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TMPRSS2 is a functional receptor for human coronavirus HKU1

Nature volume 624, pages 207–214 (2023)Cite this article

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Abstract

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2,3,4,5,6,7,8,9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell–cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.

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Fig. 1: TMPRSS2 triggers HKU1 spike fusion.
Fig. 2: Effect of WT and mutant TMPRSS2 on HKU1 cell–cell fusion and pseudovirus infection.
Fig. 3: Analysis of HKU1 spike binding to TMPRSS2.
Fig. 4: Anti-TMPRSS2 VHH nanobodies inhibit HKU1 binding to TMPRSS2, cell–cell fusion and pseudovirus infection.
Fig. 5: Live HKU1 virus infection of HBE cells.

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Data availability

Raw sequence data (human reads removed) for the HKU1 virus were deposited in the European Nucleotide Archive portal (https://www.ebi.ac.uk/ena/) under bioproject accession number PRJEB64017. The raw data of the main figures are available in the Supplementary Information. The raw data of the Extended Data are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank A. Amara, T. Bruel and N. Casartelli for critical reading of the manuscript, members of the Virus and Immunity Unit for discussions and help, N. Aulner, N. Mahtal and the UtechS Photonic BioImaging core facility (Institut Pasteur), P. Charneau for help in lentiviral pseudotype preparation and S. Pöhlmann for the kind gift of plasmids. N.S. is funded by the Ministère de l’Enseignement supérieur et de la Recherche. O.S.’s laboratory is funded by Institut Pasteur, Fondation pour la Recherche Médicale, the National Agency for AIDS Research–Emerging Infectious Diseases, the Vaccine Research Institute (ANR-10-LABX-77), Humanities in the European Research Area programme (DURABLE consortium), Labex Integrative Biology of Emerging Infectious Diseases (IBEID) (ANR-10-LABX-62-IBEID), Agence Nationale de la Recherche (ANR)/Fondation pour la Recherche Médicale Flash Covid PROTEO-SARS-CoV-2 and IDISCOVR. E.S.-L.’s lab is funded by the INCEPTION programme (Investissements d’Avenir grant no. ANR-16-CONV-0005), the National Institutes of Health Pasteur International Center for Research on Emerging Infectious Diseases programme (award no. U01AI151758), the Health Emergency Preparedness and Response European programme DURABLE (101102733) and the Labex IBEID (ANR-10-LABX-62-IBEID). H.M.’s laboratory is funded by the Institut Pasteur, the Milieu Intérieur Programme (ANR-10-LABX-69-01) and L'Institut national de la santé et de la recherche médicale, REACTing and European Union (Rapid European COVID-19 Emergency Response (RECOVER)) grants. M.M.R. was supported by the Pasteur-Paris University International Doctoral Programme and the Institut Pasteur Department of Virology ‘Bourse de Soudure’ fellowship. F.R.’s laboratory is funded by ANR grant nos. ANR-13-ISV8-0002-01 and ANR-10-LABX-62-10 IBEID, Wellcome Trust collaborative grant no. UNS22082 and the Institut Pasteur and the Centre national de la recherche scientifique. Work with UtechS Photonic BioImaging is funded by grant no. ANR-10-INSB-04-01 and Région Ile-de-France programme DIM1-Health. The funders of this study had no role in study design, data collection, analysis and interpretation or writing of this Article.

Author information

Author notes

  1. These authors contributed equally: Julian Buchrieser, Olivier Schwartz

Authors and Affiliations

  1. Virus & Immunity Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France

    Nell Saunders, Maaran Michael Rajah, Jeanne Postal, Francoise Porrot, Florence Guivel-Benhassine, Augustin Martin, Ludivine Grzelak, Hunter Hoover-Watson, Julian Buchrieser & Olivier Schwartz

  2. Structural Virology Unit, Institut Pasteur, Université de Paris Cité, CNRS UMR 3569, Paris, France

    Ignacio Fernandez, Rischa Maya Oktavia, Annalisa Meola, Olivia Ahouzi & Felix Rey

