the present study。

结果，结果

SARS-CoV-2Is Required for Efficient Proteolytic Cleavage of the Spike Protein

在SARS-Cov-2infection，wegenerated S protein mutants with altered S1/S2 cleavage site in SARS-Cov-2infection，wegenerated S protein mutants with altered S1/S2 cleavage sites（火焰2A）.In particular，we exchanged the multibasic cleavage site against its monobasic counterparts present in SARS-S or RaTG13-S（火焰2A，RaTG13is abat coronavirus closely related to SARS-COV-2[Zhou et al。，2020b]）。This resulted in mutants SARS-2-S（SARS）and SARS-2-S（RaTG）。Moreover，we either deleted all arginines in the S1/S2site of SARS-2-S or inserted an additional arginine residue（jointly with an alanine to lysine exchange），giving rise to mutants SARS-2-S（delta）and SARS-2-S（opt），respectively.Finally，we introduced the S1/S2 SARS-S（火焰2A），which yielded the mutants SARS-S（SARS-2）and SARS-S（RaTG）。

An external file that holds a picture，illustration，etc。Object name is gr2_lrg.jpg

SARS-2-S Is Cleaved by Furin，and Cleavage Is Required for Syncytium Formation and Entry into Human Lung Cells

（A）Overview of the SARS-S and SARS-2-S 1/1/S2 mutants analyzed。

（B）Analysis of furin-mediated S protein priming。Rhabdoviral particles harboring the indicated S proteins containing a C-terminal V5tag for detection were lysed and subjected to western blot analysis.Detection of vesicular stomatitis virus matrix protein（VSV-M）served as control。

（C）Rhabdoviral particles bearing MERS-S，SARS-S，or SARS-2-Sequipped with aV5 or HA epitope tag their C terminus（orno glycoprotein at all，control）were produced in the absence or presence of furin inhibitor（FI，decanoyl-RVKR-CMK；1μM or 10μM）and analyprocessing alysis。Detection of VSV-M served as control。

（D）Syncytium formation assay:Vero or Vero-TMPRSS 2cells were transfected to express the indicated S proteins（orno S protein，empty vector，control）.At24h post transfection，cells were incubated in the presence or absence of trypsin（1μg/mL）foran additional24h before they wexed，fixed，stained with May-Gruenwald and Giemsa solution，and analyzed by bright field microscopy（scale bars，200μm）。White arrowheads indicate syncytia.For（B）–（D）–，representative data from three（B and C）or four（D）independent experiments are shown。

（E）Transduction of Vero（TMPRSS 2）-什么）and Calu-3（TMPRSS 2+）cells with rhabdoviral particles bearing the indicated S proteins or vesicular stomatitis virus glycoprotein（VSV-G）。At16h post transduction，virus-encoded firefly luciferase was quantified in cell lysates.Presented are the mean data from three independent experiments。Transduction efficiency is shown relative to that measured for particles not bearing a viral glycoprotein。Error bars indicate the standard error of the mean.Statistical significance was tested byone-way analysis of variance with Dunnett’s post test（p>0.05，ns；∗∗∗p≤0.001）。

The effects of the above described S1/S2 mutations on viral entry were examined using vesicular stomatitis virus（VSV）particles bearing S proteins because these particles are safe and adequately reflect coronavirus entry into target cells。VSV particles bearing S proteins with aC-terminal antigenic tabled that all S proteins were readily incorporated into VSV particles.SARS-2-SWT（wild type）was efficiently cleaved at the S1/S2site（火焰2B），in keeping with published data（Hoffmann et al。，2020单击功能区上，Walls et al。，2020）。SARS-S and RaTG 13-S abrogated cleavage，and this effect was also seen when the multibasic motif was deleted（火焰2B）.Moreover，insertion of an additional arginine residue jointly with an alanine-to-lysine exchange at the S1/S2site did not appreciably increase cleavability.Finally，insertion of the S1/S2site of SARS-2-S into SARS-S increased S protein cleavability whility insertion of the RaTG13 S1/Site火焰2B.These results indicate that the presence of several arginine residues at the S1/S2site is required for efficient SARS-2-S proteolytic processing in human cells and also confers high cleavability to SARS-S。

