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Nectin-4 mediates rubella virus entry in airway and placental cells

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Data availability

The proteomics data have been deposited into the ProteomeXchange Consortium (identifier PXD067722; http://www.proteomexchange.org/) via the jPOST partner repository (JPST004036; https://jpostdb.org/)61. The FASTQ files for CRISPR–Cas9 screens have been deposited into the DNA Data Bank of Japan (accession number DRA022489). The statistical analysis data for the proteomics and CRISPR–Cas9 screens have been appended as Supplementary Tables. Gene expression data used for candidate selection were obtained from the Cancer Cell Line Encyclopedia (https://sites.broadinstitute.org/ccle). Subcellular localization data were obtained from the COMPARTMENTS database (https://compartments.jensenlab.org/). All other data supporting the findings of this study are available in the paper and its Supplementary Information. Source data are provided with this paper.

Code availability

No custom code was used in this study.

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Acknowledgements

We thank M. Howarth, J. Icenogle, F. Seki, B. Stringer, A. Ting and D. Trono for providing materials; members of A.R.’s Laboratory, especially M. Tahara, Y. Hatayama, N. Shimasaki, Y. Nakai, T. Kameya, F. Mizukoshi, N. Shiomatsu and N. Otsuki for their feedback, insight and assistance; A. Ninomiya for technical advice; and staff at Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Funding

This work was supported by JSPS KAKENHI grant number JP22K0986 (to Y. Mori), AMED under grant numbers 24fk0108628 and 25fk0108719 (to Y. Mori), the Joint Research Project of the Research Institute for Microbial Diseases, The University of Osaka (JRPRIMD25B5 and JRPRIMD26B6 to Y. Mori) and the Research Center for Biomedical Engineering (to Y. Mori).

Author information

Author notes

  1. These authors contributed equally: Yoshio Mori, Masafumi Sakata, Yuichiro Nakatsu

Authors and Affiliations

  1. Department of Bioinformatics and Integrative Omics, National Institute of Infectious Diseases, Japan Institute for Health Security, Musashimurayama, Japan

    Yoshio Mori, Masafumi Sakata, Satoshi Suzuki, Mayuko Nishi, Misato Nagai & Akihide Ryo

  2. Department of Respiratory Viruses, National Institute of Infectious Diseases, Japan Institute for Health Security, Musashimurayama, Japan

    Yuichiro Nakatsu, Miyuki Kawase, Masatoshi Kakizaki, Takashi Okura, Kazuya Shirato & Akihide Ryo

  3. Department of Diagnostic and Therapeutic Systems Engineering, Laboratory for Biomaterials and Bioengineering (LBB), Institute of Integrated Research (IIR), Institute of Science Tokyo, Chiyoda, Japan

    Takeshi Hori & Hirokazu Kaji

  4. Center for Biological Safety and Research (CBSR), National Institute of Health Sciences (NIHS), Kawasaki, Japan

    Takeshi Hori

  5. Advanced Medical Research Center, Yokohama City University, Yokohama, Japan

    Yoko Ino & Yayoi Kimura

  6. Molecular Genetic Research, Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, Sagamihara, Japan

    Yoko Ino

  7. Department of Academic–Industrial Partnerships Promotion, Center for Clinical Sciences, Japan Institute for Health Security, Shinjuku, Japan

    Kento Fukano

  8. Department of Virology II, National Institute of Infectious Diseases, Japan Institute for Health Security, Shinjuku, Japan

    Kento Fukano, Hideki Aizaki & Masanori Isogawa

  9. Division of Infectious Diseases, International Research Institute of Disaster Science, Tohoku University, Sendai, Japan

    Hironori Hayashi & Eiichi N. Kodama

  10. Division of Biomedical Measurements and Diagnostics, Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan

    Kazutaka Murayama

  11. Department of Infectious Diseases, Graduate School of Medicine and Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan

    Eiichi N. Kodama

  12. Laboratory of Virus Control, Center for Infectious Disease Education and Research (CiDER), The University of Osaka, Suita, Japan

