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Design of customized coronavirus receptors

Nature volume 635, pages 978–986 (2024)Cite this article

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Abstract

Although coronaviruses use diverse receptors, the characterization of coronaviruses with unknown receptors has been impeded by a lack of infection models1,2. Here we introduce a strategy to engineer functional customized viral receptors (CVRs). The modular design relies on building artificial receptor scaffolds comprising various modules and generating specific virus-binding domains. We identify key factors for CVRs to functionally mimic native receptors by facilitating spike proteolytic cleavage, membrane fusion, pseudovirus entry and propagation for various coronaviruses. We delineate functional SARS-CoV-2 spike receptor-binding sites for CVR design and reveal the mechanism of cell entry promoted by the N-terminal domain-targeting S2L20-CVR. We generated CVR-expressing cells for 12 representative coronaviruses from 6 subgenera, most of which lack known receptors, and show that a pan-sarbecovirus CVR supports propagation of a propagation-competent HKU3 pseudovirus and of authentic RsHuB2019A3. Using an HKU5-specific CVR, we successfully rescued wild-type and ZsGreen-HiBiT-incorporated HKU5-1 (LMH03f) and isolated a HKU5 strain from bat samples. Our study demonstrates the potential of the CVR strategy for establishing native receptor-independent infection models, providing a tool for studying viruses that lack known susceptible target cells.

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Fig. 1: Modular design of CVRs for efficient coronavirus entry.
Fig. 2: Effects of binding epitopes on receptor function.
Fig. 3: NTD-mediated sarbecovirus entry promoted by S2L20-CVR.
Fig. 4: CVR-supported entry and amplification of various coronaviruses.

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Data availability

The cryo-EM maps and models have been deposited to the Electron Microscopy Data Bank and Protein Data Bank (PDB) with accession numbers EMD-45174 and 9C44 (global refinement of the S2L20-bound spike trimer), and EMD-45175 and 9C45 (local refinement of the NTD and S2L20 Fab variable regions). The accession numbers (NCBI GenBank or GISAID) and protein sequence information of receptor, virus, antibodies, domains and reporter genes are provided in the Methods and supplementary tables. All other data supporting the findings of this study are available with the Article and the Supplementary Information. All reagents generated in this study are available from H.Y. with a completed materials transfer agreement. Source data are provided with this paper.

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Acknowledgements

The authors are grateful for the funding support from the Basic Science Center Program of the National Natural Science Foundation of China (NSFC) (32188101 to H.Y.), National Key R&D Program of China (2023YFC2605500 to Z.-L.S. and H.Y.), NSFC Excellent Young Scientists Fund (82322041 to H.Y.), other NSFC projects (32270164, 32070160 to H.Y., 323B2006 to C.-B.M. and 32300141 to H.G.), Fundamental Research Funds for the Central Universities (2042023kf0191 and 2042022kf1188 to H.Y.), and Natural Science Foundation of Hubei Province (2023AFA015 to H.Y.), and China Postdoctoral Science Foundation (2023M733708 to H.G.). This study was also supported by the National Institute of Allergy and Infectious Diseases (P01AI167966, DP1AI158186 and 75N93022C00036 to D.V.), an Investigators in the Pathogenesis of Infectious Disease Awards from the Burroughs Wellcome Fund (D.V.), the University of Washington Arnold and Mabel Beckman CryoEM center and the National Institute of Health grant S10OD032290 (to D.V.). D.V. is an Investigator of the Howard Hughes Medical Institute and the Hans Neurath Endowed Chair in Biochemistry at the University of Washington. The authors thank X.-X. Wang for providing the human sera collected post-vaccination (SARS-CoV-2 CoronaVac, Sinovac) and collected by Beijing Youan Hospital (approval number LL-2021-042-K); K. Cai for providing sera collected from Wuhan COVID-19 convalescents (identification number 2021-012-01); Z.-H. Qian for providing BANAL-20-52 related spike-expressing plasmids; L. Lu for providing EK1-related peptides; P. Zhang and B.-C. Xu for their help with the ultracentrifugation and electron microscopic analysis; Y.-J. Li for assisting in establishing the yeast cloning methods; Y. Chen for providing the MHV-A59 strain; C.-G Wu for assissting in isolating the HKU5_PaGD2014/15 stain; Q. Ding for providing the SARS-CoV-2 (ΔN-GFP) reverse genetics related plasmids; and K. Lan and the ABSL-3 facility and other core facilities of the Key Laboratory of Virology, Wuhan University.