Furin Cleaves the SARS-CoV-2 Spike Protein at the S1/S2 Site，and Cleavage Is Required for Effficient Cell-Call Fusion

We next investigated which protease is required for S protein processing at the S1/S2site.The S1/S2motif matches the minimal furin sequence RXR and is closely related to the furin consensus sequence RX[K/R]R.Therefore，we analyzed whether decanoyl-RVKR-CMK，a furin inhibitor，blocks SARS-2-S processing at the S1/S2site.Decanoyl-RVKR-CMK inhibited processing of MERS-S，which is known to depend on furin（Gierer et al。，2015单击功能区上，Millet and Whittaker，2014），in a concentration-dependent manner and had no effect on SARS-S expression（火焰2C），as expected.Processing of SARS-2-Swas also inhibited，indicating that furin cleaves SARS-2-Sat the S1/S2site.Inorder to determine wheter cleavage at the S1/S2site is required for SARS-2-S-driven cell-cell fusion，we studied S-protein-dependent formation of multinucleated giantceyts（C）。不同步were observed in the absence of S protein expression while MERS-S WT expression ression ression ression ression in syncytium formation，which was increased upon addition of trypsin or expression of TMPRSS 2（火焰2D）.Expression of SARS-S WT or SARS-S harboring the S1/S2site of RaTG13-S did not induce syncytium formation in the absence of protease，but modest multikaryon formation was detected in the presence of trypsin or TMPRSS 2。In contrast，SARS-S harboring the SARS-2-S1/S2site induced syncytia in the absence of protease，and syncytium formation was markedly increased by trypsin and，particularly， TMPRSS 2.SARS-2-S expression triggered syncytium formation that was strongly increased by trypsin and TMPRSS 2.Syncytium formation was clearly less prominent and required the presence of trypsin or TMPRSS 2when the SARS-2-S1/S2site was replaced by that of SARS-Sor RaG13-S.Moover， deletion of the multibasic motif resulted in a spike protein that was no longer able to induce syncytium formation even in the presence of trypsin or TMPRSS 2.Finally，the addition of an arginine residue to the S1/S2site of SARS-2-Sjointly with alanine-to-lysine exge strongly increasyncytation， indicating that viral variants with optimized S1/S2sites might show augmented cell-cell spread and potentially altered pathogenicity.Thus，the S1/S2site of SARS-2-Sis required for cell-cell fusion，and this process can be augmented by adding basic residues to the S1/S2site。

SARS-CoV-2 Spike Protein at the S1/S2 Site Is Required for Viral Entry into Human Lung Cells

We finally examined the importance of the S1/S2site for S-protein-mediated virus-cell fusion.Blockade of SARS-2-S cleavage at the S1/S2site（mutants SARS-2-S），SARS-2-S（RaTG），and SARS-2-S（delta）abrogated entry into the TMPRSS 2+human lung cell line Calu-3（火焰2E），in which the cathepsin B/L-dependent S protein activation pathway is not sufficiently available（Park et al。，2016）。In contrast，entry into TMPRSS 2-什么Vero cells，which is known to be cathepsin B/L dependent，was not affected by these mutations（火焰1E），in keeping with results reported by Walls and colleagues（Walls et al。，2020）。Optimization of the S1/S2site did not increase entry into the cell lines tested；it slightly decreased entry into both Vero and Calu-3cells，for，at present，unclear reasons.Finally，alterations of the S1/S2site of SARS-S did not augment entry efficiency.Collectively， these results demonstrate that a multibasic S1/S2site is essential for SARS-2-S-driven entry into human lung cells while a monobasic site is sufficient for SARS-S。