  3. Humoral Immunology Unit, Institut Pasteur, Université de Paris Cité, INSERM U1222, Paris, France

    Cyril Planchais & Hugo Mouquet

  4. Pathogenesis of Vascular Infections Unit, Institut Pasteur, INSERM, Paris, France

    Vincent Michel

  5. Nanoimaging core, Institut Pasteur, Université de Paris Cité, INSERM U1222, Paris, France

    Eduard Baquero Salazar

  6. Pasteur-TheraVectys Joint Lab, Institut Pasteur, Université de Paris Cité, Paris, France

    Catherine Blanc

  7. Antibody Engineering Platform, C2RT, Institut Pasteur, Université de Paris Cité, CNRS UMR 3528, Paris, France

    Gaëlle Chauveau-Le Friec, Véronique Meriaux & Pierre Lafaye

  8. G5 Evolutionary Genomics of RNA Viruses, Institut Pasteur, Paris, France

    Matthieu Prot, Deborah Delaune & Etienne Simon-Lorière

  9. Institut de Recherche Biomédicale des Armées, Brétigny-sur-Orge, France

    Deborah Delaune

  10. Department of Medical Microbiology and Infection Prevention, Amsterdam UMC, Molecular Diagnostic Unit, University of Amsterdam, Amsterdam, The Netherlands

    Marion Cornelissen

  11. Amsterdam Institute for Infection and Immunity, Amsterdam, The Netherlands

    Marion Cornelissen, Martin Deijs & Lia van der Hoek

  12. Department of Medical Microbiology and Infection Prevention, Amsterdam UMC, Laboratory of Experimental Virology, University of Amsterdam, Amsterdam, The Netherlands

    Martin Deijs & Lia van der Hoek

  13. National Reference Center for viruses of respiratory infections, Institut Pasteur, Paris, France

    Etienne Simon-Lorière

  14. Vaccine Research Institute, Creteil, France

    Olivier Schwartz

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  1. Nell Saunders
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Contributions

The experimental strategy was designed by N.S., I.F., C.P., M.M.R., H.M., F.R., J.B. and O.S. Data acquisition and analysis were performed by N.S., I.F., C.P., V.M., M.M.R., E.B.S., F.P., F.G.-B., J.P., C.B., A. Martin, L.G., R.M.O., A. Meola, O.A., H.H.-W., M.P., D.D., E.S.-L. and J.B. Vital materials and expertise were provided by G.C.-L.F., M.C., M.D., V.M., L.V.D.H. and P.L. The manuscript was written by N.S., J.B. and O.S. and edited by N.S., I.F., C.P., E.B.S., H.M., F.R., P.L., J.B. and O.S. All authors read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Julian Buchrieser or Olivier Schwartz.

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Competing interests

N.S., J.B., O.S., I.F., E.B.S., F.R. and P.L. have a provisional patent on anti-TMPRSS2 nanobodies. The other authors declare no competing interests.

Peer review

Peer review information

Nature thanks Zhengli Shi, Hyeryun Choe 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 a. Binding of the 5 anti-TMPRSS2 VHH on recombinant TMPRSS2 measured by BLI. b, c. Comparison of one commercial TMPRSS2 antibody and of the dimeric anti-TMPRSS2 VHH A01-Fc.

293 T cells were transfected with WT TMPRSS2. 24 h later, cells were analysed by flow-cytometry. Staining with the commercial antibody was performed on fixed cells (b) while surface staining with the VHH was performed on live cells (c).

Extended Data Fig. 2 Effect of TMPRSS2 and other proteases on HKU1 cell-cell fusion. a, b. Surface expression and fusogenic activity of HKU1 spikes.

