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Id Subject Object Predicate Lexical cue
T1 0-113 Sentence denotes Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV
T2 115-123 Sentence denotes Abstract
T3 124-286 Sentence denotes Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002–2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019.
T4 287-354 Sentence denotes However, little is currently known about the biology of SARS-CoV-2.
T5 355-695 Sentence denotes Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S.
T6 696-798 Sentence denotes Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions.
T7 799-979 Sentence denotes Further studies using recovered SARS and COVID-19 patients’ sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other.
T8 980-1071 Sentence denotes Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.
T9 1073-1104 Sentence denotes SARS-CoV-2 has spread globally.
T10 1105-1332 Sentence denotes Here, the authors characterize the entry pathway of SARS-CoV-2, show that the SARS-CoV-2 spike protein is less stable than that of SARS-CoV, and show limited cross-neutralization activities between SARS-CoV and SARS-CoV-2 sera.
T11 1334-1346 Sentence denotes Introduction
T12 1347-1492 Sentence denotes Coronaviruses (CoVs) infect human and animals and cause varieties of diseases, including respiratory, enteric, renal, and neurological diseases1.
T13 1493-1578 Sentence denotes They are classified into four genera, alpha-CoV, beta-CoV, gamma-CoV, and delta-CoV2.
T14 1579-1674 Sentence denotes Since beginning of this century, there have already been three zoonotic outbreaks of beta-CoVs.
T15 1675-1867 Sentence denotes In 2002–2003, severe acute respiratory syndrome coronavirus (SARS-CoV)3,4, a lineage B beta-CoV, emerged from bat and palm civet5,6, and infected over 8000 people and caused about 800 deaths7.
T16 1868-2210 Sentence denotes In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV), a lineage C beta-CoV, was discovered as the causative agent of a severe respiratory syndrome in Saudi Arabia8, currently with 2494 confirmed cases and 858 deaths9, it remains endemic in Middle East, and dromedary camel is considered as the zoonotic reservoir host of MERS-CoV.
T17 2211-2335 Sentence denotes At the end of 2019, a novel coronavirus, named SARS-CoV-2, was found in patients with severe pneumonia in Wuhan, China10–12.
T18 2336-2386 Sentence denotes Viruses were isolated from patients and sequenced.
T19 2387-2554 Sentence denotes Phylogenetical analysis revealed that it is a lineage B beta-CoV and closely related to a SARS-like (SL) CoV, RaTG13, discovered in a cave of Yunnan, China, in 201313.
T20 2555-2672 Sentence denotes They share about 96% nucleotide sequence identities, suggesting that SARS-CoV-2 might have emerged from a Bat SL-CoV.
T21 2673-2770 Sentence denotes However, the intermediate host or whether there is an intermediate host remains to be determined.
T22 2771-2917 Sentence denotes CoV uses its spike glycoprotein (S), a main target for neutralization antibody, to bind its receptor, and mediate membrane fusion and virus entry.
T23 2918-3064 Sentence denotes Each monomer of trimeric S protein is about 180 kDa, and contains two subunits, S1 and S2, mediating attachment and membrane fusion, respectively.
T24 3065-3217 Sentence denotes In the structure, N- and C- terminal portions of S1 fold as two independent domains, N-terminal domain (NTD) and C-terminal domain (C-domain) (Fig. 1a).
T25 3218-3312 Sentence denotes Depending on the virus, either NTD or C-domain can serve as the receptor-binding domain (RBD).
T26 3313-3473 Sentence denotes While RBD of mouse hepatitis virus (MHV) is located at the NTD14, most of other CoVs, including SARS-CoV and MERS-CoV use C-domain to bind their receptors15–19.
T27 3474-3732 Sentence denotes MHV uses mouse carcinoembryonic antigen related cell adhesion molecule 1a (mCEACAM1a) as the receptor20, and the receptors for SARS-CoV and MERS-CoV are human angiotensin-converting enzyme 2 (hACE2)21 and human dipeptidyl peptidase 4 (hDPP4)22, respectively.
T28 3733-3992 Sentence denotes While S proteins of SARS-CoV-2 share about 76% and 97% of amino acid identities with SARS-CoV and RaTG13, respectively, the amino acid sequence of potential RBD of SARS-CoV-2 is only about 74% and 90.1% homologous to that of SARS-CoV and RaTG13, respectively.
T29 3993-4069 Sentence denotes Recently, Zhou et al.13 reported that SARS-CoV-2 uses hACE2 as the receptor.
T30 4070-4134 Sentence denotes Fig. 1 Incorporation of SARS-CoV-2 S protein into pseudovirions.
T31 4135-4199 Sentence denotes a Diagram of full-length SARS-CoV-2 S protein with a 3xFLAG tag.
T32 4200-4450 Sentence denotes S1, receptor-binding subunit; S2, membrane fusion subunit; TM, transmembrane domain; NTD, N-terminal domain; pFP, potential fusion peptide; HR-N, heptad repeat-N; HR-C, heptad repeat-C; b–f Detection of CoVs S protein in cells lysate by western blot.
T33 4451-5061 Sentence denotes Mock, 293T cells transfected with empty vector. b Mouse monoclonal anti-FLAG M2 antibody; c Polyclonal goat anti-MHV-A59 S protein antibody AO4. d Polyclonal rabbit anti-SARS S1 antibodies T62. e Mouse monoclonal anti-SARS S1 antibody. f Mouse monoclonal anti-MERS-CoV S2 antibody. g–j Detection of CoVs S protein in pseudovirions by western blot.Gag-p24 served as a loading control. g Anti-FLAG M2. h Polyclonal goat anti-MHV-A59 S protein antibody AO4. i Polyclonal rabbit anti-SARS S1 antibodies T62. j Polyclonal anti-Gag-p24 antibodies. uncleaved S protein, about 180 kDa; cleaved S protein, about 90 kDa.
T34 5062-5107 Sentence denotes Experiments were done twice and one is shown.
T35 5108-5155 Sentence denotes Source data are provided as a Source Data file.
T36 5156-5302 Sentence denotes CoV S proteins are typical class I viral fusion proteins, and protease cleavage is required for activation of the fusion potential of S protein23.
T37 5303-5498 Sentence denotes A two-step sequential protease cleavage model has been proposed for activation of S proteins of SARS-CoV and MERS-CoV24,25, priming cleavage between S1 and S2 and activating cleavage on S2’ site.
T38 5499-5756 Sentence denotes Depending on virus strains and cell types, CoV S proteins may be cleaved by one or several host proteases, including furin, trypsin, cathepsins, transmembrane protease serine protease-2 (TMPRSS-2), TMPRSS-4, or human airway trypsin-like protease (HAT)25–32.
T39 5757-5888 Sentence denotes Availability of these proteases on target cells largely determines whether CoVs enter cells through plasma membrane or endocytosis.
T40 5889-5985 Sentence denotes However, whether any of these proteases could promote virus entry of SARS-CoV-2 remains elusive.
T41 5986-6205 Sentence denotes In this study, using a lentiviral pseudotype system, we determine cell type susceptibility, virus receptor, entry pathway, and protease priming for SARS-CoV-2, and identify several potential drug targets for SARS-CoV-2.
T42 6206-6308 Sentence denotes We demonstrate limited cross-neutralization between convalescent sera from SARS and COVID-19 patients.
T43 6310-6317 Sentence denotes Results
T44 6319-6353 Sentence denotes Expression of SARS-CoV-2 S protein
T45 6354-6727 Sentence denotes To enhance expression of the S protein of SARS-CoV-2 in mammalian cells, a codon-optimized cDNA encoding the S protein and 3xFLAG tag was synthesized, and to facilitate incorporation of S protein into lentiviral pseudovirons, the last 19 amino acids containing an endoplasmic reticulum (ER)-retention signal from the cytoplasmic tail of the S protein was removed (Fig. 1a).
T46 6728-6765 Sentence denotes The construct was named SARS-CoV-2 S.