    Shuhei Taguwa & Yoshiharu Matsuura

  13. Laboratory of Virus Control, Research Institute for Microbial Diseases (RIMD), The University of Osaka, Suita, Japan

    Shuhei Taguwa & Yoshiharu Matsuura

  14. Center for Advanced Modalities and DDS (CAMaD), The University of Osaka, Suita, Japan

    Shuhei Taguwa & Yoshiharu Matsuura

  15. Department of Infectious Diseases and Host Defense, Graduate School of Medicine, Gunma University, Maebashi, Japan

    Tatsuki Takahashi & Wataru Kamitani

Authors

  1. Yoshio Mori
  2. Masafumi Sakata
  3. Yuichiro Nakatsu
  4. Takeshi Hori
  5. Yoko Ino
  6. Satoshi Suzuki
  7. Mayuko Nishi
  8. Miyuki Kawase
  9. Masatoshi Kakizaki
  10. Misato Nagai
  11. Kento Fukano
  12. Hideki Aizaki
  13. Hironori Hayashi
  14. Kazutaka Murayama
  15. Eiichi N. Kodama
  16. Shuhei Taguwa
  17. Takashi Okura
  18. Tatsuki Takahashi
  19. Wataru Kamitani
  20. Kazuya Shirato
  21. Masanori Isogawa
  22. Yayoi Kimura
  23. Hirokazu Kaji
  24. Yoshiharu Matsuura
  25. Akihide Ryo

Contributions

Y. Mori, S.T., Y. Matsuura and A.R. conceived the project. Y. Mori, M.S., Y.N., T.H., Y.K., H.K. and A.R. designed the experiments. Y. Mori and Y.I. performed the proximity biotin labelling experiments using APEX2–VLPs. Y.I. and Y.K. performed LC–MS/MS and data analyses. Y. Mori, M.S. and M. Nagai performed CRISPR–Cas9 screening, viral attachment and internalization assays. Y. Mori, M.S. and Y.N. performed RuV infection experiments. Y. Mori and M.S. performed VSVpv infection, subgenomic replicon and inhibitor addition assays. Y. Mori, S.S., H.H., K.M. and E.N.K. prepared and purified recombinant proteins. Y. Mori, S.S. and M. Nagai performed ELISA-based binding assays. Y. Mori, K.F., H.A. and M.I. conducted SPR experiments. Y. Mori, M.S. and M. Kakizaki performed flow cytometry and cell sorting. M.S., M. Kawase, M. Kakizaki and K.S. prepared ALI-cultured respiratory epithelial cells and performed infection experiments. M. Nishi performed immunohistochemistry. Y.N., M.S. and T.H. prepared differentiated trophoblasts and performed infection experiments. T.O., T.T., W.K. and K.S. prepared recombinant RSV. Y. Mori, M.S., Y.N., Y.I., T.H., K.F., H.A., S.S., S.T., M.I., Y.K., H.K., Y. Matsuura and A.R. interpreted the experimental data. Y. Mori, Y.I. and A.R. wrote the original manuscript. Y. Mori, K.M. and A.R. wrote the revised manuscript. Y. Mori, M.S. and Y.N. generated figures. All authors read, edited and approved the final version of the manuscript before submission.

Corresponding authors

Correspondence to Yoshio Mori or Akihide Ryo.

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Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Roberto Cattaneo, Claudia Claus and Rowan Karvas for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

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Extended data figures and tables

Extended Data Fig. 1 Preparation of APEX2-VLP and proximity biotin labeling with APEX2-VLP on the surface of cells susceptible to RuV.