a. 293 T cells were transfected with plasmids encoding for HKU1A or HKU1B spikes and stained 24 h later with mAb10, a pan-anti-coronavirus spike antibody. Data are representative of 3 independent experiments. b. Cell-cell fusion mediated by HKU1A or HKU1B spikes. 293 T GFP-Split cells were transfected with HKU1 spikes and TMPRSS2, fusion was visualized by the appearance of GFP+ cells. Data are representative of 6 independent experiments. Scale bar: 400 μm c. Cell-cell fusion mediated by the SARS-CoV-2 spike. 293 T GFP-Split cells were transfected with SARS-CoV-2 spike, in the presence of ACE2 or TMPRSS2, fusion was visualized by the appearance of GFP+ cells. Data are mean ± SD of 3 independent experiments d. U2OS cell-cell fusion mediated by HKU1A spike. U2OS GFP-Split cells were transfected with HKU1A spike and TMPRSS2, fusion was visualized by the appearance of GFP+ cells 24 h later. Data are mean ± SD of 3 independent experiments. e. Expression levels of myc-tagged proteases. 293 T cells were transfected with a control plasmid or the indicated myc-tagged TMPRSS constructs and stained 24 h later with myc antibody 9E10. Left: Representative dot plots. Right: Percentage of positive cells. Data are mean ± SD of 3 independent experiments. f. Surface expression of tagged and untagged TMPRSS2. 293 T cells were transfected with WT TMPRSS2 (TMPRSS2-Untagged) or a myc-tagged TMPRSS2 and surface stained for TMPRSS2 using VHH-A01-Fc. Left: Representative dot plots. Right: Percentage of positive cells. Data are mean ± SD of 3 independent experiments. g, h, i. Surface expression of APN, DPP4 and ACE2. 293 T cells were transfected with APN, DPP4 or ACE2 plasmids, and surface stained with the respective antibodies 24 h later. Left: Representative dot plots. Right: Percentage of positive cells. Data are mean ± SD of 3 (TMPRSS2, APN, DPP4) or 4 (ACE2) independent experiments. j. Images of time lapse microscopy of HKU1-mediated cell-cell fusion (extracted from Supp. video 1). 293 T cells were transfected either with TMPRSS2-scarlet-I or HKU1A S-NeonGreen. After 24 h, cells were mixed and imaged every 2.5 min for 2 h at 37 °C.

Extended Data Fig. 3 Knock out and silencing of TMPRSS2 in Caco2 cells. a. Linear diagram of the organization of the TMPRSS2 gene.

The CRISPR-Cas9 targeting site is underlined and the proto-spacer recognition motif (PAM) is in bold. Rectangles represent exons, black for 5’ and 3’ untranslated regions, grey for coding regions. b. Sequence analysis of the knock-out Caco2 clone. Both alleles compared to the original wild-type sequence are shown. The knockout clone harbors an out-of-frame deletion and an insertion, resulting in a frameshift on both alleles. c. Validation of TMPRSS2 knockout by TMPRSS2 surface staining. Representative dot plots of VHH anti-TMPRSS2 (VHH-A01-Fc) surface staining of a WT and KO Caco2 obtained following CRISPR gene targeting. d. Role of endogenous TMPRSS2 on cell-cell fusion assessed by siRNA. 293 T donor cells expressing HKU1A spike were mixed with Caco2 acceptor cells, silenced or not for TMPRSS2. Left: experimental design. Middle: relative expression of TMPRSS2 in Caco2 cells, assessed by RT-qPCR. Data were normalized to β-Tubulin levels. Relative mRNA expression normalized to siCtrl condition (2−ΔΔCT) was plotted. Right: fusion was quantified by measuring the GFP area after 20 h of coculture. Data are mean ± SD of 3 independent experiments. Statistical analysis: two-sided unpaired t-test compared to siCtrl cells. ***p < 0.0001.

Extended Data Fig. 4 Effect of mutant TMPRSS2 on cell-cell fusion and pseudovirus infection a. Enzymatic activity of WT and mutant TMPRSS2.

