Abstract
Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), a severe and sometimes life-threatening disease characterized by viral invasion of the central nervous system with symptoms of neuroinflammation1,2. As with other orthoflaviviruses—enveloped, arthropod-borne RNA viruses—host factors required for TBEV entry remain poorly defined. Here we used a genome-scale CRISPR–Cas9-based screen to identify LRP8, an apolipoprotein E and reelin receptor with high expression in the brain, as a TBEV receptor. LRP8 downregulation reduced TBEV infection in human cells, and its overexpression enhanced infection. LRP8 bound directly to the TBEV E glycoprotein and mediated viral attachment and internalization into cells. An LRP8-based soluble decoy blocked infection of human cell lines and neuronal cells and protected mice from lethal TBEV challenge. LRP8’s role as a TBEV receptor has implications for TBEV neuropathogenesis and the development of antiviral countermeasures.
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Data availability
All data associated with this study are provided in the Article and its Supplementary Information. FASTQ files for CRISPR screens are available at the SRA BioProject under accession number PRJNA1226671: ‘Genome-wide CRISPR screen for tick-borne encephalitis virus host dependency factors’. The following sequences available at NCBI GenBank were used in this study: flavivirus genome sequences (KC806252, DQ401140.3, NC_003687, EF571853.1, OP037819.1, U27495); open reading frames encoding flavivirus C/prM/E (NC_001672, NC_003687.1, AGI15884, KF667312.1, KF769016.1); LRP8 (NM_004631, NM_053073.3); and PCDH1 (NM_002587). Source data are provided with this paper.
Code availability
Custom bash and MATLAB scripts for NGS data processing and CellProfiler pipelines are available at GitHub (https://github.com/chandranlab/mittler_2024).
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Acknowledgements
We thank K. Paez, E. Valencia and M. Ramirez, K. Dempsey, C. O’Brien, A. I. Kuehne and N. M. Josleyn for laboratory management and technical assistance; K. Cogliano for programmatic and administrative support; K. Stiasny for providing TBEV monoclonal antibodies B2 and B4; the members of the Einstein Epigenomics Core and Computational Genomics Core for NGS analysis and data analysis support; the staff at the Einstein Flow Cytometry Core Facility (supported by the Einstein National Cancer Institute’s cancer center support grant P30CA013330) for surface flow cytometry sorting of cell lines; the members of the Einstein Macromolecular Therapeutics Development Facility for protein characterization by size-exclusion chromatography; and the staff at the Karolinska Institutet ANA Futura BSL3 Core Facility (supported by the Infrastructure Board at Karolinska Institutet) for enabling virus studies under high-containment. US National Institutes of Health grant R01AI132633 (K.C.); Marianne and Marcus Wallenberg Foundation (S.G.-R.); Region Stockholm (Clinical Research Appointment) (S.G.-R.); Swedish Research Council (Dnr 2020-06249 and 2021-06602) (S.G.-R.); Swedish Research Council (Dnr 2020-06224) (A.K.Ö.); US National Institute of Allergy and Infectious Diseases grants R01AI165932 and R01AI174584 (B.M.); Congressionally Directed Medical Research Programs grant HT9425-24-1-0873 (C.F.); US National Institute of Allergy and Infectious Diseases grant R21AI182834 (C.K.M.); and French National Research Agency grants ANR-18-CE92-0006, ANR-22-CE35-0004 and ANR-10-LABX-62-10 IBEID (F.A.R.). Opinions, conclusions, interpretations and recommendations are those of the authors and are not necessarily endorsed by the US Department of the Army, the US Department of Defense or the US Department of Health and Human Services.
