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Multiple LDLR family members act as entry receptors for yellow fever virus

Nature volume 649, pages 173–182 (2026)Cite this article

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Abstract

Infection by yellow fever virus (YFV), the prototype Orthoflavivirus, induces a febrile syndrome in humans that can progress to liver failure, haemorrhage and death1. Despite decades of study, the entry receptors for YFV remain unclear. Here, using a surface protein-targeted CRISPR–Cas9 screen, we identified LRP4, a low-density lipoprotein receptor (LDLR) family member, as a candidate entry receptor for YFV. Genetic ablation of LRP4 impaired YFV infection of cells and, reciprocally, complementation or ectopic expression of LRP4 increased infection. Related viruses in the YFV antigenic complex also showed LRP4-dependent infection. LRP4 promoted YFV entry into cells through LDLR type A (LA) domain binding to domain III of the YFV envelope protein. Soluble LRP4–Fc decoy receptors neutralized YFV infection in cell culture and reduced viral burden in vivo. As we observed residual YFV infection in LRP4-deficient cells, we evaluated whether other LDLR family members promote YFV entry. This approach identified LRP1 and VLDLR as additional receptors for YFV infection in cell culture. LRP1–Fc, LRP4–Fc and VLDLR–Fc decoys protected mice from YFV challenge, and LRP1–Fc decoys inhibited YFV infection and liver pathogenesis in mice engrafted with human hepatocytes. A genetic deficiency of LRP1 in primary human hepatocyte cultures also resulted in reduced YFV infection. Our findings establish a role for multiple LDLR family members in YFV entry, infection and pathogenesis, which has implications for receptor use and countermeasure development for multiple emerging orthoflaviviruses.

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Fig. 1: LRP4 promotes YFV infection.
Fig. 2: The LBD of LRP4 supports YFV infection and binds to YFV virions.
Fig. 3: LRP4 Fc decoy inhibits YFV infection in vitro and in vivo.
Fig. 4: YFV can use multiple LDLR family members for infection.
Fig. 5: LRP1-CL-I–Fc protein protects hFRG mice from YFV infection.

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Data availability

Data supporting the findings of this study are available within the Article and its Supplementary Information, or in files uploaded to the Washington University School of Medicine’s Bernard Becker Medical Library (https://doi.org/10.17632/xnpzsx3729). Reagents will be made available on request after completion of a materials transfer agreement. Raw CRISPR screen sequencing data have been uploaded to NCBI (BioSample: SAMN50433716. Accession: SRX29989461 and SRX29989462). Source data are provided with this paper.

Code availability

A Python script for analysis of SPR data has been uploaded to GitHub (https://github.com/kaszubat/YFV-LRP4-SPR-Analysis).

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Acknowledgements

This research was funded by NIH grants (U01 AI073755 and U19 AI181960 to M.S.D., J.E.C. and D.H.F.) and contracts (75N93019C00062 and 75N93022C00035 to D.H.F.), as well as the NIH Director’s Early Independence Award (DP5 OD029608) to A.L.B. We thank N. Perlmutter for help in generating LRP4 orthologues and T. Pierson for providing the plasmids for production of YFV RVPs. Some graphics in the figures were made using BioRender, using an institutional license to Washington University in St. Louis.

Author information

Authors and Affiliations

  1. Department of Medicine, Washington University School of Medicine, St Louis, MO, USA

    Zhenlu Chong  (种振路), Sathvik Palakurty, Alan Sariol, Pengfei Li, Saravanan Raju, David A. Price, Daisy W. Leung & Michael S. Diamond

  2. Department of Pathology & Immunology, Washington University School of Medicine, St Louis, MO, USA

    Sean Hui, Sathvik Palakurty, Tomasz Kaszuba, Michael N. Nguyen, Saravanan Raju, Paige D. Hall, Christopher A. Nelson, Israel Baltazar-Perez, Gaya K. Amarasinghe, Daved H. Fremont & Michael S. Diamond

  3. Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI, USA

    Xueer Qiu & Adam L. Bailey

  4. Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO, USA

    Paul W. Rothlauf, Sean P. J. Whelan, Daved H. Fremont & Michael S. Diamond

  5. Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA

    James E. Crowe Jr.