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Author notes

  1. These authors contributed equally: Peng Liu, Mei-Ling Huang, Hua Guo, Matthew McCallum

Authors and Affiliations

  1. State Key Laboratory of Virology, College of Life Sciences, TaiKang Center for Life and Medical Sciences, Wuhan University, Wuhan, China

    Peng Liu, Mei-Ling Huang, Jun-Yu Si, Yuan-Mei Chen, Chun-Li Wang, Xiao Yu, Lu-Lu Shi, Qing Xiong, Cheng-Bao Ma, Fei Tong, Chen Liu, Ye-hui Sun, Xiao Yang, Ming Guo, Jing Li & Huan Yan

  2. Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China

    Hua Guo, Jing Chen & Zheng-Li Shi

  3. Department of Biochemistry, University of Washington, Seattle, WA, USA

    Matthew McCallum, John E. Bowen & David Veesler

  4. Humabs BioMed SA, subsidiary of Vir Biotechnology, Bellinzona, Switzerland

    Davide Corti

  5. Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA

    David Veesler

  6. Guangzhou Laboratory, Guangzhou International Bio Island, Guangzhou, China

    Zheng-Li Shi

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Contributions

H.Y., P.L., M.-L.H., H.G. and M.M. conceived the study and designed the experiments. J.L. conducted the first experiment of this study. P.L., M.-L.H., H.G., M.M., J.-Y.S., Y.-M.C., C.-L.W., X. Yu, C.L., L.-L.S., Y.-h.S., X. Yang and J.L. established the assays and methods, designed and cloned receptor and protein constructs and conducted related experiments. P.L., M.-L.H., C.-L.W., L.-L.S., C.-B.M., Q.X., F.T. and C.L. screened the nanobodies. P.L., M.-L.H. and L.-L.S. conducted 229E and MHV authentic virus-related experiments at Wuhan University. P.L. and H.G. conducted the RsHuB2019A and HKU5 authentic virus and HKU5 negative staining for electron microscopy imaging at the Wuhan Institute of Virology. J.C. and P.L. constructed the infectious clones of HKU5 and HKU5-ZGH, respectively. M.G. conducted the SARS-CoV-2 authentic virus infection-related experiments in ABSL-III at Wuhan University. M.M. carried out cryo-EM sample preparation, data collection and processing of the S2L20-bound SARS-CoV-2 spike dataset. M.M. and D.V. built and refined the structure. M.M. and J.E.B. carried out antibody-mediated triggering assays. D.C. provided critical reagents. H.Y., Z.-L.S., D.V., P.L., M.-L.H., M.M., C.-L.W. and X. Yu analysed the data. H.Y., P.L. and M.-L.H. prepared the original draft of the manuscript. H.Y., D.V., Z.-L.S., P.L. and M.-L.H. revised the manuscript with input from all other authors. H.Y., Z.-L.S. and D.V. obtained funding to support this study. H.Y. supervised the project.

Corresponding authors

Correspondence to David Veesler, Zheng-Li Shi or Huan Yan.

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Competing interests

H.Y. has submitted a patent application to the China National Intellectual Property Administration for the utilization of artificial viral receptors and their applications.

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Nature thanks Shibo Jiang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Development and optimization of a modular design strategy for CVR with a type I transmembrane topology.