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Authors and Affiliations
Contributions
B.M. and J.H. generated and validated the CRISPR–Cas9 KO library in A549 cells. R.V. performed the CRISPR–Cas9 genetic screen and NGS, the latter with support from E.M.; K.C. and E.M. analysed NGS data. D.H. and A.L.T. established the single-cycle RVP system. A.L.T., J.J. and E.M. generated cell lines, RVPs and recombinant proteins and performed infectivity studies with RVPs. P.-T.-H.T., C.F., J.J., E.M., M.L., E.C., E.R.W., W.C. and C.K.M. performed infectivity studies with authentic viruses. E.K. generated cell lines lacking expression of LDLR homologues with assistance of J.B. R.M.O., G.B.-S., F.A.R., E.R., A.K.Ö. and M.M.S. provided critical reagents. A.L.T. and E.M. designed and executed immunoprecipitation, ELISA and BLI experiments. V.K.M., A.L.T. and E.M. designed and executed MST experiments. P.-T.-H.T. performed virus attachment and internalization experiments, including analysis of confocal microscopy data. C.F. performed in vivo challenge studies with assistance from R.R.B., as well as ISH studies with support from X.Z., J.L.R. and S.K.; P.-T.-H.T. generated and characterized iPS-cell-derived neurons with critical support by M.S. and A.F.; A.K.Ö., A.S.H., M.S., C.F., M.K., A.F., E.M., F.A.R., E.G., G.B.-S., J.K., K.C. and S.G.-R. provided project supervision and participated in study conceptualization. K.C. and S.G.-R. acquired funding. C.F., E.M., A.L.T., P.-T.-H.T. and K.C. wrote the original draft of the manuscript and all of the authors participated in reviewing and editing.
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K.C. and A.S.H. are scientific advisors to and hold equity in Integrum Scientific. K.C. holds equity in Eitr Biologics. A.F. is the chief scientific officer of CCRM Nordic. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Analysis of rank uniqueness of top hits in CRISPR/Cas9 screen.
The rank uniqueness of the top 100 gene hits in our screen relative to the top 100 hits in each of 25 previously published flavivirus screen datasets was analysed on the GeneRaMeN webserver13. Rank uniqueness was determined using the Robust Rank Aggregation (RRA) method with a significance cutoff of p = 0.05. A heatmap of the top 50 unique ranks in each screen is shown. The colour scale indicates the rank of each hit in its original screen. The gray boxes indicate genes that were not present in the gene list for the screen. Both hit genes and screens are clustered according to similarity. The top 10 hits of our screens are indicated in red.
Extended Data Fig. 2 Establishment and characterization of cell lines with reduced or increased LRP8 expression.
a, A549 single-cell LRP8-knockout (KO) clones were generated by CRISPR/Cas9 engineering. Sequences of LRP8-KO alleles in clones 1 and 2 are shown. The sgRNA target sequence is highlighted in blue. b, A549 LRP8-KO and wild-type (WT) cell lines were immunostained with an anti-LRP8 antibody and visualized by fluorescence microscopy. Experiments were performed three times with similar results. Scale bar, 20 μm. c (left panel), WT and A549 LRP8-KO cell lines as well as A549 LRP8-KO cells overexpressing Flag-tagged LRP8 were immunostained with an anti-Flag mAb and visualized by fluorescence microscopy. The experiment was performed twice and representative images from one experiment are shown. Scale bar, 20 μm. (Right panel), LRP8 expression in indicated cells was determined by western blotting using LRP8-specific antibodies. The housekeeping protein β-actin was included as a loading control. Data from one experiment representative of two independent experiments are shown. Mr, relative molecular weight (K denotes ×1,000). d, A549 cells were treated with siRNAs targeting LRP8, VLDLR, and LRP1 or non-targeting (NT) siRNAs, respectively. Reduction of LRP8, VLDLR and LRP1 expression was determined by western blotting. The housekeeping protein GAPDH was included as a loading control. Experiments were performed two times with similar results. Mr, relative molecular weight (K denotes ×1,000). e (left panel), A549 clones overexpressing Flag-tagged LRP8 and WT cell lines were either subjected to immunostaining with an anti-LRP8 antibody to detect cell surface-localized LRP8 (‘Surface’ staining) or alternatively permeabilized and immunostained with an anti-Flag mAb to detect LRP8 throughout the cell (‘Intracellular’ staining). Images from one experiment representative of three independent experiments with similar results are shown. Scale bar, 20 μm. (Right panel), LRP8 expression in WT A549 cells and cells overexpressing Flag-tagged LRP8 was determined by western blotting using LRP8-specific antibodies. The housekeeping protein β-actin was included as a loading control. The experiment was performed twice and representative results from one experiment are shown. Mr, relative molecular weight (K denotes ×1,000). For gel source data, see Supplementary Information Fig. 2.