  6. Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, St Louis, MO, USA

    Daved H. Fremont

  7. Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St Louis, MO, USA

    Michael S. Diamond

  8. Center for Vaccines and Immunity to Microbial Pathogens, Washington University School of Medicine, St Louis, MO, USA

    Michael S. Diamond

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  1. Zhenlu Chong  (种振路)
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Contributions

Z.C. and M.S.D. designed the study. Z.C. performed the CRISPR screen and target gene validation. Z.C., S.P. and P.L. performed cell culture studies. S.H., C.A.N., D.A.P. and Z.C. generated recombinant proteins. Z.C., S.R., D.W.L., P.W.R., S.P.J.W., I.B.-P. and G.K.A. generated key plasmids and proteins. S.H., Z.C., P.D.H., T.K. and M.N.N. performed protein binding studies. J.E.C. provided purified recombinant antibodies. Z.C., A.S., X.Q. and A.L.B. performed in vivo studies. D.H.F. supervised the protein biophysical studies. Z.C. and M.S.D. wrote the initial draft with all of the other authors providing comments.

Corresponding author

Correspondence to Michael S. Diamond.

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Competing interests

M.S.D. is a consultant or on a scientific advisory board for Inbios, IntegerBio, Akagera Medicines, GlaxoSmithKline, Merck and Moderna. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Vir Biotechnology, Bavarian Nordic and Moderna. J.E.C. is a former member of the scientific advisory boards of Gigagen (Grifols) and BTG International, has consulted for Moderna, is founder of IDBiologics and receives royalties from UpToDate. The Crowe laboratory received unrelated sponsored research agreements from IDBiologics during the conduct of the study.

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Extended data figures and tables

Extended Data Fig. 1 CRISPR–Cas9 screen identifies LRP4 as a factor required for efficient YFV infection.

a, HAP1 ΔHS cells were transduced with the cell surface library containing 4,630 sgRNAs, selected with puromycin, and inoculated with YFV-17D at an MOI of 10. Surviving cells were expanded, and 3 weeks later, genomic DNA from cells was harvested and sequenced for sgRNA abundance. b, Next-generation sequencing confirmation of LRP4 gene editing in HAP1 ΔHS cells. Allele frequency is indicated next to the sequence. c, HAP1 ΔHS (control), HAP1 ΔHS ΔLRP4 (ΔLRP4) and LRP4-complemented HAP1 ΔHS ΔLRP4 (ΔLRP4 + LRP4) cells were analysed for surface expression of LRP4 by flow cytometry using an anti-Flag mAb. d, Next-generation sequencing confirmation of LRP4 gene editing in 293 T cells. Allele frequency is indicated next to the sequence. e, 293 T (control), ΔLRP4 and LRP4-complemented ΔLRP4 293 T (ΔLRP4 + LRP4) cells were inoculated with YFV-17D (MOI of 20) for 16 h, and infection was measured by flow cytometry (anti-E mAbs). f, Flag-LRP4 expression levels on the surface of control, ΔLRP4, and LRP4-complemented ΔLRP4 293 T (ΔLRP4 + LRP4) cells were analysed by flow cytometry (anti-Flag mAb). g-h, Control and Flag-LRP4 expressing K562 cells were analysed for surface expression of LRP4 by flow cytometry (g, anti-LRP4 mAb; h, anti-Flag mAb). i, ENTV (MOI of 5) was incubated with 200 ng/mL of E60 or isotype control mAb at for 1 h at 37 °C and then used to infect K562 cells. Infection levels were measured by flow cytometry at 24 h (anti-E mAbs). Data are mean ± s.d. (e and i) of 3 experiments, each performed in triplicate. Statistical analysis: one-way ANOVA with Dunnett’s post-test (e) or two-tailed unpaired t test (i). The diagram in a was created in BioRender. Diamond, M. (2025) https://BioRender.com/zw9zpr4.