(a) Schematic diagram illustrating the preparation of subgenomic RNA-containing VLPs or empty VLPs with DogTag inserted into the envelope proteins. BHK-SGR-NL cells were transfected with a plasmid expressing the structural protein precursor CE2E1 to produce VLPs containing SGR (SGR-VLP). Plasmids encoding C-DTE2-E1, which contains an in-frame DogTag insertion between Gly1 and Leu2 of the E2 protein, and C-E2-DTE1, which contains an in-frame DogTag insertion between Asn213 and Gln214 of the E1 protein, were also expressed. Expi293F cells were utilized to produce large quantities of empty VLPs. (b) A series of SGR-VLPs encoding NanoLuc as a reporter were inoculated into Vero cells to assess infectivity. As a negative control, culture medium collected from BHK-SGR-NL cells expressing mCherry instead of CE2E1 was used. Infectivity was determined by measuring the relative activity of NanoLuc (n = 3 biologically independent cultures). (c) Western blot analysis to detect the E2 (left panel) or E1 (right panel) protein of SGR-VLPs. SGR-VLPs were incubated overnight at 4 °C in the presence or absence of DC-APEX2. SDS-PAGE samples were prepared by adding SGR-VLPs to SDS-PAGE sample buffer containing 2-mercaptoethanol and incubating at 95 °C for 5 min. Arrows indicate the original bands of viral proteins with an inserted DogTag, while arrowheads indicate those covalently bound to DC-APEX2. (d) Infectivity analysis of SGR-VLP mixed with PBS or DC-APEX2. SGR-VLPs were incubated overnight at 4 °C with PBS or DC-APEX2 and subsequently inoculated into Vero cells to analyze infectivity. As a negative control, culture medium collected from BHK-SGR-NL cells expressing mCherry instead of CE2E1 was used. Infectivity was determined by measuring the relative activity of NanoLuc (n = 4 biologically independent cultures). (e) Electron microscopic images of VLPs and APEX2-VLPs visualized by negative staining. (f) Following treatment of JAR4-SMS1KO22 cells under the indicated conditions, biotin-labeled proteins were detected by western blotting using HRP-conjugated streptavidin. (g) Fluorescence staining images of JAR4-SMS1KO22 cells treated with biotin labeling in the presence or absence of APEX2-VLP. RuV E1 protein, biotin, and nuclei are visualized in green, magenta, and blue, respectively. Images in left and upper right panels were obtained by confocal microscopy and are shown as z-stack projections. The boxed area in the left panel is magnified in the upper right panel. The cross-section obtained by cutting along the dotted line in the left panel is shown in the lower right panel. The plasma membranes stained with WGA are shown in gray in only the cross-section image. Data are mean ± s.d. Statistical analyses were performed using one-way ANOVA with Dunnett’s post hoc multiple comparison test (b) and two-way ANOVA with Šídák’s post hoc multiple comparison test (d). Statistical significance is indicated by asterisks: ****P < 0.0001; ***, P < 0.001; **, P < 0.01; ns, not significant. Exact P values are provided in Supplementary Table 5.

Source data

Extended Data Fig. 2 Validation of the expression of Nectin-4 or MOG in cell lines.

(a) Lysates of Raji-derived cells were analyzed by western blotting using antibodies against Nectin-4 (EPR15613-68), MOG, DYKDDDDK-tag (for FLAG-tagged SeV C protein), and GAPDH. (b) Lysates of Raji-derived cells were analyzed by western blotting using antibodies against ADAM9, DYKDDDDK-tag (for FLAG-tagged SeV C protein), and GAPDH. (c) RajiSC-EV or RajiSC-hNectin-4 cells were stained with anti-Nectin-4 goat polyclonal antibody or normal goat IgG, followed by Alexa Fluor 488-conjugated donkey anti-goat IgG, and analyzed by flow cytometry. (d) RajiSC-EV or RajiSC-hMOG cells were stained with anti-MOG mouse monoclonal or mouse IgG1 monoclonal antibody (isotype control), followed by Alexa Fluor 488-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. (e) Jurkat or Jurkat-hNectin-4 cells were stained with anti-Nectin-4 human monoclonal (Enfortumab) or human IgG1 monoclonal antibody (isotype control), followed by Alexa Fluor 488-conjugated goat anti-human IgG, and analyzed by flow cytometry. (f) Jurkat or Jurkat-hMOG cells were stained with anti-MOG mouse monoclonal or mouse IgG1 monoclonal antibody (isotype control), followed by PE-conjugated goat anti-mouse IgG, and analyzed by flow cytometry. (g, h) Cells were stained with anti-Nectin-4 human monoclonal (Enfortumab) or human IgG1 monoclonal antibody (isotype control), followed by Alexa Fluor 488-conjugated goat anti-human IgG, and analyzed by flow cytometry. Dead cells were excluded from analyses by staining with 7-Aminoactinomycin D. All cells were analyzed using a FACSCalibur flow cytometer. (i) Expression of Nectin-4 and α-tubulin in NCI-H358-NT, NCI-H358-Nectin-4KO, and NCI-H358-N4KO-N4OE cells was analyzed by western blotting using anti-Nectin-4 human monoclonal (Enfortumab) and anti-α-tubulin polyclonal antibodies.