T47 6766-6893 Sentence denotes HEK293T cells were transfected with SARS-CoV-2 S plasmid and expression of SARS-CoV-2 S protein was determined by western blot.
T48 6894-7066 Sentence denotes There were two major bands, 180 kDa, and 90 kDa, detected by mouse anti-FLAG M2 antibody (Fig. 1b, lane 2), reflecting the full-length and cleaved S proteins, respectively.
T49 7067-7141 Sentence denotes The band above 250 kDa likely results from dimeric or trimeric S proteins.
T50 7142-7268 Sentence denotes Consistent with our previous report29, MERS-CoV S protein was detected by polyclonal goat anti-MHV S antibodies AO4 (Fig. 1c).
T51 7269-7412 Sentence denotes AO4 also detected SARS-CoV-2 and SARS-CoV S proteins, suggesting the presence of a conserved immunogenic epitope among all four different CoVs.
T52 7413-7462 Sentence denotes This presumably linear epitope is likely in S229.
T53 7463-7544 Sentence denotes S1 subunits of SARS-CoV-2 and SARS-CoV share almost 64% in amino acid identities.
T54 7545-7752 Sentence denotes Nevertheless, SARS-CoV-2 S protein was barely detected by rabbit polyclonal anti-SARS S1 antibodies T62 (Fig. 1d), suggesting that the major epitope(s) for T62 antibodies include non-conserved regions of S1.
T55 7753-7885 Sentence denotes The SARS-CoV-2 S protein was not detected by either a monoclonal anti-SARS S1 antibody (Fig. 1e) or anti-MERS S2 antibody (Fig. 1f).
T56 7887-7937 Sentence denotes Pseudovirion incorporation of SARS-CoV-2 S protein
T57 7938-8113 Sentence denotes The efficiency of SARS-CoV-2 S protein incorporation into pseudovirions was evaluated using monoclonal mouse anti-FLAG M2 antibody and polyclonal goat anti-MHV S antibody AO4.
T58 8114-8445 Sentence denotes While majority of SARS-CoV S proteins incorporated into pseudovirons were full-length, at 180 kDa (Fig. 1h, i), most of SARS-CoV-2 S proteins on lentiviral pseudovirions were cleaved, about 90 kDa (Fig. 1g, h), likely reflecting presence of extra furin site (R682-R683-A684-R685, Fig. 1a) between S1 and S2 in SARS-CoV-2 S protein.
T59 8446-8653 Sentence denotes Consistent with the results in cell lysate (Fig. 1d), SARS-CoV S protein, but not SARS-CoV-2 S protein, in pseudovirions was readily detected by using polyclonal rabbit anti-SARS S1 antibodies T62 (Fig. 1i).
T60 8655-8694 Sentence denotes Human ACE2 is a receptor for SARS-CoV-2
T61 8695-8802 Sentence denotes Next, we determined whether SARS-CoV-2 S pseudovirions were able to transduce human, monkey, and bat cells.
T62 8803-8937 Sentence denotes VSV-G pseudovirons were used as a positive control, whereas bald particles with no spike proteins (mock) served as a negative control.
T63 8938-9026 Sentence denotes As expected, all cell types were effectively transduced by VSV-G pseudovirons (Fig. 2a).
T64 9027-9211 Sentence denotes Compared to mock control, SARS-CoV-2 S pseudovirions showed an over 500-fold increase in luciferase activities in Calu3 cells, at a level similar to SARS-CoV S pseudovirions (Fig. 2a).
T65 9212-9323 Sentence denotes Huh7 and Vero 81 cells also gave about 10-fold increase in luciferase activities when transduced by SARS-CoV-2.
T66 9324-9576 Sentence denotes Transduction of LLCMK2 cells was higher with SARS-CoV S pseudovirions than with SARS-CoV-2 S pseudovirions (Fig. 2a), suggesting that there might be some differences on virus entry on LLCMK2 cells mediated by S proteins between SARS-CoV-2 and SARS-CoV.
T67 9577-9686 Sentence denotes We then investigated whether any known CoV receptors might be used by SARS-CoV-2 S protein as entry receptor.
T68 9687-9985 Sentence denotes The SARS-CoV-2 S pseudovirons were used to transduce BHK cells stably expressing human aminopeptidase N (hAPN), the receptor for human CoV 229E, 293 cells stably expressing hACE2 (293/hACE2), the receptor for SARS-CoV, and HeLa cells stably expressing hDPP4 (HeLa/hDPP4), the receptor for MERS-CoV.
T69 9986-10220 Sentence denotes While BHK/hAPN and HeLa/hDPP4 cells were not susceptible for the transduction of SARS-CoV-2 S pseudovirions, 293/hACE2 cells were highly transduced by SARS-CoV-2 S pseudovirions, consistent with hACE2 as the receptor for SARS-CoV-213.
T70 10221-10306 Sentence denotes We then determined whether SARS-CoV-2 S protein could directly bind to hACE2 protein.
T71 10307-10431 Sentence denotes HEK 293T cells transiently expressing SARS-CoV-2 S protein were incubated with soluble hACE2 and analyzed by flow cytometry.
T72 10432-10654 Sentence denotes As shown in Fig. 2c, SARS-CoV-2 S protein bound to soluble hACE2 at a level similar to SARS-CoV S protein, although the mean fluorescence intensity (MFI) for SARS-CoV-2 S protein was slightly lower than SARS-CoV S protein.
T73 10655-10775 Sentence denotes To further investigate if hACE2 is the receptor for SARS-CoV-2, we performed inhibition experiments using soluble hACE2.
T74 10776-10923 Sentence denotes Soluble hACE2 proteins were pre-incubated with SARS-CoV-2 S pseudovirons on ice for 1 h, then virus-protein mixture was added onto 293/hACE2 cells.
T75 10924-11210 Sentence denotes Entry of SARS-CoV-2 S pseudovirions was significantly prevented by pre-incubation of soluble hACE2 at both 10 μg/ml and 50  μg/ml (Fig. 2d), further supporting the notion that hACE2 is the receptor and soluble hACE2 might be used as a therapeutic inhibitor against SARS-CoV-2 infection.
T76 11211-11266 Sentence denotes Fig. 2 Entry and receptor of SARS-CoV-2 S pseudovirons.
T77 11267-11332 Sentence denotes a, b Entry of SARS-CoV-2 S pseudovirions on indicated cell lines.
T78 11333-11458 Sentence denotes Cells from human and animal origin were inoculated with SARS-CoV-2 S (red), SARS-CoV S (blue), or VSV-G (gray) pseudovirions.
T79 11459-11557 Sentence denotes At 48 h post inoculation, transduction efficiency was measured according to luciferase activities.
T80 11558-11815 Sentence denotes RS, Rhinolophus sinicus bat embryonic fibroblast; BHK/hAPN, BHK cells stably expressing hAPN, the hCoV-229E receptor; 293/hACE2, 293 cells stably expressing hACE2, the SARS-CoV receptor; HeLa/hDPP4, HeLa cells stably expressing hDPP4, the MERS-CoV receptor.
T81 11816-11887 Sentence denotes Experiments were done in triplicates and repeated at least three times.
T82 11888-12015 Sentence denotes One representative is shown with error bars indicating SEM. c Binding of SARS-CoV S and SARS-CoV-2 S proteins to soluble hACE2.
T83 12016-12182 Sentence denotes HEK293T cells transiently expressing SARS-CoV and SARS-CoV-2 S proteins were incubated with the soluble hACE2 on ice, followed by polyclonal goat anti-hACE2 antibody.
T84 12183-12221 Sentence denotes Cells were analyzed by flow cytometry.
T85 12222-12339 Sentence denotes The experiments were repeated at least three times. d Inhibition of SARS-CoV-2 S pseudovirion entry by soluble hACE2.
T86 12340-12471 Sentence denotes SARS-CoV S, SARS-CoV-2 S, or VSV-G pseudovirions were pre-incubated with soluble hACE2, then mixture were added to 293/hACE2 cells.
T87 12472-12542 Sentence denotes Cells were lysed 40 h later and pseudoviral transduction was measured.