Extended Data Fig. 3 LRP8 does not support entry by divergent flaviviruses.
a, A549 WT, LRP8-KO and LRP8-overexpressing cell lines were exposed to TBEVToro and TBEVHypr. Infection levels were determined at 6 h.p.i. (TBEVHypr) and 24 h.p.i. (TBEVToro). Averages ± s.d. from three independent experiments are shown (n = 6). n indicates the number of biologically independent replicates. Comparisons are presented between WT and LRP8-KO or LRP8-overexpressing cells, respectively; data was analysed by one-way ANOVA with Dunnett’s test: ****, P < 0.0001; ***, P = 0.0001. b, A549 WT, LRP8-KO and LRP8-KO cells complemented with LRP8 cDNA were exposed to TBEVHypr and infection levels were determined at 6 h.p.i. Averages ± s.d. from two experiments are shown (n = 4), in which n indicates the number of biologically independent samples. Comparisons are presented between WT and LRP8-KO or LRP8-overexpressing cells, respectively; data was analysed by one-way ANOVA with Dunnett’s test: ****, P < 0.0001. c, A549 WT, LRP8-KO and LRP8-overexpressing cell lines were exposed to Semliki Forest virus (SFV), Sindbis virus (SINV), Langat virus (LGTV), West Nile virus (WNV), Zika virus (ZIKV), Dengue virus type-2 (DENV-2), Japanese Encephalitis virus (JEV), and Powassan virus (POWV). Infection levels were determined at 16 h.p.i. (SFV, SINV), 24 h.p.i. (LGTV, WNV, JEV), 30 h.p.i. (ZIKV), 48 h.p.i. (POWV), and 65 h.p.i. (DENV-2). Averages ± s.d. from three [n = 12 (SFV, SINV), n = 6 (LGTV, WNV, JEV, POWV)], four (n = 7; ZIKV), or six (n = 18; DENV-2) independent experiments are shown. Comparisons are shown between WT and LRP8-KO or LRP8-overexpressing cells, respectively; data was analysed by one-way ANOVA with Dunnett’s test: ****, P < 0.0001; **, P = 0.0026; *, P = 0.0107. d (left panel), Human Huh-7.5.1-SpCas9 cells were transduced to express LRP8-targeting sgRNA and reduction in LRP8 expression was determined by western blotting using an LRP8-specific antibody. The housekeeping protein β-actin was included as a loading control. Mr, relative molecular weight (K denotes ×1,000). For gel source data, see Supplementary Information Fig. 3. (Right panel), sgRNA-treated A549 cells were exposed to TBEVNeudörfl, POWV, JEV, WNV and yellow fever virus (YFV) RVPs. Cells were scored for infection at 24 h.p.i. Averages ± s.d. are shown, n = 18 from six experiments (TBEVNeudörfl), n = 9 from three experiments (POWV), n = 6 from two experiments (YFV, JEV, WNV). Comparisons are shown between control and sgRNA-treated cells and were analysed by one-way ANOVA with Dunnett’s test: ****, P < 0.0001.
Extended Data Fig. 4 Expression of LRP8 variants in A549 cells and expression, purification and characterization of soluble LRP8 and PCDH1-EC1 proteins.
a, A549 cells expressing endogenous levels of LRP8 (WT) or cells overexpressing the indicated Flag-tagged LRP8 variants were immunostained with an anti-Flag mAb and visualized by fluorescence microscopy. Experiments were performed two times with similar results. Scale bar, 20 µm. b, A549 cells expressing endogenous levels of LRP8 (WT) or cells overexpressing the indicated Flag-tagged LRP8 variants were lysed and LRP8 expression levels were visualized by western blotting. The housekeeping protein β-actin was included as a loading control. Western blot images from one experiment representative of three independent experiments are shown. Mr, relative molecular weight (K denotes ×1,000). For gel source data, see Supplementary Information Fig. 3. c, Purified soluble LRP8-LA1-2, LRP8-LA1 and PCDH1-EC1 C-terminally fused to human IgG1 Fc tags were resolved on an SDS-polyacrylamide gel under non-reducing (- dithiothreitol, DTT) or reducing (+ DTT) conditions and visualized by Coomassie blue staining. Experiments were performed three times with similar results. Mr, relative molecular weight (K denotes ×1,000). For gel source data, see Supplementary Information Fig. 3. d, Analytical size-exclusion chromatography (SEC) of proteins from c. Absorbance (milli-absorbance units, mAu) was monitored at 280 nm.