Discussion

Our results reveal commonalities between the proteolytic activation of SARS-CoV-2and MERS-CoV.Both viruses depend on furin-mediated pre-cleavage of their S proteins at the S1/S2site for subsequent S protein activation by TMPRSS 2 in lung cells，which fail to express robust levels of catsinPark et al。，2016）。Thus，inhibitors of furin and TMPRSS 2 might be considered as treatment option for COVID-19，and a TMPRSS 2 inhibitor that blocks SARS-CoV-2 infection has recently been described（Hoffmann et al。，2020）。Regarding furin inhibition，it must be taken into account that furin，unlike TMPRSS 2，is required for normal development（Roebroek et al。，1998）。Blockade of this enzyme for prolonged time periods might thus be associated with unwanted toxic effects.In contrast，a brief treatment might be well tolerated and still associated with a therapeutic benefit（Sarac et al。，2002单击功能区上，Sarac et al。，2004）。

在viral hemaglutinin protein is a central virulence factor（Luczo et al。，2015）。Thus，viruses with a monobasic cleavage site are activedby TMPRSS 2 or related proteases with an expression profile confined to the aerodigestive tract.As a consequence，viral replication is limited to these organs and does not result in severe disease.In contrast，viruses with amultibasic cleavage site are activated by ubiquitously expressed protein convertases，including furin，and can thus spread systemically and cause massive disease.In the context of coronavirus infection，protein cleavability has been identified as determinant of zoonotic potentialMenachery et al。，2020单击功能区上，Yang et al。，2014）。The presence of a high ly cleavable S1/S2site in SARS-2-S may therefore not have been unexpected.However，it is noteworthy that all SARS-CoV-2-related coronaviruses of bats and pangolins identified today harbor a monobasic cleavage site（Lam et al。，2020单击功能区上，Li et al。，2020单击功能区上，Zhang et al。，2020）。It will thus be interesting to determine how the multibasic motif was acquired by SARS-CoV-2，and a recent study suggested that a recombination event might have been responsible（Zhang et al。，2020单击功能区上，Zhou et al。，2020a）。

限制，限制

Our results demonstrate that the multibasic S1/S2 cleavage site is essential for SARS-2-S-driven entry into TMPRSS 2+lung cells.It will be interesting to extend these studies to primary human respiratory epithelial cells and to authentic SARS-Cov-2，which requires a reverse genetics system not available to the present study。