a, Schematic illustration of the four miniprotein-based CVRs. b, Immunofluorescence analysis of ACE2/CVRs expression and authentic SARS-CoV-2 infection in HEK293T cells stably expressing the receptors. Upper: Receptor expression examined by C-terminal fused 3×Flag tags. Lower: SARS-CoV-2 infection efficiency as indicated by intracellular N proteins at 24 hpi. Data representative of two independent authentic SARS-CoV-2 infection assays with similar results. c, Cartoon illustrating the framework of the CVRs for TM evaluation. d, Immunofluorescence analysis of the expression of the 31 CVRs in HEK293T cells by detecting the C-terminal fused 3×Flag tags. e, SARS-CoV-2 PSV entry efficiency promoted by CVRs carrying different TMs. The detailed information on the TMs is summarized in Supplementary Table 2. Data are presented as mean ± s.d. (n = 3 wells of independently infected cells), representative of two independent experiments with similar results. f, Schematic diagram showing the LCB1-based CVRs with indicated TM or TMC substitutions. g, h, SARS-CoV-2 PSV entry in HEK293T cells transiently expressing the indicated CVRs examined by RLU (g) or GFP (h). Data are presented as mean ± s.d. (n = 3 wells of independently infected cells), Statistical analysis was performed using One-way ANOVA analysis followed by Dunnett’s test. Data represented were performed in at least two independent experiments with similar results. i, Immunofluorescence analysis of the subcellular distribution of LCB1-MXRA8 TMC-based CVRs with or without EPM transiently expressed in HEK293T cells. The white dashed boxes highlight the cell surface distribution with a higher magnification. j, SARS-CoV-2 PSV entry efficiency in HEK293T cells transiently expressing CVRs with or without EPM. Data are mean ± s.d. (n = 3 wells of independently infected cells) and analyzed with unpaired two-tailed Student’s t-tests, representative of two independent experiments with similar results. Scale bars: 100 μm in b, d, h, and i. *P < 0.05, **P < 0.01, ****P < 0.0001.

Source Data

Extended Data Fig. 2 Exploring factors that contribute to the receptor function of CVRs with different topologies or modules.

a, Schematic diagram showing CVRs carrying LCB1 or mNb1 displayed in either type I or type II transmembrane topology. b, Evaluation of SARS-CoV-2 or MERS-CoV PSV entry efficiency supported by the indicated CVRs with different transmembrane topologies in HEK293T cells. Data are mean ± s.d. of biological triplicates examined over three independent infection assays. Unpaired two-tailed Student’s t-tests. c, Assessment of CVR expression, SARS-CoV-2 RBD-mFc binding, and PSV entry efficiency supported by the CVRs carrying varying copies of TR23 repeats transiently expressed in HEK293T cells. Data are representative of three independent experiments. Scale bars: 100 μm. d, Schematic representation of the CVRs carrying different numbers of immunoglobulin (Ig) domains (left) or an Fc mutant with abolished dimerization ability. e, Western blot analysis of CVRs expression in HEK293T cells under either reducing or non-reducing conditions, respectively. f, Assessment of SARS-CoV-2 PSV entry efficiency in HEK293T cells transiently expressing the indicated CVRs. Data are mean ± s.d. (n = 3 wells of independently infected cells.) and analyzed by unpaired two-tailed Student’s t-tests. g, Schematic representation of the CVRs carrying different numbers of Ig-like domains (left) from mCEACAM1a. h, Western blot analysis of CVRs expression in HEK293T cells. i, SARS-CoV-2 PSV entry efficiency in HEK293T cells transiently expressing the indicated CVRs. Data are mean ± s.d. (n = 3 wells of independently infected cells). One-way ANOVA analysis followed by Dunnett’s test. j, Schematic representation of the CVRs carrying different SARS-CoV-2 RBD targeting miniproteins. k, l, Expression (k) and SARS-CoV-2 entry-supporting (l) ability of different CVRs in 293T cells. Data are mean ± s.d. (n = 3 wells of independently infected cells). One-way ANOVA analysis followed by Dunnett’s test. m, Schematic diagram showing CVRs carrying different types of VBDs, the two representative VBDs for each type are indicated. n, Immunofluorescence analyzing the expression of the indicated CVRs transiently expressed in HEK293T cells by detecting the C-terminal fused 3×Flag tags, and the SARS-CoV-2 RBD binding. Scale bars: 100 μm. o, PSV entry are supported by indicated CVRs transiently expressed in HEK293T cells. Data are mean ± s.d. (n = 3 wells of independently infected cells). Data representative of two independent transfections, expression verification, and infection assays with similar results for d-f, g-i, j-l, and m-o, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, Not significant (P > 0.05).