Extended Data Fig. 5 Mapping of LRP8:E binding.
a, TBEV RVPs were added to mAb 4G2-coated plates and capture of the indicated human IgG1 proteins was measured by ELISA. A450nm, absorbance at 450 nm. Averages ± s.d. are shown from three independent experiments, n = 6, where n indicates the number of biologically independent samples. Comparisons are between EC1-Fc and LRP8-LA1-Fc or LRP8-LA1-2-Fc, respectively and were analysed by two-way ANOVA with Tukey’s correction: ****, P < 0.0001; ***, P = 0.006; *, P = 0.0427. b, Sensorgrams of binding of TBEV RVPs (quantified based on TBEV E [nM]) to sensors coated with EC1-Fc as measured by BLI. Data presented are from one representative measurement of two independent experiments (n = 2). c, Sensorgrams of binding of JEV RVPs (quantified based on JEV E [nM]) to sensors coated with LRP8-LA1-2-Fc as measured by BLI. Data presented are from one representative measurement of two independent experiments (n = 2). d, Monomeric purified TBEVNeudörfl E domain III (DIII) bearing a C-terminal hexa-histidine tag was resolved on an SDS-polyacrylamide gel (under nonreducing and reducing conditions [-/+ DTT]) and visualized by Coomassie blue staining. Experiment was performed three times with similar results. Mr, relative molecular weight (K denotes ×1,000). e, TBEVNeudörfl E DIII was captured onto anti-His mAb-coated plates and the binding of decreasing amounts of LRP8-LA1-2-Fc and -LA1-Fc was determined by ELISA. A450nm, absorbance at 450 nm. Averages ± s.d. are shown from three independent experiments, n = 6, where n indicates the number of biologically independent samples. f, LRP8-LA-1-2-Fc was coated onto plates and capture of TBEV soluble E protein (sE) was measured by ELISA. A450nm, absorbance at 450 nm. Averages ± s.d. are shown from two independent experiments, n = 4. g, TBEV sE was incubated with LRP8-LA1-2-Fc and subsequently captured with Protein G magnetic beads. Co-immunoprecipitated protein complexes and a fraction of the input material were analysed by western blot. Data from one experiment representative of two independent experiments (n = 2) are shown. Mr, relative molecular weight (K denotes ×1,000). h, TBEV and POWV-I E DIII bearing a C-terminal COMP pentamerization domain followed by a hexa-histidine tag were resolved on an SDS-polyacrylamide gel under nonreducing and reducing conditions (-/+ DTT) and visualized by Coomassie blue staining. Experiment was performed three times with similar results. Mr, relative molecular weight (K denotes ×1,000). i, Analytical SEC of COMP-tagged TBEV E DIII. Absorbance (mAu) was monitored at 280 nm. j, TBEV and POWV-I E DIII C-terminally fused to a COMP domain were captured onto anti-His mAb-coated plates and the binding of decreasing amounts of LRP8-LA1-2-Fc was determined by ELISA. A450nm, absorbance at 450 nm. Averages ± s.d. are shown from two independent experiments, n = 5. k, Binding of EC1-Fc to TBEV E DIII-COMP as measured by microscale thermophoresis (MST). Results from one experiment (shown as mean ± s.e.m. of three technical replicates) representative of two independent experiments are shown (n = 2). For gel source data, see Supplementary Information Fig. 3.