293 T cells were transfected with WT and indicated TMPRSS2 mutants. 24 h post transfection, the catalytic activity was assessed using BoC-QAR-AMC fluorogenic substrate. Data are mean ± SD of 4 independent experiments. Statistical analysis: a: one-way ANOVA with Dunnett’s multiple comparisons compared to Ctrl cells (transfected with pQCXIP-Empty). **p < 0.01. b. Effect of TMPRSS2 on HKU1 spike expressed on adjacent cells. 293 T cells were transfected either with HKU1A spike or with the different TMPRSS2. 20 h post-transfection, cells were mixed at a 1:1 ratio, and let to settle. 3 or 6 h post-mixing, cells were harvested and lysed for WB. One membrane was probed for S1, TMPRSS2 and actin, another membrane was probed for S2 and actin. For gel source data, see SI 1c, d. Representative blots of 2. Molecular weights: kDa. The arrows and # denote the uncleaved and cleaved protein products, respectively. c. Surface levels of WT and mutant TMPRSS2. 293 T cells were transfected with the indicated doses of TMPRSS2 plasmid. 18 h post-transfection they were stained intracellularly for TMPRSS2 using the commercial antibody. Left: % of TMP positive cells. Right: Median fluorescent intensity (MFI). #: indicates the chosen plasmid ratios to achieve similar TMPRSS2 levels with WT and indicated mutants. Data are mean ± SD of 3 independent experiments. d. Comparison of the anti-TMPRSS2 commercial antibody and VHH staining in 293 T cells. 293 T cells were transfected with WT, R255Q and S441A TMPRSS2 plasmids. Cells were stained 24 h later with the indicated antibodies and analysed by flow cytometry. One experiment representative of 3 is shown. Light grey curves correspond to staining with control antibodies or VHH. Parental: untransfected cells. e. Effect of WT and mutant TMPRSS2 on SARS-CoV-2 pseudovirus infection. 293 T cells expressing ACE2 were transfected with WT and indicated TMPRSS2 mutants. 24 h later, cells were infected by Luc-encoding SARS-CoV-2 pseudovirus. Luminescence was measured after 48 h. Data are mean ± SD of 4 independent experiments. Statistical analysis: RM one-way ANOVA with Geisser-Greenouse correction on log-transformed data, with Dunnett’s multiple comparisons compared to Ctrl cells (transfected with pQCXIP-Empty). **p < 0.01. f, g. Effect of SB412515, E64d or hydroxychloroquine (HCQ) on f. HKU1A or g. HKU1B pseudovirus infection. 293 T cells expressing WT or S441A TMPRSS2 were incubated for 2 h with the indicated drugs, before infection with HKU1A or HKU1B pseudoviruses. Luminescence was read 48 h post infection. Left: SB412515. Middle: E64d. Right: HCQ. Data are mean ± SD of 3 (E64d, HKU1B), 4 (E64d HKU1A, SB142515 HKU1B), 5 (HCQ HKU1A) or 6 (HCQ HKU1B, SB412515 HKU1A) independent experiments. Statistical analysis: RM two-way ANOVA with Geisser-Greenouse correction on non-normalized log transformed data, with Dunnett’s multiple comparisons compared to the non-treated conditions. h, i. Surface levels of TMPRSS2 in different cell lines stably expressing WT or S441 TMPRSS2. Cells were stained for TMPRSS2 using VHH-A01-Fc and analyzed by flow cytometry. Representative histograms are shown. h. Left: U2OS. Right: A549. Light grey: cells stained with a non-target control VHH-Fc. Dark grey: Unmodified parental cell lines. Dark and light blue: Cells transduced with either TMPRSS2 or TMPRSS2 S441A mutant. i. Caco2. Light grey: cells stained with a non-target control VHH-Fc. Blue: Unmodified parental cell line. Dark Grey: TMPRSS2 KO Caco2. Dark and light blue: TMPRSS2 KO caco2 stably transduced with TMPRSS2 WT or S441A mutant expression vectors. j. Effect of Camostat on endogenous TMPRSS2 in Caco2 cells. Caco2 cells were incubated in the presence of 100 μM Camostat for 2 h, before infection with HKU1A, HKU1B, or SARS-CoV-2 (D614G or Delta) pseudovirus. Data are normalized to the infection in the absence of the drug. Data are mean ± SD of 3 (SARS-CoV-2) or 4 (HKU1) independent experiments. Statistical analysis: RM two-way ANOVA on non-normalized log transformed data, with Sidak’s multiple comparisons compared to the non-treated conditions. k. Susceptibility of Vero E6 and Vero E6-TMPRSS2 cells to HKU1 pseudovirus infection. Left: pseudovirus infection. Right: TMPRSS2 surface levels. Dark grey: Parental cells. Dark blue: Cells transduced with TMPRSS2. Data are mean ± SD of 4 independent experiments.