T88 12543-12603 Sentence denotes Experiments were done twice and one representative is shown.
T89 12604-12653 Sentence denotes Error bars indicate SEM of technical triplicates.
T90 12654-12701 Sentence denotes Source data are provided as a Source Data file.
T91 12703-12760 Sentence denotes The SARS-CoV-2 enters 293/hACE2 cells through endocytosis
T92 12761-12843 Sentence denotes The majority of S proteins on SARS-CoV-2 S pseudovirions are cleaved (Fig. 1g, h).
T93 12844-12947 Sentence denotes We next determined whether SARS-CoV-2 S pseudovirons entered cells through endocytosis or cell surface.
T94 12948-13091 Sentence denotes HEK 293/hACE2 cells were treated with lysosomotropic agents, ammonia chloride and bafilomycin A, and their effect on virus entry was evaluated.
T95 13092-13263 Sentence denotes Consistent with previous reports, 20 mM NH4Cl and 100 nM bafilomycin A decreased entry of SARS-CoV S and VSV-G pseudovirions by over 99%, compared to no treatment control.
T96 13264-13582 Sentence denotes More than 98% reduction in transduction on 293/hACE2 cells by SARS-CoV-2 S pseudovirions was also shown when the cells were incubated with either NH4Cl or bafilomycin A (Fig. 3a), indicating that SARS-CoV-2 S pseudovirions enter 293/hACE2 cells mainly through endocytosis, despite that its spike proteins were cleaved.
T97 13583-13651 Sentence denotes Fig. 3 Endocytosis of SARS-CoV-2 S pseudovirions on 293/hACE2 cells.
T98 13652-13882 Sentence denotes a Inhibition of entry of SARS-CoV-2 S pseudovirion on 293/hACE2 by lysosomotropic agents (20 mM NH4Cl and 100 nM bafilomycin A). b Inhibition of entry of SARS-CoV, MERS-CoV, and MHV S pseudovirions by a PIKfyve inhibitor apilimod.
T99 13883-14061 Sentence denotes HeLa/mCEACAM, 293/hACE2, HeLa/hDPP4 cells were pretreated with different concentrations of apilimod and transduced with MHV S, SARS-CoV S, MERS-CoV S pseudovirions, respectively.
T100 14062-14122 Sentence denotes The luciferase activity was measured 40 h post transduction.
T101 14123-14166 Sentence denotes VSV-G pseudovirions were used as a control.
T102 14167-14238 Sentence denotes Experiments were done in triplicates and repeated at least three times.
T103 14239-14345 Sentence denotes One representative is shown with error bars indicating SEM. c Inhibition of MHV A59 infection by apilimod.
T104 14346-14465 Sentence denotes The 17Cl.1 cells were pretreated with 3, 10, 30, 100, 300 nM apilimod for 30 min and infected by MHV A59 at MOI = 0.01.
T105 14466-14559 Sentence denotes Viral infection and cell viability were determined by using qPCR and MTT assay, respectively.
T106 14560-14631 Sentence denotes Experiments were done in triplicates and repeated at least three times.
T107 14632-14794 Sentence denotes One representative is shown with error bars indicating SEM. d, e Inhibition of entry of SARS-CoV-2 S protein pseudovirions by apilimod, YM201636, and tetrandrine.
T108 14795-14962 Sentence denotes HEK 293/hACE2 cells were pretreated with either apilimod (d), YM201636 (e), or tetrandrine (f), then inoculated with SARS-CoV-2 S pseudovirons in the presence of drug.
T109 14963-15024 Sentence denotes The luciferase activity were measured 40 h post transduction.
T110 15025-15082 Sentence denotes YM201636, PIKfyve inhibitor; tetrandrine, TPC2 inhibitor.
T111 15083-15158 Sentence denotes The experiments were done in triplicates and repeated at least three times.
T112 15159-15243 Sentence denotes One representative is shown with error bars indicating SEM of technical triplicates.
T113 15244-15291 Sentence denotes Source data are provided as a Source Data file.
T114 15293-15339 Sentence denotes PIKfyve and TPC2 critical for SARS-CoV-2 entry
T115 15340-15399 Sentence denotes Phosphoinositides play many essential roles in endocytosis.
T116 15400-15532 Sentence denotes Among them, one is phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), which regulates early endosome to late endosome dynamics33,34.
T117 15533-15645 Sentence denotes Phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) is the main enzyme synthesizing PI(3,5)P2 in early endosome.
T118 15646-15727 Sentence denotes HEK 293/hACE2 cells were treated with apilimod, a potent inhibitor for PIKfyve35.
T119 15728-15965 Sentence denotes Inhibition of PIKfyve by apilimod significantly reduced entry of SARS-CoV S pseudovirions on 293/hACE2 cells in a dose dependent manner (Fig. 3b), whereas it had no effect on entry of VSV-G pseudovirions, which occurs in early endosomes.
T120 15966-16260 Sentence denotes Similar inhibitory effects were observed when HeLa/hDPP4 cells and HeLa cells stably expressing mouse carcinoembryonic antigen related cell adhesion molecule 1a (mCEACAM1a) (HeLa/mCEACAM1a) were treated with apilimod and transduced with MERS-CoV and MHV S pseudovirions (Fig. 3b), respectively.
T121 16261-16369 Sentence denotes Moreover, infection of live MHV on 17Cl.1 cells was also strongly inhibited by apilimod treatment (Fig. 3c).
T122 16370-16462 Sentence denotes No significant cell toxicity was observed on apilimod at any concentration tested (Fig. 3c).
T123 16463-16562 Sentence denotes We then determined whether apilimod could inhibit entry of SARS-CoV-2 S pseudovirions on 293/hACE2.
T124 16563-16678 Sentence denotes As expected, apilimod treatment inhibited entry of SARS-CoV-2 S pseudovirions in a dose dependent manner (Fig. 3d).
T125 16679-16791 Sentence denotes Similar effects were shown when 293/hACE2 cells were treated with YM201636, another PIKfyve inhibitor (Fig. 3e).
T126 16792-16919 Sentence denotes These results suggested that PIKfyve might be a potential general drug target for viruses that enter cells through endocytosis.
T127 16920-17027 Sentence denotes Two pore channel subtype 2 (TPC2) and TRPML1 in lysosome are two major downstream effectors of PI(3,5)P236.
T128 17028-17310 Sentence denotes While blocking TPC2 activity by tetrandrine, an inhibitor for TPC237, decreased entry of SARS-CoV-2 S pseudovirions (Fig. 3f), treatment of cells with 130, a TRPML1 inhibitor, had no effect (Supplementary Fig. 1), indicating that TPC2, not TRPML1, is important for SARS-CoV-2 entry.
T129 17312-17362 Sentence denotes Effect of cathepsin inhibitors on SARS-CoV-2 entry
T130 17363-17550 Sentence denotes Protease activation on S protein is an important step for coronavirus entry, and cathepsins in lysosome have been shown to be critical for SARS-CoV and MERS-CoV entry through endocytosis.
T131 17551-17798 Sentence denotes To investigate whether cathepsins are required for SARS-CoV-2 entry, HEK 293/hACE2 cells were treated with either a broad inhibitor for cathepsin B, H, L, and calpain (E64D), cathepsin L inhibitor (SID 26681509), or cathepsin B inhibitor (CA-074).
T132 17799-17925 Sentence denotes VSV-G pseudovirions were used as a negative control, since virus entry mediated by VSV-G does not require protease activation.
T133 17926-18117 Sentence denotes E64D treatment of 293/hACE2 cells reduced entry of SARS-CoV-2 S pseudovirions by 92.5%, indicating that at least one of cathepsins or calpain might be required for SARS-CoV-2 entry (Fig. 4a).
T134 18118-18447 Sentence denotes While cathepsin B inhibitor treatment did not show any marked effect on virus entry, cathepsin L inhibition treatment decreased entry of SARS-CoV-2 S pseudovirions into 293/hACE2 by over 76% (Fig. 4a), suggesting that cathepsin L should be essential for priming of SARS-CoV-2 S protein in lysosome for entry into 293/hACE2 cells.