Extended Data Fig. 6 LRP8 boosts TBEV E-mediated attachment to host cells.
a, A549 WT, LRP8-KO and cells overexpressing LRP8 variants (LA1-7, LA1-3, LA1-2, and LA1) were exposed to TBEVSofjin (MOI, 10 IU) at 4 °C. Cells were immunostained for TBEV E, plasma membranes were labelled with wheat germ agglutinin, and cells were imaged by fluorescent confocal microscopy. Images from one experiment representative of >10 randomly captured images of two independent experiments (n = 2) are shown. Scale bar, 50 μm. b (left panel), A549 WT, LRP8-KO and LRP8-overexpressing cells were exposed to rLGTVTBEV-93/783 (MOI, 0.5 IU) at 4 °C and the amounts of bound viral particles were determined by western blotting. GAPDH was included as a loading control. Data from one experiment representative of four independent experiments (n = 4) is shown. Mr, relative molecular weight (K denotes ×1,000). For gel source data, see Supplementary Information Fig. 4. (Right panel), E signal was quantified on a ChemiDoc XRS+ Imaging System using the onboard software. Averages ± s.d. are shown from four experiments, n = 4. WT versus LRP8-KO or LRP8-overexpressing cells, one-way ANOVA with Dunnett’s test: ****, P < 0.0001; ***, P = 0.0003; *, P = 0.0140. c, A549 LRP8-overexpressing cells were exposed to TBEVSofjin (MOI, 10 IU) at 37 °C. Cells were immunostained for TBEV E and the early endosomal marker EEA1 and imaged by fluorescent confocal microscopy. Arrowheads indicate colocalization of TBEV E and EEA1. Images from one experiment representative of >20 randomly captured images of two independent experiments (n = 2) are shown. Scale bar, 50 μm.
Extended Data Fig. 7 LRP8 selectively inhibits TBEV E-mediated infection.
a, Infection of Huh-7.5.1 cells with single-cycle JEV RVPs after pre-incubation with increasing amounts of LRP8-LA1-2-Fc, LRP8-LA1-Fc, or human IgG isotype control. Averages ± s.d. are shown from three independent experiments, n = 9, where n indicates the number of biologically independent samples. b, Infection of Huh-7.5.1 cells with TBEVHypr after pre-incubation with increasing amounts of LRP8-LA1-2-Fc. Averages ± s.d. are shown from three experiments, n = 6, where n indicates the number of biologically independent replicates. c, Infection of U-87 MG glioblastoma cells with TBEVSofjin after pre-incubation with decreasing amounts of LRP8-LA1-2-Fc. Averages ± s.d. are shown from three experiments, n = 6. n indicates the number of biologically independent samples. Comparisons are shown between cells infected with untreated and LRP8-incubated virus and were analysed by one-way ANOVA with Dunnett’s test: ****, P < 0.0001.
Extended Data Fig. 8 Characterization of neural progenitor cells and TBEV infection of neuronal cell cultures differentiated for 60 days.
a, Neural progenitor cells (NPCs) were immunostained for SOX2, NESTIN, GFAP, OLIG2, and PDGFRα and their cellular distribution was visualized by fluorescence microscopy. The number of NPCs expressing the indicated marker proteins was quantified manually or using ImageJ and a custom analysis pipeline in CellProfiler. Pooled data from one experiment is shown (n ≥ 400 individual cells per marker protein). Arrowheads indicate GFAP+ cells. Scale bar, 100 μm. b, Neural cells were differentiated in culture for 60 days, immunostained for the neuronal markers NeuN, MAP2, PSD-95, βIII-tubulin, LRP8 and GFAP, and their cellular distribution was visualized by fluorescence microscopy. Arrowheads indicate GFAP+ cells. Representative images of one experiment per condition are shown. Scale bar, 100 μm. c, The number of neural cell bodies (6.1 μm extension from the perimeter of the nuclei) from cells characterized in b and co-expressing LRP8 and βIII-tubulin or LRP8 and GFAP, respectively was quantified using ImageJ and a custom analysis pipeline in CellProfiler. Pooled data from one experiment is shown (n = 6000 individual cells per protein combination). d (left panel), Infection of neuronal cells characterized in b with TBEVSofjin (MOI, 0.02 IU) after pre-incubation with LRP8-LA1-2-Fc or human IgG (400 nM). Cells were subjected to immunostaining followed by immunofluorescence microscopy. Images from one experiment representative of two independent experiments are shown. Scale bar, 100 μm. (Right panel), TBEV-infected neural cell bodies in fluorescent images were enumerated using ImageJ and a custom analysis pipeline in CellProfiler. Pooled data from two independent experiments is shown as averages ± s.d. [n = 29 (human IgG) and n = 23 (LRP8-LA1-2-Fc), in which n represents fluorescent images]. Comparisons are presented between cells infected with human IgG- or LRP8-LA1-2-Fc-treated TBEV, respectively; data was analysed by two-tailed unpaired t-test: ****, P < 0.0001.