Source Data

Extended Data Fig. 3 Investigating receptor function of CVRs with VBDs connected in various ways.

a-c, Illustration (a), RBD binding efficiency (b), and PSV entry-supporting efficiency (c) of a SARS-CoV-2/MERS-CoV bi-specific CVR transiently expressed in HEK293T cells. Data are presented as mean ± s.d. (biological triplicates of infected cells), representative of three independent experiments with similar results. Unpaired two-tailed Student’s t-test. d-f, Illustration (d), expression (e), and PSV entry-supporting efficiencies (f) of CVRs carrying single or trimeric VBD. Data are mean ± s.d. (biological triplicates of infected cells), analyzed by unpaired two-tailed Student’s t-test. Representative of two independent experiments. g-i, Schematic illustration of bispecific adapter protein (g) and MERS-CoV PSV entry efficiency in BHK-21-hACE2 cells in the presence of indicated concentrations of adapter proteins (h11B11-mNb1) throughout the infection. Entry efficiency is examined by GFP intensity (h) or RLU (i). Data are mean ± s.d. (biological triplicates of infected cells), representative of three independent infection assays. j-l, Schematic illustration of FcγR (CD32a) mediated antibody-dependent coronavirus entry (j). CD32a expression, antibody (CB6) binding (k), and SARS-CoV-2 PSV entry (l) into HEK293T-CD32a cells pretreated with indicated concentration (con.) of the CB6 antibodies. Data are mean ± s.d. (n = 4 wells of biologically independent cells), examined over two independent experiments. m-o, Entry of pre-attached PSV promoted by soluble neutralizing antibody (Nb27-hFc) or bi-specific neutralizing antibody with membrane-associating ability (Nb27-hFc-h11B11). Schematic illustration (m), Nb27-hFc promoted entry (n), and Nb27-hFc-h11B11 promoted PSV entry (o) in Caco2 cells with virus pre-attachment by 1500 rpm centrifugation at 4 °C for 1 h. Data are mean ± s.d. (biological triplicates of infected cells). Data are representative of three independent infection assays. Scale bars: 100 μm for b, e, and k, and 200 μm for h. One-way ANOVA analysis followed by Dunnett’s test for i, l, n, and o. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, Not significant (P > 0.05).

Source Data

Extended Data Fig. 4 Comparing receptor function of CVRs with native receptors, or alternative receptors/coreceptors in different cell types.

a-c, The ability of CVRs to promote cell-cell membrane fusion, and authentic SARS-CoV-2 infection is comparable to their native receptors. Spike and receptor-mediated cell-cell fusion was demonstrated by reconstituted GFP intensity (a) and relative light unit (RLU) of Renilla luciferase activity (b). Authentic SARS-CoV-2 infection was examined by immunostaining of intracellular N proteins at 24 hpi (c). Data are mean ± s.d. (biological triplicates) for b. Data are representative of two independent fusion assays or infection assays with similar results. d, Receptor specificity of different coronavirus PSVs in HEK293T stably expressing the native receptor or the indicated CVRs. e, SARS-CoV-2 and MERS-CoV PSV entry into various cell types expressing the indicated receptors. Data representative of two independent transfection and infection assays with similar results for d and e. f, SARS-CoV-2 PSV entry efficiencies in HEK293T cells expressing different receptors or entry factors. Data are presented as mean ± s.d. (n = 3 wells of independently infected cells.), representative of two independent transfection and infection experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, Not significant (P > 0.05).

Source Data

Extended Data Fig. 5 Relationship between the binding affinity, neutralizing activity, and CVR entry-promoting efficiency of 25 SARS-CoV-2 RBD-targeting nanobody-fused receptors.

a, b, Assessment of the entry-promoting ability of 25 nanobody-CVRs in HEK293T cells, indicated by GFP (a) and the RLU (b), respectively. Data are presented as mean ± s.d. (n = 3 wells of independently infected cells), representative of two independent experiments. One-way ANOVA analysis followed by Dunnett’s test. Scale bars: 200 μm. c, Comparing RBD binding, neutralization, and PSV entry-promoting ability of different nanobody-fused proteins in HEK293T cells. RBD-mFc binding and PSV entry assays were conducted in HEK293T transiently expressing the 25 nanobody-CVRs. The SARS-CoV-2 PSV neutralization assay was performed in HEK293T-ACE2 in the presence of indicated nanobody-Fc recombinant proteins (10 μg ml−1). Data are representative results of two independent experiments with similar results and plotted by the mean (n = 3 wells of independently infected/bound cells). ***P < 0.001, ****P < 0.0001; NS, Not significant (P > 0.05).