T135 18448-18515 Sentence denotes Fig. 4 Activation of SARS-CoV-2 S protein by cathepsin and trypsin.
T136 18516-18608 Sentence denotes a Effects of cathepsin inhibitors on entry of SARS-CoV-2 S pseudovirions on 293/hACE2 cells.
T137 18609-18843 Sentence denotes HEK 293/hACE2 cells were pretreated with broad-spectrum cathepsin inhibitor E64D, cathepsin L-specific inhibitor (SID 26681509), or cathepsin B-specific inhibitor (CA-074) and then transduced with SARS-CoV-2 S and VSV-G pseudovirions.
T138 18844-18907 Sentence denotes Pseudoviral transduction was measured at 40 h post inoculation.
T139 18908-18979 Sentence denotes Experiments were done in triplicates and repeated at least three times.
T140 18980-19008 Sentence denotes One representative is shown.
T141 19009-19111 Sentence denotes Error bars indicate SEM of technical triplicates. b Cell–cell fusion mediated by SARS-CoV-2 S protein.
T142 19112-19308 Sentence denotes HEK 293T cells were transiently expressing eGFP and either SARS-CoV-2 or SARS-CoV S protein were detached with either trypsin or EDTA, and co-cultured with 293/hACE2 or 293 cells for 4 h at 37 °C.
T143 19309-19605 Sentence denotes The scale bar indicates 250 µm. c Quantitative analysis of syncytia in panel b. d, e Thermostability analysis of SARS-CoV-2 S protein. d SARS-CoV and SARS-CoV-2 S pseudovirons were incubated at 37 °C for the specified times (0 to 4 h) in the absence of serum, and then assayed on 293/hACE2 cells.
T144 19606-19908 Sentence denotes The results from infection at 0 h were set as 100%, and the experiments were repeated four times, and means with standard deviations are shown. e SARS-CoV and SARS-CoV-2 S pseudovirions without serum were incubated at the indicated temperature (37 to 51 °C) for 2 h and then assayed on 293/hACE2 cells.
T145 19909-19977 Sentence denotes The results are reported as the percentage of transduction at 37 °C.
T146 19978-20065 Sentence denotes The experiments were repeated four times, and means with standard deviations are shown.
T147 20066-20113 Sentence denotes Source data are provided as a Source Data file.
T148 20115-20164 Sentence denotes Cell–cell fusion mediated by SARS-CoV-2 S protein
T149 20165-20356 Sentence denotes Type II membrane serine proteases (TMPRSS)-mediated cleavage can activate the fusion potential of SARS-CoV and MERS-CoV S protein and induce receptor-dependent syncytium formation28,29,38,39.
T150 20357-20591 Sentence denotes To test whether SARS-CoV-2 S protein cleaved by these cellular proteases could trigger syncytium formation, we overlaid HEK 293T cells expressing SARS-CoV-2 S or control proteins on 293/hACE2 cells in presence or absence of proteases.
T151 20592-20760 Sentence denotes Expression of TMPRSS 2, 4, 11 A, 11D, and 11E on 293/hACE2 cells enhanced SARS-CoV-2 S protein-mediated cell–cell fusion similarly to SASR-CoV S (Supplementary Fig. 2).
T152 20761-20799 Sentence denotes Next we evaluated the role of trypsin.
T153 20800-20962 Sentence denotes Consistent to our previous report23, addition of trypsin triggered syncytium formation on 293/hACE2 cells by SARS-CoV S protein after 4 h incubation (Fig. 4b, c).
T154 20963-21125 Sentence denotes Large syncytia were formed at a level similar to SARS-CoV S proteins when SARS-CoV-2 S protein expressing 293T cells were added onto 293/hACE2 cells with trypsin.
T155 21126-21396 Sentence denotes Of note, SARS-CoV-2, but not SARS-CoV, S proteins induced syncytia formation on 293/hACE2 cells even in the absence of trypsin (Fig. 4c), suggesting that SARS-CoV-2 S proteins could be triggered upon the receptor binding without exogenous protease priming or activation.
T156 21397-21552 Sentence denotes During 2013–2016 Ebola virus outbreak, A82V and T544I mutations in glycoprotein (gp) of Ebola virus were linked to increase of virus transmissibility40–42.
T157 21553-21696 Sentence denotes Further study showed that A82V and T544I mutation decreases the thermostability of gp while they increases virus infectivity in cell culture42.
T158 21697-21762 Sentence denotes We then investigated the thermostability of SARS-CoV-2 S protein.
T159 21763-21921 Sentence denotes Compared to SARS-CoV S protein, SARS-CoV-2 S protein was less stable, requiring significant shorter time and lower temperature to be inactivated (Fig. 4d, e).
T160 21922-22150 Sentence denotes The native S protein is metastable, and there is an energy barrier that prevents it from undergoing conformational change before triggering, SARS-CoV-2 S protein might decrease its energy barrier by reducing its thermostability.
T161 22151-22219 Sentence denotes This might contribute to high-transmission efficiency of SARS-CoV-2.
T162 22221-22282 Sentence denotes Effect of anti-SARS S1 antibodies T62 on SARS-CoV-2 S protein
T163 22283-22514 Sentence denotes Since SARS-CoV-2 S reacted weakly with polyclonal rabbit anti-SARS S1 antibodies T62 in western blot (Fig. 1d), we investigated whether SARS-CoV-2 S protein in native conformation could be recognized by anti-SARS S1 antibodies T62.
T164 22515-22664 Sentence denotes HEK293T cells transiently expressing SARS-CoV-2 S proteins were incubated with polyclonal anti-SARS S1 antibodies T62 and analyzed by flow cytometry.
T165 22665-22707 Sentence denotes SARS-CoV S was used as a positive control.
T166 22708-22850 Sentence denotes As expected, expression of SARS-CoV S proteins on 293T cell surface were readily detected by polyclonal anti-SARS S1 antibodies T62 (Fig. 5a).
T167 22851-22986 Sentence denotes In contrast, only low level of binding of SARS-CoV-2 S protein to polyclonal rabbit anti-SARS S1 antibodies T62 was detected (Fig. 5a).
T168 22987-23273 Sentence denotes To further delineate where the conformational epitopes for this polyclonal antibodies are located, two new constructs were generated, a SARS-CoV S protein backbone with RBD from SARS-CoV-2 (SARS-CoV S/nRBD) and a SARS-CoV-2 S protein backbone with RBD from SARS-CoV (SARS-CoV-2 S/sRBD).
T169 23274-23553 Sentence denotes Replacement of SARS-CoV RBD with SARS-CoV-2 RBD (SARS-CoV S/nRBD) decreased binding of polyclonal antibodies T62 to S protein, whereas substitution of SARS-CoV-2 RBD with SARS-CoV RBD (SARS-CoV-2 S/sRBD) increased the affinity of S protein to polyclonal antibodies T62 (Fig. 5a).
T170 23554-23653 Sentence denotes All chimera proteins were expressed at levels similar to SARS-CoV-2 S protein (Fig. 5b, top panel).
T171 23654-23846 Sentence denotes These results suggest that at least one major conformational epitope for polyclonal antibodies T62 might be located in RBD where the sequences differ between SARS-CoV and SARS-CoV-2 (Fig. 5c).
T172 23847-24171 Sentence denotes Of note, while SARS-CoV-2 S/sRBD bound to polyclonal antibodies T62 better than SARS-CoV S/nRBD in flow cytometry, the signal for SARS-CoV S/nRBD was stronger than SARS-CoV-2 S/sRBD in western blot by polyclonal antibodies T62, indicating that major linear epitopes of polyclonal antibodies T62 were located on SARS-CoV NTD.
T173 24172-24330 Sentence denotes Among residues in SARS-CoV RBD critical for receptor binding and virus entry43,44, seven of them are different between SARS-CoV and SARS-CoV-2 RBDs (Fig. 5c).