Extended Data Fig. 3 Effects of hNectin-4 on replication, transcription, and translation of the RuV genome.

(a) Schematic diagram of the measurement of replication, transcription, and translation activity by the RuV subgenomic replicon (SGR). SGR-RNA, in which the structural protein gene of RuV was replaced by a fusion protein gene of puromycin-N-acetyltransferase (PAC), an FMDV 2 A protease cleavage site, and NanoLuc (NLuc), was generated by in vitro transcription. SGR-GND is an RNA replication-deficient mutant generated by substituting amino acids in the active center of RNA-dependent RNA polymerase of p90 (GDD to GND). SGR-WT RNA or SGR-GND RNA was transfected into RajiSC-EV or RajiSC-hNectin-4 cells together with mRNA for RuV C protein and Firefly Luciferase (FLuc), and incubated at 35 °C. At 72 h post-transfection, the NLuc and FLuc activities were measured. (b) Results of a reporter assay following RuV-SGR RNA transfection. SGR activity was determined as NLuc activity normalized by FLuc activity (n = 9 biologically independent cultures derived from three independent experiments). Statistical analyses were performed using two-way ANOVA with Šídák’s post hoc multiple comparison test. No significant differences (ns) were detected. Exact P values are provided in Supplementary Table 5.

Source data

Extended Data Fig. 4 Involvement of Nectin family members in cell entry by RuV.

(a) Flow cytometry analysis of cell surface expression of the Nectin family in RajiSC-EV, hNectin1-, hNectin2-, or hNectin3-overexpressing RajiSC cells. (b) RajiSC cells expressing each Nectin member were inoculated with pseudotyped VSV strains encoding the firefly luciferase gene: VSVpv-G (VSV-G), VSVpv-ΔG (∆G), and VSVpv-RuV-CE2E1 (RuV-CE2E1). After incubation for 20 h at 37 °C, relative light units (RLU) were measured (n = 3 biologically independent cultures). Data are presented as mean ± standard deviations. Statistical analyses were performed using two-way ANOVA with Dunnett’s post hoc multiple comparison test. Statistical significance is indicated by asterisks: ****, P < 0.0001; ns, not significant. Exact P values are provided in Supplementary Table 5. (c) CBB-stained image of the SDS-PAGE gel of purified human IgG1-Fc domain-fused ectodomains derived from Nectin family members. In each lane, 1 µg of protein was loaded. (d) ELISA-based binding assay. Purified Fc domain-fused Nectin ectodomains were added to wells coated with RuV particles at the indicated concentrations. Bound proteins were detected by immunochemistry (n = 3 biologically independent experiments). Data are presented as mean ± standard deviations.

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Extended Data Fig. 5 Colocalization of VLP and hNectin-4 during endocytic entry and a proposed model of Nectin-4-dependent entry of RuV.

(a) Confocal microscopy of VLP binding on the cell surface. NCI-H358 cells were labeled with anti-RuV E1 (green) and anti-Nectin-4 (magenta) antibodies after 2 h of VLP binding on ice. Slice-1 (apical) and Slice-2 (mid-section) are shown. Lower panels are magnifications of the dotted regions in Slice-1. (b) Confocal microscopy of VLP internalization into cells. NCI-H358 cells were incubated with VLPs on ice for 2 h for binding, followed by incubation at 37 °C for 1 h to induce internalization. Cells were labeled with anti-Rab5 (green), anti-RuV E1 (cyan), and anti-Nectin-4 (magenta) antibodies. The nuclei were shown in blue. The main image shows a representative mid-section slice. Right panels represent magnified views of the regions enclosed by dotted boxes 1 and 2. (c) Proposed model for RuV cell entry mediated by hNectin-4 receptor. (1) RuV initially attaches to cholesterol- and sphingomyelin-rich membrane domains, which are abundantly distributed on the target cell surface, through calcium ion-dependent interactions involving fusion loops within the E1 protein. (2) RuV subsequently binds to hNectin-4 at the cell surface via the E2E1 heterodimer, which (3) induces endocytosis of the virus–receptor complex. (4) pH-dependent conformational changes in the E1 protein within the acidic endosomal compartment trigger membrane fusion between the viral envelope and the endosomal membrane, facilitating the release of the nucleocapsid into the cytoplasm.