STAR★Methods

密钥资源表

REAGENT or RESOURCESOURCEIDENTIFIER
防波堤，防波堤
Monoclonal anti-HA antibody produced in mouseSigma-AdrichCat.#:H3663；RRID：AB_262051
Monoclonal anti-β-actin antibody produced in mouseSigma-AdrichCat.#:A5441；RRID：AB_476744
Monoclonal anti-VSV-M（23H12）antibodyKeraFastCat.#:EB0011；RRID：AB_2734773
noclonal anti-mouse，peroxidase-coupledDianovaCat.#:115-035-003；RRID：AB_1015289
Anti-VSV-G antibody（I1，produced from CRL-2700mouse hybridoma cells）ATCCCat.#CRL-2700；RRID:CVCL_G654
Bacterial and Virus Strains
VSV∗ΔG-FLucBerger Rentsch and Zimmer，2011N/A
One Shot™OmniMAX™，™2T1R校验组件E.coliThermo Fisher ScientificCat.#:C854003
Chemicals，Peptides，and Recombinant Proteins
Lipofectamine LTX with Plus ReagentThermo Fisher ScientificCat.#:15338100
Furin inhibitor，decanoyl-RVKR-CMKTocrisCat.#:3501
主-格林nwald solutionSigma-AdrichCat.#:63590
Giemsa solutionSigma-AdrichCat.#:GS500
严重公共资产
Beetle-Juice KitPJKCat.#:102511
Experimental Models:Cell Lines
293TDSMZCat.#:ACC-635；RRID:CVCL_0063
Calu-3钢轨拉深ATCC Cat#HTB-55；RRID:CVCL_0609
Vero和地制造厂ATCC Cat#CRL-1586；RRID:CVCL_0574
Vero-TMPRSS 2Hoffmann et al。，2020N/A
Oligonucleotides
SARS-S（BamHI）FCT TGGATCCGCCCACCATGTTATTTTC
TTATTATTTC		Sigma-AdrichN/A
SARS-SΔ18（XbaI）R CTTTCTAGACTTGAGCAAGAA
CCACAAGAGC		Sigma-AdrichN/A
SARS-SΔ18（-）STOP（XbaI）R CTTTCTAGACTTGAGCAAG
AACCACAAGAGC		Sigma-AdrichN/A
SARS-2-S（BamHI）FGAATTCGGATCCGCCCACCATGTCGTGT
GTTTCTGGTGCTGC		Sigma-AdrichN/A
SARS-2-SΔ18（XbaI）R AAGGCCTCTAGACTACTTGAGCA
GCTGGCCACAGC		Sigma-AdrichN/A
SARS-2-SΔ18（-）STOP（XbaI）R AAGGCCTCTAGACTTGCA
GCAGCTGCCACAGC		Sigma-AdrichN/A
SARS-S（SARS）FCAGACAAACAGCCAGACGGCCAG
AAGTACTAGCCAAAAATCTATTG		Sigma-AdrichN/A
SARS-S（SARS）RTCTGGCCCGTCTGGGCTGTTGTCT
GTGTATGGTAACTAGCAAATGC		Sigma-AdrichN/A
SARS-S（RaTG）FCAGACAAACAGCAGAAGTACTAGCCA
AAAATC		Sigma-AdrichN/A
SARS-S（RaTG）RTC T GCTGTTGTCTGTATGGTAACTA
GCACAAATGC		Sigma-AdrichN/A
SARS-2-S（SARS）FGTTTCTTTATTACGTCTGGCCAGC
CAGAGCATC		Sigma-AdrichN/A
SARS-2-S（SARS）R A CGTAATAAAGAAACTGTCTGGTAGC
TGGCACAGATG		Sigma-AdrichN/A
SARS-2-S（RaTG）FCA GACAAACAGCAGATCTGTGGCCAGC
CAGAGCATC		Sigma-AdrichN/A
SARS-2-S（RaTG）RGCTGGCCACAGATCTGCTGTGTCTG
TGTCTGTAGC		Sigma-AdrichN/A
SARS-2-S（delta）FCAACAGCCCCGCCATCTGGCCAGCC
AGAGCATC		Sigma-AdrichN/A
SARS-2-S（delta）RGCTGGCCAGATGGCTGGCTGGTTTGTC
TGGTCTGGTAGC		Sigma-AdrichN/A
SARS-2-S（opt）FCGACGAAAAAAGATCTGGCCAGCCA
GAGCATC		Sigma-AdrichN/A
SARS-2-S（opt）RTCTTTCGTCGTGCTGTTGTCTGT
CTGG		Sigma-AdrichN/A
pCG1Seq F CCTGGCAACGTGCTGGTSigma-AdrichN/A
pCG1Seq R GTCAGATGCTCAAGGGCTTCASigma-AdrichN/A
SARS-S387F TGTTATATAGCATGTAACSigma-AdrichN/A
SARS-S790F AAGCCAACTACATTTATGCSigma-AdrichN/A