Source Data

Extended Data Fig. 2 Cell surface expression of LRP4 variants and orthologs in K562 cells.

a, K562 cells ectopically expressing indicated LRP4 variants were measured by flow cytometry (anti-Flag mAb). b, Sequence alignment of LRP4 LBD from indicated species. c, K562 cells ectopically expressing LRP4 orthologs were measured by flow cytometry (anti-Flag mAb). d, MFI of Flag-LRP4 expression levels in (c).

Source Data

Extended Data Fig. 3 Binding of LRP4 to YFV.

a, SDS-PAGE gel with Coomassie staining of purified human LRP4-LBD-Fc and LDLRAD3-LA1-Fc proteins. b-c, SDS-PAGE gel with Coomassie staining of YFV-M185D/BinJ (fraction in lane 2 was used for BLI experiments) (b), YFV-ES504/BinJ (fraction in lane 3 was used for BLI experiments) and YFV-Asibi/BinJ (c) (fraction in lane 7 was used for BLI experiments). d, Micrographs of YFV-Asibi/BinJ and YFV-M185D/BinJ chimeric virions show mature and immature particles of approximately 50 nm in diameter. Scale bar, 100 nM. e-f, Inhibition of YFV-17D infection with indicated concentrations of LRP4-LBD-Fc fusion protein in 293 T (e) or Vero (f) cells was measured by flow cytometry (anti-E mAbs). g-h, Inhibition of YFV-17D infection with indicated concentrations of LRP4-Fc fusion proteins containing only one LA domain (g) or two LA domains (h) in HAP1 cells was measured by flow cytometry (anti-E mAbs). i-k, Infection of control K562 cells or those expressing indicated LRP4 variants with YFV-17D RVPs at 24 h was measured by flow cytometry. l, K562 cells ectopically expressing indicated LRP4 variants in (h-j) were measured by flow cytometry (anti-Flag mAb). Data (a-d) are representative of 2 experiments. Data (e-f and i-k) are mean ± s.d. from 3 experiments, each performed in duplicate (e-f) or triplicate (g-k). Data (g-h) are mean values from 3 experiments, each performed in triplicate. Source gel data images (a-c) are in Supplementary Fig. 1.

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Extended Data Fig. 4 Contacts between YFV E-DIII and antibody.

a, Alignment of YFV-Asibi and YFV-17D E-DIII proteins with other YFV strains and YFV complex viruses. Red dots underneath indicate mutated residues in panels b-c. b, Binding of YFV-Asibi E-DIII mutant proteins to anti-YFV Adi-49147 mAb by BLI. Biosensors were coated with Adi-49147 mAb following incubation with YFV-Asibi E-DIII mutant proteins in solution. c, Infection of YFV-17D RVPs with indicated mutations in E-DIII mutant was measured by flow cytometry on Raji-DC-SIGNR cells (GFP expression). Data (b, c) are representative of 2 experiments. The diagram in b was created in BioRender. Diamond, M. (2025) https://BioRender.com/d5zz6nu.

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Extended Data Fig. 5 LRP4 mRNA expression pattern in humans and mice.

a-b, Human LRP4 mRNA levels (a) or protein levels (b) are showed in indicated tissues. c, Mouse Lrp4 mRNA levels are showed from indicated tissues. Data were obtained from Human Protein Atlas database (https://www.proteinatlas.org/ENSG00000134569-LRP4/tissue) (a-b) or BioGPS database (http://biogps.org/#goto=genereport&id=228357) (c).