Source Data

Extended Data Fig. 6 Expression and antigen-binding ability of CVRs targeting distinct SARS-CoV-2 neutralizing epitopes.

a, Western blot analysis of the expression levels of indicated scFv-CVRs transiently expressed in HEK293T cells. Data are representative of two independent experiments. b, Binding of SARS-CoV-2 S-trimer to HEK293T cells expressing the indicated CVRs. Data are representative of three assays using independent preparations of proteins. c, Flow cytometry analysis of the binding efficiency of scFv-mFc with HEK293T cells transiently expressing the SARS-CoV-2 Spike proteins and ZsGreen simultaneously. The ZsGreen positive cells were gated for subsequent analysis of mFc binding efficiency. Data representative from a single experiment with mean values (n = 3 wells of biologically independent cells) indicated. d, Trypsin-mediated S2′ cleavage of SARS2-CoV-2 PSV in the presence of soluble receptors or CB6-scFv-mFc. The concentrated SARS-CoV-2 PSV particles were incubated with 100 μg ml−1 of soluble receptors or CB6-scFv-mFc for 1 h, followed by incubation with the indicated concentration of TPCK-treated trypsin for 30 min. Western blot analysis was conducted by detecting the S2P6 epitope in the S2 subunit. Data are representative of three independent assays with similar results.

Extended Data Fig. 7 Molecular basis of NTD-mediated coronavirus entry.

a-c, Package efficiency of PSVs carrying indicated sarbecoviruses spike glycoproteins (a) and the indicated mutants (b, c). Western blot was conducted by detecting the conserved S2P6 epitope. VSV-M serves as a loading control. Blots representative of two independent transfection assays for pseudovirus production. d, Structures of SARS-CoV-2 BA.4/5 spike trimer without antibody binding (left), or in complex with S2L20 (right). Dashed boxes highlighted the N370-glycan spatially proximate to the S2L20. e, Heatmap showing the inhibitory efficacy of indicated SARS-CoV-2 neutralizing antibodies against PSV entry in HEK293T-hACE2 or HEK293T-S2L20, with BSA as a control. Data are representative results of two independent neutralization assays and plotted by the mean (n = 3 wells of independently infected cells). f, Structures of SARS-CoV-2 BA.2 spike trimers with (upper) or without (lower) the binding of NTD-targeting 4A8, along with the side-view (top) and top-view (bottom) cryoEM structures of SARS-CoV-2 Wuhan-Hu-1 spike trimers with the binding of NTD-targeting CV3-13 and DH1052. Orange: NTD; Green: CTD; Red: S2L20; Magenta: 4A8; Blue: CV3-13; Pink: DH1052. g, CryoEM data processing workflow and validation of the S2L20-bound SARS-CoV-2 S CryoEM structure. h, Negative stain microscopy of prefusion SARS-CoV-2 S-glycoprotein (without stabilizing proline substitutions) incubated without antibody, or with S2H14, S2X28, or S2L20 as indicated. S2H14 is known to promote the transition to the postfusion state and was used as a control37. Scale bars: 10 nm. Data representative of images captured from two independent experiments with similar results. i, Top-view and side-view CryoEM structures depicting soluble mCEACAM1a (cyan) in complex with MHV spike trimer (gray). NTD and CTD of MHV S are indicated in orange and green, respectively.