SARS S1194F TGATGTAAGACAAATAGCGSigma-AdrichN/A
SARS S1575F TATTAAGAACCAGTGTGTCSigma-AdrichN/A
SARS S1987F GTGCTAGTTACCATACAGSigma-AdrichN/A
SARS S2391F CTAAAGCCAACTAAGAGGSigma-AdrichN/A
SARS S2787F TCAACTGCATTGGCAAGSigma-AdrichN/A
SARS-2-S651F CAAGATCTACAGCAAGCACCSigma-AdrichN/A
SARS-2-S1380F GTCGGCGGCAACTACAATTACSigma-AdrichN/A
SARS-2-S1992F CTGTCTGATCGGAGCCGICACSigma-AdrichN/A
SARS-2-S2648F TGAGATGATCGCCCAGTACACSigma-AdrichN/A
SARS-2-S3286F GCCATCTGCCACGGCAAAGSigma-AdrichN/A
pCG1-V5FCTACGTCCTCGGTCGATCTACGTG
AAAGCTGATCTTTCCCTCTGCC		Sigma-AdrichN/A
pCG1-V5RGBACGAGGGTTAGGATAGGCTTACCG
CATGGCCTGCAGGTTTAAACAGTCG		Sigma-AdrichN/A
pCG1-XhoIR C TCCTCGAGTTCATAAGAAGAGGGSigma-AdrichN/A
Recombinant DNA
Plasmid:pCG1-SARS-SHoffmann et al。，2013N/A
Plasmid:pCG1-SARS-S-HAHoffmann et al。，2020N/A
Plasmid:pCG1-SARS-2-SHoffmann et al。，2020N/A
Plasmid:pCG1-SARS-2-S-HAHoffmann et al。，2020N/A
Plasmid:pCG1-SARS-SΔ18Hoffmann et al。，2013N/A
Plasmid:pCG1-SARS-SΔ18-V5This paperN/A
Plasmid:pCG1-SARS-2-SΔ18This paperN/A
Plasmid:pCG1-SARS-2-SΔ18-V5This paperN/A
Plasmid:pCG1-SARS-SΔ18（SARS-2）This paperN/A
Plasmid:pCG1-SARS-SΔ18-V5（SARS-2）This paperN/A
Plasmid:pCG1-SARS-SΔ18（RaTG）This paperN/A
Plasmid:pCG1-SARS-SΔ18-V5（RaTG）This paperN/A
Plasmid:pCG1-SARS-2-SΔ18（SARS）This paperN/A
Plasmid:pCG1-SARS-2-SΔ18-V5（SARS）This paperN/A
Plasmid:pCG1-SARS-2-SΔ18（RaTG）This paperN/A
Plasmid:pCG1-SARS-2-SΔ18-V5（RaTG）This paperN/A
Plasmid:pCG1-SARS-2-SΔ18（delta）This paperN/A
Plasmid:pCG1-SARS-2-SΔ18-V5（delta）This paperN/A
Plasmid:pCG1-SARS-2-SΔ18（opt）This paperN/A
Plasmid:pCG1-SARS-2-SΔ18-V5（opt）This paperN/A
Plasmid:pCAGGS-MERS-V5Gierer et al。，2013N/A
Plasmid:pCAGGS-VSV-GBrinkmann et al。，2017N/A
Plasmid:pCAGGS-DsRedHoffmann et al。，2013N/A
Plasmid:pCG1Roberto CattaneoN/A
Plasmid:pCG1-V5This paperN/A
软件和警报
Hidex Sense Microplate Reader SoftwareHidex Deutschland Vertrieb GmbHhttps://www.hidex.de/
Chemoster Imager Software（版本v.3.23）Intas Science Imaging Instruments GmbHhttps://www.intas.de/
ZEN imaging softwareCarl Zeisshttps://www.zeiss.com/
Clustal OmegaEuropean Molecular Biology Laboratory–European Bioinformatics Institute（EMBL-EBI）https://www.ebi.ac.uk/Tols/msa/clustalo/打开；Madeira et al。，2019
Adobe Photoshop CS5扩展版（版本12.0 3 32）Adobehttps://www.adobe.com/
GraphPad Prism（版本8.3.0（538））GraphPad Softwarehttps://www.graphpad.com/
YASARA（版本19.1.27）YASARA Biosciences GmbHhttp://www.yasara.org/打开；Krieger and Vriend，2014
Microsoft Office Standard2010（版本14.0.7232.5000）微软公司https://products.office.com/home
Other
Prefusion structure of SARS-Cov spike glycoprotein（5X5B）元宝。，2017https://www.rcsb.org/structure/5X5B