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Extended Data Fig. 6 LRP4-Fc decoy molecules serum levels and activity.

a, AG129 mice were given 500 μg of LRP4-LBD-Fc, LRP4-LA (6-8)-Fc or LDLRAD3-LA1-Fc protein by i.p. at 4 h before infection. Mice were then inoculated with 105 PFU of YFV-17D by intraperitoneal injection. At 5 days post-infection, viral RNA levels in indicated tissues were measured by qRT-PCR (n = 7, 7, and 9, respectively). b, Indicated LRP4-Fc decoy neutralization activity in vitro and serum levels at day +1 after injection. c, Binding of YFV-M185D/BinJ to indicated LRP4 single LA domain Fc fusion proteins by BLI. Biosensors were coated with indicated Fc-fusion proteins following incubation with YFV-M185D/BinJ. d, Inhibition of YFV-17D infection with indicated concentrations of LRP4-LA (2-4)-3X-Fc fusion protein in HAP1 cells was measured by flow cytometry (anti-E mAbs). e, 500 μg of LRP4-LA (2-4)-Fc or LDLRAD3-LA1-Fc protein were incubated with 105 PFU of YFV-17D at 37 °C for 1 h and then inoculated into AG129 mice via intraperitoneal injection. Mice were also given 500 μg of LRP4-LA (2-4)-Fc or LDLRAD3-LA1-Fc protein at 1- and 2-days after infection (n = 12 and 8, respectively). At 3 d.p.i., viral RNA levels in indicated tissues were measured. f, Wild-type and Lrp4hypo/hypo mice were inoculated in the footpad with 102 PFU of VEEV ZPC-738. At 4 d.p.i., tissues were collected, and viral RNA levels tissues were measured (n = 7 and 11, respectively). Data in (a, e, and f) are pooled from 2 experiments. Bars indicate median values. Data in (c) are representative of 2 experiments. Data in (d) are from 3 experiments, each performed in triplicate. Statistical analysis: one-way ANOVA with Dunnett’s post-test (a, c); two-tailed Mann-Whitney test (e), ns, not significant. The diagram in c was created in BioRender. Diamond, M. (2025) https://BioRender.com/v82694b.

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Extended Data Fig. 7 Ectopic expression of LDLR family members.

a, Diagram of LDLR family members. The indicated LBD domains were expressed ectopically using lentiviruses. b, Expression of indicated LDLR family members in K562 cells was measured by flow cytometry (anti-Flag mAb). The diagram in a was created in BioRender. Diamond, M. (2025) https://BioRender.com/xga4f13.

Extended Data Fig. 8 LRP1 and VLDLR mRNA expression pattern in humans and mice.

a-b, human LRP1 (a) or mouse Lrp1 (b) mRNA levels are showed in indicated tissues. Data were obtained from Human Protein Atlas database (https://www.proteinatlas.org/ENSG00000123384-LRP1/tissue) (a) or BioGPS database (http://biogps.org/#goto=genereport&id=16971) (b). c-d, human VLDLR (c) or mouse Vldlr (d) mRNA levels are showed in indicated tissues. Data were obtained from Human Protein Atlas database (https://www.proteinatlas.org/ENSG00000147852-VLDLR/tissue) (c) or BioGPS database (http://biogps.org/#goto=genereport&id=22359) (d).

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Extended Data Fig. 9 LRP1 and VLDLR interact with YFV virions.