Extended Data Fig. 5 Effect of neuraminidase on HKU1 pseudovirus infection.

U2OS-TMPRSS2 cells were treated with indicated concentration of neuraminidase from Arthrobacter ureafaciens for 24 h. a. HKU1A and HKU1B pseudovirus infection in neuraminidase treated cells. Cells were infected with HKU1 A or B pseudoviruses. Luminescence was read 48 h post infection. Data were normalized to the non-treated condition. Data are mean ± SD of 3 (4 mU/mL) or 4 (0, 20, 100 mU/mL) independent experiments. Statistical analysis: Mixed-effect analysis with Geisser-Greenhouse corrections (handles missing values) on non-normalized log transformed data, with Dunnett’s multi-comparison test compared to non-treated cells. b. Surface levels of TMPRSS2 in neuraminidase treated cells. Left: % of TMP positive cells. Right: Median fluorescent intensity (MFI). Data are mean of 2 independent experiments. c. Sambucus Nigra Lectin (SNA) binding on neuraminidase treated cells. Treated cells were stained with fluorescent Lectin SNA that preferentially binds α-2,6- over α-2,3 linked sialic acids. Left: Representative histograms. Right: MFI. Data are mean of 2 independent experiments. d. Siglec-E binding on neuraminidase treated cells. Treated cells were stained with recombinant Siglec-E-Fc protein that preferentially binds α2,8- over α2,3- and α2,6-linked sialic acids. Left: Representative histograms. Right: MFI. Data are mean ± SD of 3 independent experiments.

Extended Data Fig. 6 Binding of HKU and SARS-CoV-2 spikes to TMPRSS2 or ACE2. a. Binding of the indicated recombinant spikes to 293 T cells expressing TMPRSS2.

Cells were transfected with WT or mutant TMPRSS2 and incubated or not overnight with 10 µM of Camostat. The spikes were then incubated for 0.5 h and their binding was revealed with streptavidin-647 and measured by flow cytometry. The % of cells binding to TMPRSS2 was quantified. Data are mean ± SD of 3 (HKU1A) or 4 (HKU1B, SARS-CoV-2) independent experiments. Two Way ANOVA with Dunnett’s multiple comparisons compared to control cells with or without Camostat. Exact p-values: HKU1A: TMPRSS2 WT-: *0.029, TMPRSS2 WT + :*** < 0.0001, TMPRSS2 R255Q-: **0.0010, TMPRSS2 R255Q + : **0,0013, TMPRSS2 S441A-: ***0,0001, TMPRSS2 S441A + : ***<0.0001. HKU1B: ***<0.0001. b. Binding of the indicated recombinant spikes to 293 T cells expressing ACE2. Cells were transfected with ACE2. The spikes were then incubated for 0.5 h and their binding was revealed with streptavidin-647 and measured by flow cytometry. The % of cells binding to ACE2 was quantified. Data are mean of 2 independent experiments. c. Binding of the indicated soluble spikes on immobilized ACE2 measured by ELISA. d. Binding of S441A TMPRSS2 to HKU1A, HKU1B or SARS-CoV-2 RBD measured by BLI. The response was measured at the indicated concentrations of spikes. Left: HKU1A. Middle: HKU1B. Right: SARS-CoV-2. One representative experiment of 4 is shown. e. Determination of the affinity of HKU1A and B RBD for TMPRSS2 using the steady state method. Circles: Experimental values. Black: Fitting of the experimental data f. Binding of ACE2 to SARS-CoV-2 or HKU1B RBD quantified by BLI, at different concentrations of spikes. g. Binding of S441A TMPRSS2 to HKU1B mutants. Response was measured by BLI at different concentrations of spikes. Left: HKU1B RBD mutant W515A. B: HKU1B RBD mutant R517A. h. Determination of the affinity of HKU1B-R517A RBD for TMPRSS2 using the steady state method. Circles: Experimental values. Black: Fitting of the experimental data. i. Cell surface levels of WT and mutant HKU1 spikes. 293 T were transfected with the indicated WT or mutant HKU1A or B spikes, expression was measured by flow cytometry after 24 h, using the anti-spike mAb10.