资源可用性

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact，Stefan Plann（spoehlmann@dpz.eu）。

实际可用性

All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement。

数据和代码可用性

The study did not generate unique datasets or code。

方法详细信息

Cell cultures

293T（human，kidney）and Vero（African green monkey，kidney）cells were cultivated in Dulbecco’s Modified Eagle Medium（PAN-Biotech）supplemented with10%fetal bovine serum（Biochrom），100U/mL of penicillin and 0.1mg/mL of streptomycin（PAN-Biotech）。Vero cells that stably express human TMPRSS 2 have been described previously（Hoffmann et al。，2020）and were cultivated in the presence of 10μg/mL blasticidin（Invivogen）。Calu-3（human，lung；kindly provided by Stephan Ludwig，Westfälische Wilhelms-Universität，uenster/Germany）cells were cultivated in Minimum Essential Medium（Thermo Fisher Scientific）supplemented with 10%fetal bovine serum（Biochrom），100U/mL of penicillin and 0.1mg/mL of streptomycin（PAN-Biotech），1xnon-essential amino acid solution（Fromx stock，PAA and 10mrom）Scientific）。全中央焊接at37℃和5%CO2in a humidified atmosphere。

Plasmids

高速plasmids for full-length vesicular stomatitis virus（VSV）glycoprotein（VSV-G），Middle-East respiratory syndrome coronavirus spike glycoprotein（MERS-S）containing aC-terminal V5epitope，Severe acute respiratory syndrome coronavirus spike glycoprotein（SARS-S）and severe acute respiratory syndrome coronavirus2spike glycoprotein（SARS-2-S）both equipped with aC-terminal hemaglutinin（HA）epitope been described previouslyBrinkmann et al。，2017单击功能区上，Hoffmann et al。，2020）。Empty pCG1expression vector was kindly provided by Roberto Cattaneo，Mayo Clinic，Rochester，MN/USA。Based on the SARS-S and SARS-2-S expression plasmids we cloned mutated versions with alterations at the S1/S2 cleavage site:We generated SARS-S containing the cleavisite of SARS-2，SARS-S bat/Yunnan/RaTG13/2013（RaTG；GISAID:EPI_ISL_402131），SARS-S（RaTG）。Further，we generated SARS-2-S harboring the S1/S2 cleavage site of SARS-S，SARS-2-S（SARS）or RaTG-S，SARS-2-S（RaTG）。Finally，we constructed SARS-2-S variants in which either the multibasic motif was deleted，SARS-2-S（delta），or in which the proline residue preceding the multibasic motif was mutated to arginine and the alanine residue within the minimal furin motif was changed to lysine in order to increase the basic environment at the S1/S2site，SARS-2-S（opt）。18 amino acids attheir respective C terminus as this has been shown to improve coronavirus spike protein incorporation into VSV particles and thus transduction（Schwegmann-We乇els et al。，2009年）。Further，for each construct an untagged variant as well as version containing a C-terminal V5epitope tag was constructed。

预应力型和阻力检测装置

A previously published protocol was employed to produce VSV pseudotype particles（VSVpp）carrying foreign viral glycoproteins in their envelope（Berger Rentsch and Zimmer，2011单击功能区上，Kleine-Weber et al。，2019）。First，293T cells were transfected with expression plasmid for the respective spike glycoprotein or VSV-G or empty expression vector by calcium-phosphate precipitation.At16h posttransfection，中央电源，中央电源∗ΔG-fLuc（kindly provided by Gert Zimmer，Institute of Virology and Immunology，Mittelhäusern/Switzerland），areplication-deficient VSV vector that lacks the genetic infor VSV-G and encodes for egFP and firefly luciferase，inoculum was removed and cells were washed with phosphate-buffered saline（PBS）before medium containing anti-VSV-G antibody（I1，mouse hybridoma supernatant from CRL-2700；ATCC）was added to all cells except for those expressing VSV-G（here，medium without antibody was added）.Cells were further incubated for16h，before the VSVpp containing supernatants were harvested，freed from cellular debris by centrifugation and used for experiments。

For transduction，target cells were grown in 96-well plates until they reached50%-80%confluency.The culture supernatant was removed by aspiration and 100μl/well of the respective pseudotype were added（quadruplicate samples）。At16h posttransduction，culture supernatants were aspirated and cells lysed in1x cell culture lysis reagent（prepared from5x stock，Promega）for20min at room temperature。The lysates were then transferred to white，opaque-walled96-well plates and luciferase activity was quantified by measuring luminescence upon addition of a substrate（PJK）using a Hidex Sense plate luminometer（Hidex）。