T174 24331-24428 Sentence denotes We then mutated each of them in SARS-CoV S protein to the corresponding residues in SARS-CoV-2 S.
T175 24429-24637 Sentence denotes With exception of SARS-CoV mutant Y442L, which had reduced protein expression (Supplementary Fig. 3), binding of SARS-CoV S mutants to polyclonal rabbit anti-SARS S1 antibodies T62 was not affected (Fig. 5d).
T176 24638-24736 Sentence denotes This suggests that these residues might not be direct binding sites for polyclonal antibodies T62.
T177 24737-24857 Sentence denotes Next, we tested whether polyclonal rabbit anti-SARS S1 antibodies T62 could inhibit entry of SARS-CoV-2 S pseudovirions.
T178 24858-25019 Sentence denotes CoV S protein pseudovirions were incubated with polyclonal antibodies T62 on ice for 1 h, and their transduction was measured according to luciferase activities.
T179 25020-25144 Sentence denotes As expected, polyclonal antibodies T62 neutralized SARS-CoV S pseudovirion effectively in a dose dependent manner (Fig. 5e).
T180 25145-25299 Sentence denotes In contrast, even at a concentration of 50 μg/ml, polyclonal antibodies T62 did not have marked effect on transduction by SARS-CoV-2 S proteins (Fig. 5e).
T181 25300-25373 Sentence denotes Fig. 5 Characterization of polyclonal rabbit anti-SARS S1 antibodies T62.
T182 25374-25484 Sentence denotes a Binding of polyclonal rabbit anti-SARS S1 antibodies T62 to SARS-CoV-2, SARS-CoV S, and chimeric S proteins.
T183 25485-25779 Sentence denotes HEK293T cells transiently expressing either SARS-CoV-2 S, SARS-CoV S, SARS-CoV S/nRBD, or SARS-CoV-2 S/sRBD proteins were incubated with polyclonal rabbit anti-SARS-CoV S1 antibody T62 for 1 h on ice, followed by a FITC-conjugated secondary antibody, then cells were analyzed by flow cytometry.
T184 25780-25926 Sentence denotes Experiments were done three times and one representative is shown. b Expression of SARS-CoV-2 S, SARS-CoV S, or chimeric S proteins on 293T cells.
T185 25927-26104 Sentence denotes Cells from panel A were lyzed and blotted with anti-FLAG M2 antibody and polyclonal anti-SARS S1 antibody T62. c Amino acid sequence alignment of SARS-CoV and SARS-CoV-2 S RBDs.
T186 26105-26399 Sentence denotes Stars (*) indicate the seven critical residues different between SARS-CoV-2 and SARS-CoV RBDs. d Binding of polyclonal rabbit anti-SARS S1 antibodies T62 to mutant SARS-CoV S proteins. e Neutralization of SARS-CoV-2 S and SARS-CoV S pseudovirions by polyclonal rabbit anti-SARS S1 antibody T62.
T187 26400-26565 Sentence denotes Pseudovirons were pre-incubated with serially diluted polyclonal rabbit anti-SARS S1 antibodies T62 on ice, then virus-antibody mixture was added on 293/hACE2 cells.
T188 26566-26615 Sentence denotes Pseudoviral transduction was measured 40 h later.
T189 26616-26705 Sentence denotes Experiments were done in triplicates and repeated twice, and one representative is shown.
T190 26706-26755 Sentence denotes Error bars indicate SEM of technical triplicates.
T191 26756-26803 Sentence denotes Source data are provided as a Source Data file.
T192 26805-26859 Sentence denotes Cross-neutralization of SARS and COVID-19 patient sera
T193 26860-27118 Sentence denotes As rabbit polyclonal anti-SARS S1 antibodies did not show significant neutralization activity against SARS-CoV-2 S pseudovirions, we then asked whether sera from recovered SARS and COVID-19 patients could neutralize SARS-CoV S and SARS-CoV-2 S pseudovirions.
T194 27119-27331 Sentence denotes We obtained sera from one recovered SARS patient and five recovered COVID-19 patients and tested their neutralization activities against transduction on 293/hACE2 cells by SARS-CoV and SARS-CoV-2 S pseudovirions.
T195 27332-27534 Sentence denotes Serum from recovered SARS patient showed strong inhibition on transduction by SARS-CoV S pseudovirions, and it had modest neutralization activity against SARS-CoV-2 S pseudovirons (Fig. 6a and Table 1).
T196 27535-27734 Sentence denotes In contrast, sera from all five COVID-19 patients neutralized SARS-CoV-2 S pseudovirions effectively, and none of them had an effect on transduction by SARS-CoV S pseudovirions (Fig. 6b and Table 1).
T197 27735-27797 Sentence denotes Fig. 6 Limited cross-neutralization of SARS and COVID-19 sera.
T198 27798-27866 Sentence denotes All sera were incubated on 56 °C for 30 min to eliminate complement.
T199 27867-28050 Sentence denotes SARS-CoV S and SARS-CoV-2 S pseudovirons were pre-incubated with serially diluted SARS patient serum (a) or COVID-19 patient sera (b) for 1 h on ice and then added on 293/hACE2 cells.
T200 28051-28100 Sentence denotes Pseudoviral transduction was measured 40 h later.
T201 28101-28190 Sentence denotes Experiments were done in triplicates and repeated twice, and one representative is shown.
T202 28191-28240 Sentence denotes Error bars indicate SEM of technical triplicates.
T203 28241-28288 Sentence denotes Source data are provided as a Source Data file.
T204 28289-28371 Sentence denotes Table 1 Neutralization activities of antisera from SARS-CoV and COVID-19 patients.
T205 28372-28435 Sentence denotes Patient serum SARS-CoV S pseudovirion SARS-CoV-2 S pseudovirion
T206 28436-28459 Sentence denotes SARS-CoV patient >80 40
T207 28460-28496 Sentence denotes COVID-19 patient #1 Not detected >80
T208 28497-28532 Sentence denotes COVID-19 patient #2 Not detected 40
T209 28533-28569 Sentence denotes COVID-19 patient #3 Not detected >80
T210 28570-28606 Sentence denotes COVID-19 patient #4 Not detected >80
T211 28607-28643 Sentence denotes COVID-19 patient #5 Not detected >80
T212 28644-28811 Sentence denotes SARS-CoV and SARS-CoV-2 S pseudovirions were pre-incubated with serially diluted patient sera for 1 h on ice, then virus-antibody mixture was added on 293/hACE2 cells.
T213 28812-28882 Sentence denotes Cells were lysed 40 h later and pseudovirus transduction was measured.
T214 28883-28997 Sentence denotes The highest dilution of the serum sample that decreased transduction by 50% or more was considered to be positive.
T215 28998-29045 Sentence denotes Source data are provided as a Source Data file.
T216 29047-29057 Sentence denotes Discussion
T217 29058-29190 Sentence denotes About 70% of the emerging pathogens infecting humans originate from animals, and CoVs are among the forefronts of these pathogens45.
T218 29191-29390 Sentence denotes The newly emerged SARS-CoV-2 infects human and causes severe pneumonia, and as of 10 February, 2020, the current outbreak has spread to 25 countries with over 40,000 confirmed cases and 900 deaths46.
T219 29391-29434 Sentence denotes However, little is known about its biology.
T220 29435-29631 Sentence denotes Since the virus is categorized as a biosafety level 3 (BSL3) agent, according to WHO guideline, we developed a pseudotype system with S protein of SARS-CoV-2 to study virus entry in BSL2 settings.
T221 29632-29763 Sentence denotes Understanding how SARS-CoV-2 enters cell will provide valuable information for virus pathogenesis, vaccine design, and drug target.
T222 29764-29912 Sentence denotes Utilizing this pseudotype system, we screened a panel of human and monkey cell lines for their susceptibility of SARS-CoV-2 S-mediated transduction.