Extended Data Fig. 7 Characterization of anti-TMPRSS2 VHHs. a. Binding of VHHs on TMPRSS2-expressing cells.

293 T cells were transfected with TMPRSS2 S441A. Binding of the indicated VHHs (0.5 μM) was assessed by flow cytometry. Left: representative dot plots. Right: Quantification of the percentage of positive cells and MFI (Median Fluorescent Intensity of positive cells). Data are mean ± SD of three independent experiments b. Effect of VHHs or Camostat on TMPRSS2 enzymatic activity. Recombinant soluble TMPRSS2 was incubated with the indicated VHHs at different concentrations or with Camostat (1 µM). The initial rate of enzymatic activity, measured with a fluorescent substrate is plotted. One representative experiment of 3 is shown. c. Effect of VHHs on HKU1A or HKU1B cell-cell fusion. 293 T GFP-Split cells were co-transfected with TMPRSS2 and HKU1 spike in the presence of indicated amounts of VHH. Fusion was quantified by measuring the GFP area after 20 h. Data were normalized to the non-treated condition for each spike. d. Effect of VHHs on HKU1A or HKU1B pseudovirus infection. 293 T cells transfected with S441A TMPRSS2 were incubated with the indicated amounts of VHH for 2 h and infected by Luc-encoding pseudovirus. Luminescence was read 48 h post infection. Data were normalized to the non-treated condition for each virus. Data are from one experiment representative of 3. Statistical analysis: c: One Way ANOVA with Dunnett’s multiple comparisons compared to cells stained with a non-target antibody (VHH93). d: One Way ANOVA on log-transformed data with Dunnett’s multiple comparisons compared to cells stained with a non-target antibody (VHH93).

Extended Data Fig. 8 Isolation and characterization of a live HKU1B virus. a. Viral RNA copies in the supernatant of HBE cells were quantified by RT-qPCR.

The values at day 0 are the content of the input. Left: initial amplification (first passage). Right: Second amplification of the first passage virus (harvested at day 4) used at two dilutions (1/10 and 1/100) in duplicates. b. Reads coverage and frequency of minor variants frequencies of the isolate sequencing data (first passage virus). Intra-sample single-nucleotide variants frequencies were estimated using iVAR70. The genome organization of HKU1 is shown below the plot. c, d. Phylogenetic analysis of HKU1. Maximum likelihood phylogenies of human coronavirus HKU1 (n = 48) estimated using IQ-TREE v2 with 1000 replicates from c. the complete genome and d. spike coding sequences. The tree is midpoint rooted and ultrafast bootstraps values are shown on the main branches. A similar topology was obtained when rooting the tree using another embecovirus (OC43). The tip name corresponding to the newly isolated virus sequence is highlighted in bold and underlined. The tips names corresponding to the recombinant spikes used in this study are shown in bold.

Extended Data Table 1 Affinity of the nanobodies for TMPRSS2 S441A and summary of their effect
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Supplementary Information sections 1–6 and Supplementary Table 1.

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Supplementary Video 1

Representative video of time-lapse microscopy of TMPRSS2-scarlet-I (red) and HKU1A S-NeonGreen (green) expressing cells. 293T cells were transfected with either TMPRSS2-scarlet-I or HKU1A S-NeonGreen. At 24 h posttransfection, the cells were mixed and imaged every 150 s for 2.5 h at 37 °C, 5% CO2.

Supplementary Video 2

Representative video of time-lapse microscopy of TMPRSS2-scarlet-I (red) and HKU1A S-NeonGreen (green) expressing cells. 293T cells were transfected either with TMPRSS2-scarlet-I or HKU1A S-NeonGreen. At 24 h posttransfection, the cells were mixed and imaged every 150 s for 2.5 h at 37 °C, 5% CO2.

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Saunders, N., Fernandez, I., Planchais, C. et al. TMPRSS2 is a functional receptor for human coronavirus HKU1. Nature 624, 207–214 (2023). https://doi.org/10.1038/s41586-023-06761-7

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