Western blot analysis

For the analysis of S protein processing，we subjected VSVpp harboring V5-or HA-tagged S proteins to SDS-PAGE and western blot analysis.For this，we loaded 1mL VSVpp onto50μlofa20%（w/v）sucrose cushion and performed high-speed centrifugation（25.00g for at 4 min）。Next，we removed1mL of supernatant，added50μl of2xSDS-sample buffer and incubated the samples for 15min at96°C。Thereafter，the samples were subjected to SDS-PAGE and protein transfer to nitrocellulose membranes by western blot.The membranes were subsequently blocked in 5%skim milk solution（PBS containing 0.05%Tween-20[PBS-T]and5%skim milk powder）for1h at room temperature。Theblots were then incubated over night at4°C with primary antibody solution，EB0011,1:2500）.Following this incubation，the blots were washed3x with PBS-T before they were incubated for1h at room temperature with peroxidase-coupled goat anti-mouse antibody（Dianova，115-035-003,1:10000）。Finally，the blots were again washed and imaged.For this，an in house-prepared enhanced chemiluminescent solution（0.1M Tris-HCl[pH8.6]，250μg/mL luminol，1mg/mL para-hydroxycoumaric acid，0.3%H2O2）and the ChemoCam imaging system along with the ChemoStar Professional software（Intas Science Imaging Instruments GmbH）were used。

同步formation assay

Vero or Vero-TMPRSS 2 cells were grown on coverslips seeded in 24-well plates and transfected with S protein expression plasmids（1μg/well）using Lipofectamine2000LTX with Plus reagent（Thermo Fisher Scientific）and Optimem medium（GIBCO）。After6h the transfection solutions were aspirated and the cells further incubated for24h in standard culture medium。Next，the medium was changed to serum free medium±1μg/mL bovine trypsin（Sigma-Aldrich）and the cells were incubated for additional24h.Then，the cells were washed with PBS，fixed with4%paraformaformadehyde solution for20min at room temperature，washed again，air-dried and incubated for30min with May-Gruenwald solution（Sigma-Aldrich）。Thereafter，the cells were washed three times with deionized water，air-dried and incubated for30min with1:10diluted Giemsa solution（Sigma-Aldrich）。After an additional washing interval with deionized water，the samples were air-dried and analyzed by bright-field microscopy using a Zeiss LSM800confocal laser scanning microscope and the ZEN imaging software（both from Zeiss）。