Extended Data Fig. 9 Murine LRP8 ortholog boosts TBEV infection.
a, Amino-acid sequence alignment of LRP8’s ligand-binding domain (LBD) of human (HsLRP8) and murine (MmLRP8) orthologs is shown. Signal sequences (HsLRP8: aa 1–32; MmLRP8: aa 1–26) are not shown, and residue numbering reflects the mature sequences. The LA repeats are marked. Orange shading, divergent residues. b, Overexpression of murine and human LRP8 orthologs in A549 WT cells was determined via western blotting. The housekeeping protein β-actin was included as a loading control. Experiment was performed two times with similar results. Mr, relative molecular weight (K denotes ×1,000). For gel source data, see Supplementary Information Fig. 4. c, A549 WT and LRP8-overexpressing cell lines were exposed to TBEV RVPs and infection levels were determined at 24 hpi. Averages ± s.d. from four independent experiments are shown (n = 12; n indicates the number of biologically independent samples). Comparisons are presented between WT and LRP8-overexpressing cells, respectively; data was analysed by one-way ANOVA with Dunnett’s test: ****, P < 0.0001.
Extended Data Fig. 10 Weight changes, clinical scores and in situ hybridization scores of the TBEV-challenged mice study cohorts.
a, C57BL/6 mice were challenged with TBEVHypr (1,000 PFU, i.p.) pre-complexed with LRP8-LA1-2-Fc, EC1-Fc or vehicle (PBS) and monitored daily for weight loss for 28 days. Averages from two independent experiments are shown and data are represented as mean ± s.d., n = 20 mice per group. b, Clinical scores of challenged animals in a were monitored for 28 days. Averages from two experiments are shown, n = 20 mice per group. c, C57BL/6 mice were administered LRP8-LA1-2-Fc, EC1-Fc or vehicle 6 h prior to TBEVHypr challenge (1,000 PFU, i.p.) and were subsequently monitored daily for weight loss for 28 days. Averages from two independent experiments are shown and data are represented as mean ± s.d., n = 19 mice (EC1-Fc) and n = 20 mice (vehicle, LRP8-LA1-2-Fc). d, Clinical scores of challenged animals in c were monitored for 28 days. Averages from two experiments are shown, n = 19 mice (EC1-Fc) and n = 20 mice (vehicle, LRP8-LA1-2-Fc). e, In situ hybridization (ISH) scores from challenged animals in a. On day 6 post TBEVHypr challenge, 5 animals per treatment group were sacrificed, brain tissues harvested and subjected to ISH. TBEV RNA level scoring: +++, RNA-positive cells frequently detected; ++, RNA-positive cells occasionally detected; +, RNA-positive cells rarely detected; -, no RNA-positive cells detected. f, ISH scores from challenged animals in c. Mice brain tissues were harvested, prepared for ISH and scored as described in e.
Supplementary information
Supplementary Figs. 1–4
Supplementary Figs. 1–4.
Supplementary Table 1
CRISPR–Cas9 screen: MAGeCK data analysis from Fig. 1a.
Supplementary Table 2
CRISPR–Cas9 screen: sgRNA summary from Fig. 1a.
Supplementary Table 3
Analysis of rank uniqueness of top hits in CRISPR–Cas9 screen from Extended Data Fig. 1.
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Mittler, E., Tse, A.L., Tran, PTH. et al. LRP8 is a receptor for tick-borne encephalitis virus. Nature (2025). https://doi.org/10.1038/s41586-025-09500-2
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DOI: https://doi.org/10.1038/s41586-025-09500-2
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