Extended Data Fig. 6 Establishment of RajiSC-hN4-GPI cells, which express the hNectin-4 mutant with transmembrane and cytoplasmic domains swapped for a GPI anchor attachment signal (hN4-GPI).

(a) The amino acid sequence of hNectin-4 from position 340 onward is shown. Transmembrane domains and linear amino acid motifs were predicted using SOSUI and ELM, respectively. For hNectin-4, the putative transmembrane domain is underlined, putative binding motifs for the sigma (ELME000526) and mu subunits (ELME000120) of the adaptor protein complexes are shown in pink and blue, respectively, and binding sites to the Afadin PDZ domain are shown in gray. The amino acid sequence shown in green for hN4-GPI is the GPI anchor attachment signal derived from human CD55. (b) RajiSC-EV, RajiSC-hNectin-4, or RajiSC-hN4-GPI cells were stained with anti-Nectin-4 human monoclonal (Enfortumab) or human IgG1 monoclonal (isotype control) antibodies, followed by Alexa Fluor 488-conjugated goat anti-human IgG, and analyzed by flow cytometry using a FACSCalibur flow cytometer. (c) Western blot analysis of Nectin-4 expression in RajiSC-EV, RajiSC-hNectin-4, and RajiSC-hN4-GPI cells. Nectin-4 and GAPDH were detected using anti-Nectin-4 rabbit monoclonal antibody and anti-GAPDH mouse monoclonal antibody, respectively. Asterisks indicate non-specific signals.

Extended Data Fig. 7 Preparation of soluble forms of hNectin-4 ectodomains and hMOG fused with human IgG1 Fc domain.

(a) Schematic diagram of hNectin-4 structure. Nectin-4 is a single-pass type I transmembrane protein of the immunoglobulin superfamily and contains one V-set (V) and two C2-set (C2) domains in the ectodomain. TM, Transmembrane domain; CT, Cytoplasmic tail. (b) Schematic diagram of a series of soluble hNectin-4 ectodomains fused with the human IgG1 Fc domain. The recombinant Nectin-4 proteins consist of either one V domain, two C2 domains, or both, with the interleukin-2 signal sequence (IL2ss) fused to the N-terminus and the human IgG1 F domain fused to the C-terminus, designated hN4-VCC-Fc, hN4-V-Fc, and hN4-CC-Fc. As a control, IL2ss-fused Fc domain (Fc) was also prepared. (c) SDS-PAGE and CBB staining of the purified recombinant proteins: hN4-VCC-Fc, hN4-V-Fc, hN4-CC-Fc, and Fc. In each lane, 1 µg of protein was loaded. (d) Schematic diagram of hMOG structure. hMOG is also a single-pass type I transmembrane protein of the immunoglobulin superfamily and contains a single V-set (V) domain in the ectodomain. TM, Transmembrane domain; CP, Cytoplasmic domain; MAD, Membrane-associated domain. (e) Schematic diagram of a soluble hMOG-V domain fused with the human IgG1 Fc domain. (f) SDS-PAGE and CBB staining of the purified recombinant proteins, Fc and hMOG-V-Fc. In each lane, 1 µg of protein was loaded. (g) Western blot analysis of the purified recombinant proteins, Fc and hMOG-V-Fc. The proteins were detected with anti-MOG monoclonal antibody or anti-human IgG. (h) ELISA-based binding assay. Purified Fc domain-fused ectodomains of hNectin-4 and hMOG were added to wells coated with RuV particles at the indicated concentrations. Bound proteins were detected by immunochemistry (n = 3 biologically independent experiments). Data are presented as mean ± standard deviations.

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Extended Data Fig. 8 Cell surface expression of the RuV E2 and E1 proteins in 293 T cells.

(a) Schematic diagram of RuV structural protein expression constructs designed for cell surface expression and examination of their binding with hN4-VCC-Fc. The construct expressing E2-E1 precursor and E2 contains the transmembrane domain of the C protein added as a signal sequence, while the construct expressing E1 contains the transmembrane domain of E2 added as a signal sequence. E1-GPI construct expresses the E1 protein ectodomain with the human CD55 signal sequence and GPI anchor attachment signal added to the N-terminus and C-terminus, respectively. (b) 293 T cells transfected with plasmids expressing a series of RuV structural proteins E2E1 or empty vector (EV) were stained with anti-RuV E2 protein mouse monoclonal or mouse IgG1 monoclonal antibody (isotype control) (upper panels), or anti-RuV E1 protein mouse monoclonal (clone 85) or mouse IgG2a monoclonal antibody (isotype control) (lower panels), followed by Alexa Fluor 488-conjugated goat anti-mouse IgG, and analyzed by flow cytometry using a FACSCalibur flow cytometer.