Sequence analysis and protein models

Sequence alignments were performe using the Clustal Omega online tool（https://www.ebi.ac.uk/Tols/msa/clustalo/）。Protein models were designed using the YASARA software（http://www.yasara.org/index.html）。SARS-2-S template（PDB:5X5B元宝。，2017）using the SWISS-MODEL online tool（https://swissmodel.expasy.org/）。The following sequences information were obtained from National Center for Biotechnology Information（NCBI）database:SARS-CoV BJ01（GenBank:AY278488.2），SARS-COV CUHK-W1（GenBank:AY278554.2），SARS-Cov Frankfurt-1（GenBank:AY291315.1），SARS-COV Tor2（GenBank:CS050815.1），SARS-COV Urbani（GenBank:AY278741.1），civet SARS-Cov SZ16（GenBank:AY304488.1），civet SARS-Cov civet020（GenBank:AY572038.1），raccoon dog SARS-COV A030（GenBank:AY687357.1），bat SARSr-COV BtKY72/KEN（GenBank:KY352407.1），bat SARSr-COV BM48-31/BGR/2008（GenBank:GU190215.1），bat SARSr-COV Rs4231（GenBank:KY417146.1），bat SARSr-COV WIV16（GenBank:KT444582.1），bat SARSr-COV Rs4874（GenBank:KY417150.1），bat SARSr-CoV SL-CoVZC45（GenBank:G772933.1），bat SARSr-CoV SL-CoVZXC21（GenBank:G772934.1），bat SARSr-Cov LYRa11（GenBank:KF569996.1），bat SARSr-COV LYRa3（GenBank:KF569997.1），bat SARSr-COV WIV1（GenBank:KF367457.1），bat SARSr-COV RsSHC014（GenBank:KC881005.1），bat SARSr-COV Rs3367（GenBank:KC881006.1），bat SARSr-Cov Cp/Yunnan2011（GenBank:JX999988.1），bat SARSr-COV Rp/Sianxi2011（GenBank:JX999987.1），bat SARSr-Cov HKU3-1（GenBank:DQ022305.2），bat SARSr-COV Rm1（GenBank:DQ412043.1），bat SARSr-COV Rp3（GenBank:DQ071615.1），bat SARSr-COV Rf1（GenBank:DQ412042.1），bat SARSr-COV279（GenBank:DQ648857.1），bat SARSr-COV273（GenBank:DQ648856.1），bat SARSr-Cov YN2013（GenBank:KJ473816.1），bat SARSr-COV Rs/HuB2013（GenBank:KJ473814.1），bat SARSr-COV Rs/GX2013（GenBank:KJ473815.1），bat SARSr-COV Rf/SX2013（GenBank:KJ473813.1），bat SARSr-COV Rf/JL2012（GenBank:KJ473811.1），bat SARSr-COV Rf/HeB2013（GenBank:KJ473812.1），bat SARSr-Cov YNLF/34C（GenBank:KP886809.1），bat SARSr-Cov YNLF/31C（GenBank:KP886808.1），bat SARSr-COV Rs672（GenBank:FJ588686.1），bat SARSr-COV Rs7327（GenBank:KY417151.1），bat SARSr-COV Rs4084（GenBank:KY417144.1），bat SARSr-COV Rs9401（GenBank:KY417152.1），bat SARSr-COV Rs4247（GenBank:KY417148.1），bat SARSr-COV Rs4255（GenBank:KY417149.1），bat SARSr-COV Rs4081（GenBank:KY417143.1），bat SARSr-COV Rs4237（GenBank:KY417147.1），bat SARSr-CoV As 6526（GenBank:KY417142.1），bat SARSr-COV Rf4092（GenBank:KY417145.1），bat SARSr-COV Longquan-140（GenBank:KF294457.1），bat SARSr-COV Rs806（GenBank:FJ588692.1），bat SARSr-Cov Anlong-103（GenBank:KY770858.1），bat SARSr-COV JTMC15（GenBank:KU182964.1），bat SARSr-COV16BO133（GenBank:KY938558.1），bat SARSr-COVB15-21（GenBank:KU528591.1），pangolin coronavirus MP789（GenBank:T084071.1）。In addition the following sequences information were obtained from the Global Initiative on Sharing All Influenza Data（GISAID）database:SARS-CoV-2（GISAID:EPI_ISL_404895），bat SARSr-CoV RaTG13（GISAID:EPI_ISL_402131）。

质量和统计分析

If not stated otherwise，statistical significance was tested by one-way analysis of variance with Dunnet’s posttest（GraphPad Prism7.03）。Only p values of 0.05 or lower were considered statistically significant（p>0.05[ns，not significant]，p≤0.05[∗]，p≤0.01[∗∗]，p≤0.001[∗∗∗]）。For all statistical analyses，the GraphPad Prism7software package was used（GraphPad Software）。

Acknowledgments

We thank Inga Nehlmeier for technical assistance.We gratefully acknowledge the authors and the originating and submitting laboratories for their sequence and metadata shared through GISAID，on which this research is based.This work was supported by the Bundesministerium für Bildung und ForschuBMg，RAPID Consortium，01KI 1723D to S.P.）.We further like to thank Andrea Maisner and Stephan Ludwig for providing the Vero cells and Calu-3cells，respectively。

Author Contributions

Conceptualization，M.H.and S.P.；Formal Analysis，M.H.and S.P.；Investigation，M.H.and H.K.-W；Writing–Original Draft，M.H.and S.P.；Writing–Review&Editing，all authors；Funding Acquisition，S.P。

中断，中断

The authors declare not competing interests

注释，注释

Published:May1200

参考，参考

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