T223 29913-30080 Sentence denotes In line with SASR-CoV-2 causing respiratory infections in humans, we found that human lung cancer cell line Calu3 is highly susceptible to SARS-CoV-2 S-mediated entry.
T224 30081-30303 Sentence denotes LLCMK2 cells, a rhesus monkey kidney epithelium cell line, exhibited different susceptibility to SARS-CoV S and SARS-CoV-2 S transduction, but the reasons for this are currently not known and require further investigation.
T225 30304-30513 Sentence denotes Both S proteins use hACE2 as the receptor for binding and entry13,21, which we further confirmed by flow cytometry analysis and competitive inhibition experiment using soluble hACE2 in this study (Fig. 3b, c).
T226 30514-30662 Sentence denotes While full-length S proteins of SARS-CoV-2 and SARS-CoV S share almost 76% identities in amino acid sequences, the NTDs show only 53.5% of homology.
T227 30663-30746 Sentence denotes Of note, NTDs of different CoVs S proteins have been shown to bind different sugar.
T228 30747-30907 Sentence denotes While NTD of MERS-CoV prefers α2,3-linked sialic acid over α2,6-linked sialic acid47, NTDs of human CoVs OC43 and HKU1 bind to 9-O-acetylated sialic acids48,49.
T229 30908-30963 Sentence denotes No sugar binding has been reported for NTD of SARS-CoV.
T230 30964-31090 Sentence denotes Whether or not NTD of SARS-CoV-2 binds to sugar and whether sugar binding of NTD affects virus entry remains to be determined.
T231 31091-31249 Sentence denotes Successful conformational changes of S proteins, leading to membrane fusion, not only require receptor binding, but also need appropriate protease activation.
T232 31250-31413 Sentence denotes There is a furin site between S1 and S2 (amino acids 682-685, RRAR) subunits in SARS-CoV-2 S protein, similar to what happens in high-pathogenic influenza viruses.
T233 31414-31549 Sentence denotes Whether the presence of this furin site has any effect on viral pathogenesis and virus spreading among humans remains to be determined.
T234 31550-31680 Sentence denotes Although majority of SARS-CoV-2 S proteins in pseuodvirions were cleaved, endocytosis is the main entry pathway on 293/hACE2 cell.
T235 31681-31849 Sentence denotes In our experiments, lysosomal cathepsin L, not B, appeared critical for SARS-CoV-2 S protein activation, similar to what was reported for SARS-CoV and MERS-CoV28,29,50.
T236 31850-31948 Sentence denotes Trypsin could also activate SARS-CoV-2 S protein efficiently and induce large syncytium formation.
T237 31949-32156 Sentence denotes To our surprise, we found that, even without trypsin, SARS-CoV-2 S protein could trigger syncytium formation on 293/hACE2 cells, a phenomena similar to what was observed in MHV S protein in certain aspect51.
T238 32157-32297 Sentence denotes This led us to speculate that SARS-CoV-2 S protein is capable of triggering protease-independent and receptor-dependent syncytium formation.
T239 32298-32439 Sentence denotes Such a mechanism might enhance virus spreading through cell–cell fusion and this might partially explain rapid progress of the diseases10,11.
T240 32440-32618 Sentence denotes However, we have not excluded a potential role for other endogenous proteases in HEK293T cells for syncytium formation and we have also not evaluated entry into other cell types.
T241 32619-32650 Sentence denotes This requires further research.
T242 32651-32898 Sentence denotes Recent studies showed that early to late endosome maturation is regulated by PI(3,5)P2, and inhibition of PIKfyve, the key enzyme synthesizing PI(3,5)P2, and TPC2, a downstream effector in lysosome, significantly reduced virus entry of MERS-CoV52.
T243 32899-33151 Sentence denotes We confirmed this in this study, and further showed that blocking PIKfyve and TPC2 also strongly inhibited entry mediated by SARS-CoV-2 S protein, indicating that PI(3,5)P2 pathway might be considered as potential general drug target for CoV infection.
T244 33152-33331 Sentence denotes CoV S protein is one of the key components determining virus virulence, tissue tropism and host range, and it is also a main target for neutralizing antibodies and vaccine design.
T245 33332-33560 Sentence denotes Although the S proteins of SARS-CoV-2 and SARS-CoV are highly homologous, polyclonal rabbit anti-SARS S1 antibodies T62 did not bind to SARS-CoV-2 S protein well, and poorly neutralized SARS-CoV-2 S protein-mediated virus entry.
T246 33561-33674 Sentence denotes Further analysis reveals that major immune-epitopes for T62 antibodies likely lie in the region of RBD (Fig. 4a).
T247 33675-33800 Sentence denotes We further evaluated cross-neutralization of SARS-CoV and SARS-CoV-2 using convalescent sera from SARS and COVID-19 patients.
T248 33801-34044 Sentence denotes We only saw moderate cross-neutralization activities between SARS-CoV-2 and SARS-CoV convalescent sera, suggesting that those previously recovered from SARS-CoV infection may not be fully protected against SARS-CoV-2 infection, and vice versa.
T249 34045-34269 Sentence denotes While this manuscript was under review, Hoffmann et al.53 reported that human ACE2 is the entry receptor of SARS-CoV-2, and that serine protease TMPRSS2 is important for SARS-CoV-2 S activation, consistent with our findings.
T250 34270-34475 Sentence denotes In agreement with our work, sera from convalescent SARS patients neutralized SARS-CoV-2 S pseudovirion entry with lower efficiency than SARS-CoV S pseudovirion entry, showing moderate cross-neutralization.
T251 34476-34667 Sentence denotes In conclusion, we demonstrated that SARS-CoV-2 S protein entry on 293/hACE2 cells is mainly mediated through endocytosis, and that PIKfyve, TPC2, and cathepsin L are critical for virus entry.
T252 34668-34787 Sentence denotes We further found that SARS-CoV-2 S protein could trigger syncytia in 293/hACE2 cells independent of exogenous protease.
T253 34788-34903 Sentence denotes Finally, there was limited cross-neutralization activity between convalescent sera from SARS and COVID-19 patients.
T254 34904-35028 Sentence denotes Our findings provide potential targets for development of drugs and vaccines against this newly emerging lineage B beta-CoV.
T255 35030-35037 Sentence denotes Methods
T256 35039-35062 Sentence denotes Constructs and plasmids
T257 35063-35332 Sentence denotes Codon-optimized cDNA (sequence shown in Supplementary Table 2) encoding SARS-CoV-2 S glycoprotein (QHU36824.1) with C-terminal 19 amino acids deletion was synthesized and cloned into eukaryotic cell expression vector pCMV14-3×Flag between the Hind III and BamH I sites.
T258 35333-35798 Sentence denotes Plasmids encoding SARS-CoV S glycoprotein, MERS-CoV S glycoprotein and MHV S glycoprotein (sequences shown in Supplementary Table 2) were constructed by inserting DNA fragment encoding codon-optimized SARS-CoV S protein (AAP13441.1) lacking the last 19 amino acids (aa), MERS-CoV S protein (AFS88936.1) lacking the last 16 aa but with a FLAG tag at the C terminus, or full-length MHV S protein (AAU06356.1) into pcDNA3.1between BamH I and Not I sites, respectively.
T259 35799-35909 Sentence denotes The VSV-G encoding plasmid and lentiviral packaging plasmid psPAX2 were obtained from Addgene (Cambridge, MA).
T260 35910-36037 Sentence denotes The pLenti-GFP lentiviral reporter plasmid that expresses GFP and luciferase was generously gifted by Fang Li, Duke University.
T261 36038-36105 Sentence denotes All primers used in this study are listed in Supplementary Table 1.
T262 36107-36117 Sentence denotes Cell lines
T263 36118-36560 Sentence denotes Human embryonic kidney cell line 293 (#CRL-1573) and 293T expressing the SV40 T-antigen (#CRL-3216), human airway epithelial cell line Calu3 (#HTB-55), human alveolar epithelial cell line A549 (#CCL-185), human fibroblasts derived from lung tissue MRC5 (#CCL-171), African green monkey kidney cell line Vero E6 (#CRL-1586) and Vero 81 (#CCL-81), and human cervical carcinoma cell line HeLa(#CCL-2) were obtained from ATCC (Manassas, VA, USA).