Extended Data Fig. 9 Preparation of ALI-cultured HBTECs.

Immunofluorescence staining of ALI-cultured HBTEC cross-sections. Acetylated α-tubulin (cilia marker), mucin 5AC (goblet cell marker), CC10 (club cell marker), and cytokeratin 5 (basal cell marker) were immunostained using corresponding monoclonal antibodies. Nuclei were counterstained with DAPI. Images were obtained by confocal microscopy and are shown as z-stack projections. Target proteins and nuclei are displayed in green and blue, respectively. Images are oriented with the apical surface at the top and basolateral surface at the bottom.

Extended Data Fig. 10 Characteristics of differentiated STB-like ST(2D) cells and apical-out spherical trophoblast organoids.

(a) Immunofluorescence staining of TS cells CT27 and differentiated CT27-ST(2D) cells. E-cadherin (CTB marker) and SDC1 and hCGβ (STB markers) were immunostained using corresponding monoclonal antibodies. Nuclei were counterstained with DAPI. Images were acquired using fluorescent microscopy, and target proteins and nuclei are displayed in green and blue, respectively. (b) Representative bright-field image of CT27-organoids. (c) Immunofluorescence staining of an apical-out trophoblast organoid derived from three independent TS cells, CT27, CT29 and CT30. Nectin-4, SDC1 (STB marker), and E-cadherin (CTB marker) were immunostained with corresponding monoclonal antibodies. Nuclei were counterstained with Hoechst 33342. The cells were observed by confocal microscopy, and the single X–Y plane images are displayed. Nectin-4, SDC1, E-cadherin, and nuclei are displayed in green, magenta, gray, and blue, respectively. (d) Western blotting using anti-Nectin-4 rabbit monoclonal antibody and anti-hCGβ polyclonal antibody was performed to detect endogenous Nectin-4 and hCGβ, respectively, in three independent lines of TS cells, CT27, CT29 and CT30, and their differentiated ST(2D) cells. As an internal control, GAPDH was detected using HRP-conjugated anti-GAPDH antibody. (e) Western blotting using anti-Nectin-4 rabbit monoclonal antibody was performed to detect endogenous Nectin-4 in three independent lines of TS cells and their differentiated organoids. As an internal control, GAPDH was detected using HRP-conjugated anti-GAPDH antibody. (f) After incubation with anti-Nectin-4 antibody, Enfortumab, or human IgG1 (isotype control) at a concentration of 20 µg/mL, TS cells or ST(2D) cells derived from two independent lines, CT29 and CT30, in a 96-well culture plate were inoculated with 1 × 105 PFU of RuV-NLuc per well. At 2 days post-inoculation, NLuc activity was measured, and that in the isotype control-treated cells was represented as 100% (n = 12 biologically independent cultures derived from three independent experiments). (g) After incubation with anti-Nectin-4 antibody, Enfortumab, or human IgG1 (isotype control) at a concentration of 40 µg/mL, trophoblast organoids derived from two independent lines, CT29 and CT30, on agarose gels were inoculated with 1 × 106 PFU of RuV-NLuc per well. At 2 days post-inoculation, NLuc activity was measured, and that in the isotype control-treated cells was represented as 100% (n = 6 biologically independent cultures derived from three independent experiments). Data are presented as mean ± standard deviations. Statistical analyses were conducted using two-way ANOVA with Sidak’s post hoc multiple comparison tests (f and g). Significant differences are indicated by asterisks: ***, P < 0.001; **, P < 0.01; *, P < 0.05. Exact P values are provided in Supplementary Table 5.

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Mori, Y., Sakata, M., Nakatsu, Y. et al. Nectin-4 mediates rubella virus entry in airway and placental cells. Nature (2026). https://doi.org/10.1038/s41586-026-11248-2

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