Source Data

Extended Data Fig. 8 Generation and characterization of VBDs used for CVRs customized for various coronaviruses.

a, Workflow demonstrating the customization of nanobody-based CVRs for specific coronaviruses. b, Coronavirus CTD or S1 binding in HEK293T cells transiently expressing the corresponding CVRs. Dashed lines indicate thresholds for positive ratio calculation. Data are presented as mean ± s.d. (n = 3 wells of biologically independent cells), representative of two independent experiments. c, The pan-sarbecovirus entry-promoting ability of CVR-Nb27 was evaluated by six different sarbecoviruses in 293T cells. Data are presented as mean ± s.d. (biological quadruples of infected cells), representative of two independent infection assays. Unpaired two-tailed Student’s t-tests. d, BLI analyses of binding kinetics of immobilized RBD and S1-hFc (for 229E) of the indicated coronaviruses with the indicated monomeric nanobodies. e, a summary of binding kinetics of nanobodies bound to the immobilized virus antigens. f, g, Expression (f) and entry-supporting efficiency (g) of the CVRs with or without EPM transiently expressed in the HEK293T cells. EPM: endocytosis prevention motif. Data are mean ± s.d. (n = 3 independently infected cells). Unpaired two-tailed Student’s t-tests. Experiments were performed twice with similar results, and representative data were shown. h-k, HKU1 specific 2D1-CVR exhibited a comparable receptor function to TMPRSS2. The expression (h) and HKU1-PSV entry promoting efficiency (h, i) of the two receptors were examined. The amplification of propagation-competent rVSV-HKU1-GFP in Caco2 cells expressing 2D1-CVR and TMPRSS2 was demonstrated by GFP (j) and RNA accumulation (k). Scale bars: 100 μm for f, h, and j. Data are mean ± s.d. (n = 3 biologically independent wells of cells) analyzed by unpaired two-tailed Student’s t-tests for i. Data presented are RNA copies of two independently infected cells with each point representing the mean of technical duplicates (RT-qPCR) for k. Experiments presented were independently performed twice with similar results for h-j and single time for k. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, Not significant (P > 0.05).

Source Data

Extended Data Fig. 9 Neutralization and inhibition assays based on CVR-expressing HEK293T cells.

a, Comparison of neutralization profiles of sera collected from COVID-19 convalescents (left) or vaccinated individuals (right) based on HEK293T cells expressing ACE2 or two different CVRs. Serum dilution: 1:200. Heatmap plotted by the mean values (biologically triplicates of infected cells), which are representative results out of two independent experiments. b, c, Neutralization assays of several broadly neutralizing antibodies against PSV entry of representative coronaviruses in HEK293T stably expressing the indicated CVRs. Neutralization curves (b) and a summary of IC50 (c) against each virus are shown. The RBD-targeting REGN 19033 (REGN) was employed as a control. /: no inhibition detected. Data are mean ± s.d. (biologically triplicates of infected cells). d, The IC50 of selected entry inhibitors against SARS-CoV-2 PSV entry was determined in both HEK293T-ACE2 or HEK293T-LCB1-CVR cells. Data are mean ± s.d. (biologically triplicates of infected cells). e, Inhibitory efficacy of inhibitors against PSV entry of SARS-CoV-2-D614G, HKU1, HKU3 and HKU5 in HEK293T cells stably expressing the indicated CVRs. Data are mean ± s.d. (biologically triplicates of infected cells), representative of two infection inhibition assays with similar results. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, Not significant (P > 0.05).

Source Data

Extended Data Fig. 10 Characterization of CVR-promoted amplification of replication-competent pcVSV-CoVs or authentic coronaviruses.