T264 36561-36706 Sentence denotes Human carcinoma cell line derived from hepatocyte Huh7 was kindly provided by Dr. Wei Yang (Chinese Academy of Medical Sciences, Beijing, China).
T265 36707-36803 Sentence denotes Bat embryo fibroblast cells RS were isolated mid-gestation fetuses from Rhinolophus sinicus bat.
T266 36804-37086 Sentence denotes HEK 239 cells stably expressing recombinant human ACE2 (293/hACE2), baby hamster kidney fibroblasts stably expressing recombinant human APN (BHK/hAPN), HeLa cells stably expressing recombinant human DPP4 (HeLa/hDPP4) were established in our lab by overexpression of these receptors.
T267 37087-37270 Sentence denotes All above cells were maintained in Dulbecco’s MEM containing 10% fetal bovine serum and 100 unit penicillin, 100 μg streptomycin, and 0.25 μg Fungizone (1% PSF, Gibco) per milliliter.
T268 37271-37386 Sentence denotes Rhesus monkeys kidney cell line LLC-MK2 (#CCL-7) from ATCC was maintained in Opti-MEM containing 10% FBS and 1%PSF.
T269 37388-37413 Sentence denotes Antibodies and inhibitors
T270 37414-37712 Sentence denotes Broad-spectrum cysteine protease inhibitor E64D (#HY-100229), Cathepsin L-specific inhibitor SID 26681509 (#HY-103353), Cathepsin B-specific inhibitor CA-074 (#HY-103350), PIKfyve inhibitors apilimod (#HY-14644) and YM201636 (#HY-13228), were purchased from Med Chem Express (MCE, New Jersey, USA).
T271 37713-37808 Sentence denotes Calcium channel blocker tetrandrine (#S2403) was purchased from Selleck Chemicals (Texas, USA).
T272 37809-37941 Sentence denotes Endosome acidification inhibitors NH4Cl (#A9434) and bafilomycin A (#19-148) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
T273 37942-38239 Sentence denotes Rabbit polyclonal against SARS S1 antibodies (#40150-T62), mouse monoclonal against MERS-CoV S2 antibody (#40070-MM11), mouse monoclonal against SARS S1 antibody (#40150-MM02), rabbit polyclonal against HIV-1 Gag-p24 antibody (11695-RP01) were purchased from Sino Biological Inc. (Beijing, China).
T274 38240-38341 Sentence denotes Goat polyclonal against human ACE2 antibody (#AF933) was purchased from R&D Systems (Minnesota, USA).
T275 38342-38414 Sentence denotes Mouse monoclonal anti-FLAG M2 antibody was purchased from Sigma-Aldrich.
T276 38415-38595 Sentence denotes Donkey Anti-Rabbit IgG (#711-035-152), Goat Anti-Mouse IgG (#115-035-146), and Rabbit Anti-Goat IgG (#305-035-003) were purchased from Jackson ImmunoResearch (West Grove, PA, USA).
T277 38596-38742 Sentence denotes Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG were purchased from ZSGB-Bio LLC (Beijing, China).
T278 38744-38800 Sentence denotes Production of SARS-CoV-2 S pseudovirions and virus entry
T279 38801-38992 Sentence denotes Pseudovirions were produced by co-transfection 293T cells with psPAX2, pLenti-GFP, and plasmids encoding either SARS-CoV-2 S, SARS-CoV S, VSV-G, or empty vector by using polyetherimide (PEI).
T280 38993-39146 Sentence denotes The supernatants were harvested at 40, 64 h post transfection, passed through 0.45 μm filter, and centrifuged at 800 × g for 5 min to remove cell debris.
T281 39147-39282 Sentence denotes To transduce cells with pseudovirions, cells were seeded into 24-well plates and inoculated with 500 μl media containing pseudovirions.
T282 39283-39343 Sentence denotes After overnight incubation, cells were fed with fresh media.
T283 39344-39475 Sentence denotes About 40 h post inoculation, cells were lysed with 120 μl medium containing 50% Steady-glo (promega) at room temperature for 5 min.
T284 39476-39639 Sentence denotes The transduction efficiency was measured by quantification of the luciferase activity using a Modulus II microplate reader (Turner Biosystems, Sunnyvale, CA, USA).
T285 39640-39718 Sentence denotes All experiments were done in triplicates, and repeated at least twice or more.
T286 39720-39772 Sentence denotes Detection of S protein of SARS-CoV-2 by western blot
T287 39773-39876 Sentence denotes The spike glycoprotein of SARS-CoV-2 in cells and on pseudovirions were detected by using western blot.
T288 39877-40139 Sentence denotes Briefly, cells transfected with plasmids encoding spike glycoprotein of SARS-CoV-2, SARS-CoV, MERS-CoV, and MHV were lysed at 40 h post transfection by RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP40, 1×protease inhibitor cocktail).
T289 40140-40353 Sentence denotes To pellet down pseudovirions, the viral supernatants were centrifuged at 25,000 rpm for 2 h in a Beckman SW41 rotor at 4 °C through a 20% sucrose cushion, and virus pellets were resuspended into 30 μl RIPA buffer.
T290 40354-40476 Sentence denotes The samples were boiled for 10 min and separated in a 10% SDS-PAGE gel and transferred to nitrocellulose filter membranes.
T291 40477-40707 Sentence denotes After blocked by 5% milk, the membranes were blotted with primary antibodies, followed by incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (1:5000) and visualized with Chemiluminescent Reagent (Bio-Rad).
T292 40708-41227 Sentence denotes MHV S proteins were detected using polyclonal goat anti-MHV S antibody AO4 (1:2000); SARS-CoV S proteins were blotted with either polyclonal anti-SARS S1 antibodies T62 (1:2000) (Sinobiological Inc, Beijing, China) or mouse monoclonal against SARS S1 antibody MM02 (1:1000) (Sinobiological Inc, Beijing, China), MERS-CoV and SARS-CoV-2 S proteins were detected using mouse monoclonal anti-MERS S (1:1000) (Sinobiological Inc, Beijing, China) and anti-FLAG M2 antibody (1:1000) (Sigma, St. Louis, MO, USA), respectively.
T293 41229-41283 Sentence denotes Effects of agents and inhibitors on pseudovirion entry
T294 41284-41721 Sentence denotes HEK 293/hACE2 cells were pretreated with either lysosomotropic agents (endosome acidification inhibitor NH4Cl and bafilomycin A; PIKfyve inhibitor apilimod and YM201636; calcium channel blocker tetrandrine) or cathepsin inhibitors (cathepsin L inhibitor SID26881509, cathepsin B inhibitor CA-074, and cathepsin inhibitor E64D) for 1 h at 37 °C, then spin-inoculated with pseudovirions in the presence of inhibitor at 1200 × g for 30 min.
T295 41722-41801 Sentence denotes After overnight incubation, cells were fed with fresh medium without inhibitor.
T296 41802-41887 Sentence denotes Cells were lysed at 48 h post inoculation and their luciferase activity was measured.
T297 41889-41937 Sentence denotes Flow cytometric analysis of S protein expression
T298 41938-42070 Sentence denotes Briefly, 293T cells were transfected with 2 μg of plasmids encoding either SARS-CoV-2 S, SARS-CoV S or MERS-CoV S protein using PEI.
T299 42071-42138 Sentence denotes Forty hours later, cells were detached by using PBS with 1 mM EDTA.
T300 42139-42404 Sentence denotes After washing, cells were incubated with polyclonal rabbit anti-SARS S1 antibodies T62 (1:200 dilution) (Sinobiological Inc., Beijing, China) for 1 h on ice, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200) (ZSGB-Bio LLC, Beijing, China) for 1 h.
T301 42405-42448 Sentence denotes Cells were then analyzed by flow cytometry.