a, LRP1, LRP4 and VLDLR KO efficiency (as indicated by sequences of wild-type allele) in HepG2 cells was analysed by next-generation deep sequencing. b, Diagram of LRP1-CL-I-Fc, VLDLR-LBD-Fc and VLDLR-LA (1-2)-Fc proteins. c, SDS-PAGE gel with Coomassie staining of purified human LRP1-CL-I-Fc and VLDLR-Fc proteins. d, Binding of YFV virions to VLDLR-LBD-Fc fusion protein by BLI. Biosensors were coated with VLDLR-LBD-Fc fusion protein and incubated with YFV-M185D/BinJ virions in solution. e, Inhibition of YFV-17D infection with indicated concentrations of VLDLR-LBD-Fc fusion protein in HepG2 cells was measured by flow cytometry (anti-E mAbs). f, Infection of control K562 cells or those expressing indicated VLDLR variants with YFV-17D RVPs was measured by flow cytometry at 24 h. g, Expression of indicated VLDLR tandem LA domain repeats in (f) was measured by flow cytometry (anti-Flag mAb). h, 500 μg of LRP1-CL-I-Fc or LDLRAD3-LA1-Fc protein was incubated with 105 PFU of YFV-17D at 37 °C for 1 h and then inoculated into anti-IFNAR1 antibody treated C57BL/6J mice via intraperitoneal injection. Mice were also given 500 μg doses of LRP1-CL-I-Fc or LDLRAD3-LA1-Fc protein at 1 and 2 dpi (n = 10 and 7, respectively). At 3 d.p.i., viral RNA levels in indicated tissues were measured. i, 500 μg of LDLRAD3-LA1-Fc or VLDLR-LA (1-2)-Fc proteins were incubated with 105 PFU of YFV-M185D at 37 °C for 1 h and then inoculated into anti-IFNAR1 antibody treated C57BL/6J mice via intraperitoneal injection. Mice were also given 500 μg of the same Fc proteins at 1 and 2 dpi (n = 10 and 8, respectively). At 3 d.p.i., viral RNA levels in indicated tissues were measured. j, 500 μg of LDLRAD3-LA1-Fc or 1:1:1 mixture (167 μg of each) of LRP1-CL-I-Fc, LRP4-LA (2367)-Fc and VLDLR-LA (1-2)-Fc proteins were incubated with 105 PFU of YFV-M185D at 37 °C for 1 h and then inoculated into anti-IFNAR1 antibody treated C57BL/6J mice via intraperitoneal injection. Animals also were given 500 μg of the same Fc proteins at 1 and 2 dpi (n = 10 and 9, respectively). At 3 d.p.i., viral RNA levels in indicated tissues were measured. Data (c, d) are representative of 2 experiments. Data (e-f) are from 3 experiments, each performed in triplicate. Data (h-j) are pooled from 2 experiments, and bars indicate median values. Statistical analysis: two-tailed Mann-Whitney test (h-j), ns, not significant. Source gel data image (c) is in Supplementary Fig. 1. The diagrams in b,d were created in BioRender. Diamond, M. (2025) https://BioRender.com/psypg0x; Diamond, M. (2025) https://BioRender.com/v82694b.

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Extended Data Fig. 10 LRP1-CL-I-Fc decoy protects hFRG mice against YFV infection in vivo.

a, Serum levels of VLDLR-LA (1-2)-Fc and LRP1-CL-I-Fc proteins were measured at 1 day post i.p. injection by ELISA. b, Individual hFRG mouse weight change in LDLRAD3-LA1-Fc or LRP1-CL-I-Fc treated groups after YFV infection. c, LRP1 KO efficiency (as indicated by sequences of wild-type allele) in PHH was analysed by next generation deep sequencing. Data (b) are from 2 experiments. d, Flow cytometry contour plots showing YFV infection in control and LRP1 KO PHH.

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Supplementary information

Supplementary Figure 1 (download TIF )

Original source data of SDS–PAGE gels. The gels in a–c correspond to those in Extended Data Fig. 3a–c. The gel in d corresponds to data in Extended Data Fig. 9c. The red boxes indicate the gel regions that were used to create the Extended Data Figure panels.

Reporting Summary (download PDF )

Supplementary Table 1 (download XLSX )

sgRNA sequences in surfaceome library for used for gene editing.

Supplementary Table 2 (download XLSX )

MAGeCK analysis of genes in CRISPR–Cas9 screen.

Supplementary Table 3 (download XLSX )

Nucleotide sequence of LDLR family members used for expression and targeted screen in K562 cells.

Supplementary Table 4 (download XLSX )

Unnormalized infection data of control cells.

Peer Review File (download PDF )

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Chong, Z., Hui, S., Qiu, X. et al. Multiple LDLR family members act as entry receptors for yellow fever virus. Nature 649, 173–182 (2026). https://doi.org/10.1038/s41586-025-09689-2

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