a, Genetic organizations and workflow for generating replicable pcVSV-HKU3 or pcVSV-HKU5. b, Successful rescue (P0) and amplification (P1) of pcVSV-HKU3 assisted by VSV-G. Representative images of an experiment that was conducted for a single time. c, d, Trypsin-enhanced cell-cell fusion (c) and VSV-G-independence (d) of pcVSV-HKU3 infection in Caco2-Nb27 (MOI: 0.001). Data are representative of four independent infection assays with similar results. e, f, Accumulation of pcVSV-HKU3 (e) or pcVSV-HKU5 (f) RNA copies in the supernatant at indicated time points. g, pcVSV-HKU5 mediated cell-cell fusion in Caco2 or Caco2-1B4 cells at MOI = 0.1. TPCK-Try + : 20 μg ml−1 TPCK-treated Trypsin in DMEM + 2% FBS. Representative of two independent experiments. h, TCID50 determination assay for RsHuB2019A in Caco2-Nb27 cells by the Red-Muench method. Caco2-Nb27 cells were inoculated with a 10-fold serial dilution of RsHuB2019A containing supernatant (Passage 6). The TCID50 was determined using immunofluorescence to detect the presence of N protein expression of the inoculated cells at 4 dpi. i, Trypsin-dependent amplification of RsHuB2019A in Huh-7 cells. The RsHuB2019A genomic RNA copies in the supernatant collected at indicated time points of infected Huh-7 cells were quantified by RT-qPCR using RdRp-specific primers. Inoculation was conducted at an MOI of 0.0001, with or without trypsin treatment. Try: 100 μg ml−1 Trypsin in DMEM. j, Genetic organizations of the HKU5 (HKU5-1 LMH03f) ΔORF5-ZsGreen-HiBiT (HKU5-ZGH). k-m, Supernatant RNA copies of HKU5-ZGH (k) in Caco2-1B4 cells, ZsGreen-HitBit signal (l), and increase in ZsGreen intensity (P0) (m). Representative of two independent infection assays are shown. Data are mean ± s.d. (n = 3 independently infected wells of cells) for l. n, Isolation of HKU5 (strain PaGD2014/15) from bat samples by Caco2-1B4 cells and its trypsin-dependent propagation. o, Vero E6 and Caco2 cells were infected with HKU5-1 (LMH03f) with or without trypsin treatment. Data are representative of three independent experiments. p, Sequencing results show L76R and K519T mutations in HKU5-1 spikes after ten passages in Caco2-1B4 cells. q, HKU5-1 after ten passages carrying mutations remains unable to infect Caco2 cells without exogenous trypsin treatment. Caco2-1B4 with CVR expression was included as a positive control. r, Efficacy of indicated antiviral reagents against HKU5-1 infection in Caco2-1B4 cells assessed by intracellular N proteins at 48 hpi. s, Efficient inhibition of HKU5 entry by EK1 and EK1C4 peptides in Caco2-1B4. t, Inhibitory effect of selected anti-viral reagents against authentic HKU5-ZGH infection in Caco2-1B4. Inhibitors were coincubated with either the cells or the viruses for 1 h and present in the culture medium during infection. The HiBiT-based luciferase activity was determined at 48 hpi to assess the inhibitory effect of selected anti-viral reagents against the infection of authentic HKU5-ZGH in Caco2-1B4. Data are representative of two independent infection assays for r-t. u, Overview of the protease cleavage sites of selected coronaviruses. The residue responsible for reduced endosomal cysteine protease activity (ECP) is marked in red, numbering based on SARS-CoV-2. The HKU5 infection efficiencies in n, o, q, r, and s were assessed using rabbit polyclonal antibodies targeting the HKU5 N protein (Cy3) at 48 hpi. Data presented are RNA copies of two independently infected cells with each point representing the mean of technical duplicates (RT-qPCR) for e, f, i, and k, representative of two independent experiments. Scale bars: 125 μm for all images.

Source Data

Extended Data Fig. 11 Schematic diagram of the modular design strategy for CVRs and the potential applications of this technique.

a, Workflow outlining the process of creating artificial receptor scaffolds (ARS) and viral binding domains (VBDs) to construct functional customized viral receptors (CVRs) for establishing in vitro and in vivo infection models. b, The crucial role of CVRs in bridging infection models and virus strains, along with the potential applications of this technique in both basic research and applied science.

Extended Data Table 1 CryoEM data collection, processing, and model refinement statistics
Full size table

Supplementary information

Supplementary Figures

Supplementary Figs 1 and 2 present uncropped immunoblots from the Main and Extended Data Figs, respectively; Supplementary Fig. 3 describes gating strategies for flow cytometry analysis.

Reporting Summary

Supplementary Table 1

Virus and receptor gene information.

Supplementary Table 2

Domain sequences for CVR design.

Supplementary Table 3

Information regarding nanobodies used in this study.

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Liu, P., Huang, ML., Guo, H. et al. Design of customized coronavirus receptors. Nature 635, 978–986 (2024). https://doi.org/10.1038/s41586-024-08121-5

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