T302 42450-42459 Sentence denotes MTT assay
T303 42460-42504 Sentence denotes MTT assay was used to assess cell viability.
T304 42505-42684 Sentence denotes Briefly, cells were seeded into a 96-well plate at a cell density of 3000 per well and allowed to adhere for 24 h, followed by treatment with serially diluted inhibitors for 12 h.
T305 42685-42782 Sentence denotes Two microliters of 5 mg/ml MTT was added to the medium 24 h later and incubated for 4 h at 37 °C.
T306 42783-42843 Sentence denotes After removing the culture medium, 100 µl of DMSO was added.
T307 42844-42953 Sentence denotes The absorbance was measured at 570 nm using a microplate spectrophotometer (Multiskan FC, Thermo Scientific).
T308 42954-43106 Sentence denotes All experiments were performed in triplicate, and the cell viability of cells was calculated as the ratio of each experimental condition to the control.
T309 43108-43130 Sentence denotes Cell–cell fusion assay
T310 43131-43217 Sentence denotes HEK-293T cells were co-transfected with plasmids encoding CoV S glycoprotein and eGFP.
T311 43218-43472 Sentence denotes For trypsin-dependent cell–cell fusion, cells were detached with trypsin (0.25%) at 40 h post transfection and overlaid on an 80% confluent monolayer of 293/hACE2 cells at a ratio of approximately one S-expressing cell to three receptor-expressing cells.
T312 43473-43582 Sentence denotes For trypsin-independent cell–cell fusion, cells were detached with 1 mM EDTA and overlaid on 293/hACE2 cells.
T313 43583-43732 Sentence denotes After 4 h incubation, images of syncytia were captured with a Nikon TE2000 epifluorescence microscope running MetaMorph software (Molecular Devices).
T314 43733-43807 Sentence denotes All experiments were done in triplicate and repeated at least three times.
T315 43809-43858 Sentence denotes Expression and purification of soluble human ACE2
T316 43859-44042 Sentence denotes DNA fragments encoding residues 19-615 of human hACE2 with N-terminal FLAG and 6×his tags were cloned between Sal I and Hind III of modified pFASTBac1 vector with gp67 signal peptide.
T317 44043-44214 Sentence denotes The soluble receptors were expressed in High Five insect cells using the bac-to-bac system (Invitrogen) and purified using nickel affinity and ion-exchange chromatography.
T318 44216-44243 Sentence denotes Soluble hACE2-binding assay
T319 44244-44347 Sentence denotes HEK-293T cells were transfected with plasmids encoding SARS-CoV-2 S, SARS-CoV S, or MERS-CoV S protein.
T320 44348-44498 Sentence denotes Cells were detached with 1 mM EDTA 40 h post transfection, washed twice with 3% FBS in 1×PBS, and incubated with 5 μg/ml soluble hACE2 for 1 h on ice.
T321 44499-44760 Sentence denotes After washing three times with 3% FBS in 1×PBS, cells were incubated with polyclonal goat anti-human ACE2 antibody (1:200) (R&D Systems, MN, USA) for 1 h, followed by FITC-conjugated rabbit anti-goat secondary antibody (1:500) (Jackson ImmunoResearch, PA, USA).
T322 44761-44819 Sentence denotes After washing, cells were then analyzed by flow cytometry.
T323 44821-44851 Sentence denotes Soluble hACE2 inhibition assay
T324 44852-44995 Sentence denotes Briefly, lentiviruses pseudotyped with SARS-CoV-2 S, SARS-CoV S or VSV-G were pre-incubated with serially diluted soluble hACE2 for 1 h on ice.
T325 44996-45111 Sentence denotes The mixture were then added on 293/hACE2 cells, followed by centrifugation inoculation for 1 h at room temperature.
T326 45112-45190 Sentence denotes Cells were fed with fresh medium 6 h later and lysed at 48 h post inoculation.
T327 45191-45264 Sentence denotes Pseudoviral transduction was measured according to luciferase activities.
T328 45266-45298 Sentence denotes Pseudovirus neutralization assay
T329 45299-45553 Sentence denotes SARS-CoV S, SARS-CoV-2 S, and VSV-G pseudovirions were pre-incubated with serially diluted either polyclonal rabbit anti-SARS S1 antibodies T62 or patient sera for 1 h on ice, then virus-antibody mixture was added onto 293/hACE2 cells in a 96-well plate.
T330 45554-45620 Sentence denotes After 6 h incubation, the inoculum was replaced with fresh medium.
T331 45621-45715 Sentence denotes Cells were lysed 40 h later and pseudovirus transduction was measured as previously described.
T332 45716-45811 Sentence denotes Prior to experiments, patient sera were incubated at 56 °C for 30 min to inactivate complement.
T333 45813-45829 Sentence denotes Serum collection
T334 45830-46054 Sentence denotes Five patients were hospitalized with pneumonia in Hengshui Third People’s Hospital, Hengshui, Heibei province, China, and their respiratory specimens were collected for coronavirus detection and were positive for SARS-CoV-2.
T335 46055-46156 Sentence denotes After all patients recovered, their sera were collected right before discharge with informed consent.
T336 46157-46262 Sentence denotes Serum from a recovered SARS patient was also collected at two years after recovery with informed consent.
T337 46264-46281 Sentence denotes Reporting summary
T338 46282-46398 Sentence denotes Further information on research design is available in the Nature Research Reporting Summary linked to this article.
T339 46400-46425 Sentence denotes Supplementary information
T340 46427-46452 Sentence denotes Supplementary Information
T341 46453-46470 Sentence denotes Reporting Summary
T342 46472-46483 Sentence denotes Source Data
T343 46484-46659 Sentence denotes Source Data Peer review information Nature Communications thanks Thomas Gallagher and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
T344 46660-46795 Sentence denotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
T345 46796-46830 Sentence denotes These authors contributed equally:
T346 46831-46863 Sentence denotes Xiuyuan Ou, Yan Liu, Xiaobo Lei.
T347 46865-46890 Sentence denotes Supplementary information
T348 46891-46975 Sentence denotes Supplementary information is available for this paper at 10.1038/s41467-020-15562-9.
T349 46977-46993 Sentence denotes Acknowledgements
T350 46994-47380 Sentence denotes This work was supported by grants from Chinese Science and Technology Key Projects (2020YFC0841000), National Natural Science Foundation of China (31670164 and 31970171 to Z.Q., and 81930063 to J.W.), Chinese Science and Technology Key Projects (2017ZX0103004), COVID-19 Emergency Fund from CAMS (2020HY320001), and the CAMS Innovation Fund for Medical Sciences (2016-12M-1-014 to J.W.)
T351 47382-47402 Sentence denotes Author contributions
T352 47403-47487 Sentence denotes Z.Q. and J.W. conceived the projects, Z.Q., J.W., and Q.J. coordinated the projects.
T353 47488-47589 Sentence denotes Z.Q., J.W., and Q.J. designed the experiments with the help of X.O., Y.L., X.L., L.R., L.G., and Z.X.
T354 47590-47636 Sentence denotes X.O., Y.L., X.L., P.L., D.M., R.G., T.C., J.H.
T355 47637-47683 Sentence denotes Z.M., and X.C., performed all the experiments.
T356 47684-47786 Sentence denotes Z.Q., J.W., and Q.J. analyzed the data with help of X.O., Y.L., X.L., L.R., L.G., Z.X., J.C., and K.H.
T357 47787-47850 Sentence denotes Z.Q., and J.W. wrote the manuscript with help of other authors.
T358 47852-47869 Sentence denotes Data availability
T359 47870-47961 Sentence denotes The data supporting the findings of this study are available from the authors upon request.
T360 47962-48121 Sentence denotes The source data underlying Figs. 1b–j, 2a, b, d, 3a–f, 4a, c–e, 5b, e and 6a, b and Table 1 and Supplementary Figs. 1 and 3 are provided as a Source Data file.
T361 48123-48142 Sentence denotes Competing interests
T362 48143-48186 Sentence denotes The authors declare no competing interests.