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LDLRAD3 is a receptor for Venezuelan equine encephalitis virus

Abstract

Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans1. VEEV is a biodefence concern because of its potential for aerosol spread and the current lack of sufficient countermeasures. The host factors that are required for VEEV entry and infection remain poorly characterized. Here, using a genome-wide CRISPR–Cas9-based screen, we identify low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3)—a highly conserved yet poorly characterized member of the scavenger receptor superfamily—as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation with LDLRAD3. LDLRAD3 binds directly to VEEV particles and enhances virus attachment and internalization into host cells. Genetic studies indicate that domain 1 of LDLRAD3 (LDLRAD3(D1)) is necessary and sufficient to support infection by VEEV, and both anti-LDLRAD3 antibodies and an LDLRAD3(D1)–Fc fusion protein block VEEV infection in cell culture. The pathogenesis of VEEV infection is abrogated in mice with deletions in Ldlrad3, and administration of LDLRAD3(D1)–Fc abolishes disease caused by several subtypes of VEEV, including highly virulent strains. The development of a decoy-receptor fusion protein suggests a strategy for the prevention of severe VEEV infection and associated disease in humans.

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Fig. 1: LDLRAD3 is required for efficient VEEV infection in cells.
Fig. 2: LDLRAD3 modulates VEEV attachment and internalization.
Fig. 3: Direct binding of LDLRAD3 to VEEV.
Fig. 4: LDLRAD3 is required for VEEV pathogenesis in mice.

Data availability

All data that support the findings of this study are available within the Article and its Supplementary Information. The Supplementary Tables provide data for the CRISPR–Cas9 screen. Any other relevant data are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This study was supported by NIH grants R01 AI143673 (M.S.D. and D.H.F.), U19 AI142790 (M.S.D. and D.H.F.), U19 AI142759 (M.S.D.), R01 AI095436 (W.B.K.), HHSN272201700060C (D.H.F.), T32 AI007172 (N.M.K. and K.B.), Defense Reduction Threat Agency grants HDTRA1-15-1-0013 (M.S.D.), and HDTRA1-15-1-0047 (W.B.K). We thank G. Bu for discussions, J. Lai for anti-E1 human monoclonal antibodies, N. Ihenacho for technical assistance, M. Elam-Noll for animal husbandry, the Washington University Morphology Core, K. Carlton and J. Mascola for a gift of the VEEV VLPs and S. Whelan for providing comments on the manuscript.

Author information

Affiliations

Authors

Contributions

H.M. performed CRISPR–Cas9 screening, validation and infection studies with assistance from A.S.K., J.T.E. and A.S. A.S.K., K.B. and C.A.N. generated recombinant proteins and performed binding experiments. C.S. created SINV chimaeras and other GFP-expressing alphavirus reagents. N.M.K., J.B.C. and T.C.G. performed the experiments in mice. A.S.K. and L.B.T. designed and analysed the LDLRAD3-deficient mice. H.M., A.S.K., N.M.K., J.T.E., W.B.K., D.H.F. and M.S.D. designed experiments. H.M., A.S.K., N.M.K., J.T.E., K.B. and C.A.N. performed data analysis. H.M., A.S.K. and M.S.D. wrote the initial draft, and the other authors provided editing comments.

Corresponding author

Correspondence to Michael S. Diamond.

Ethics declarations

Competing interests

M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals and Carnival Corporation, and is on the Scientific Advisory Board of Moderna and Immunome. The Diamond laboratory at Washington University School of Medicine has received unrelated sponsored research agreements from Moderna, Vir Biotechnology and Emergent BioSolutions. D.H.F. is a founder of Courier Therapeutics.

Additional information

Peer review information Nature thanks Suresh Mahalingam, Berend Jan Bosch and Jan Carette for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 CRISPR–Cas9-based screen identifying LDLRAD3 as a required factor for VEEV infectivity.

a, ∆B4galt7 N2a cells were transfected separately with two half libraries containing 130,209 sgRNAs, puromycin-selected and then inoculated with SINV–VEEV–GFP (TrD strain) at an MOI of 1. After 24 h, GFP-negative cells were sorted, expanded in the presence of anti-VEEV monoclonal antibodies (VEEV-57, VEEV-67 and VEEV-68 (2 μg/ml)) and re-inoculated with SINV–VEEV–GFP. The infection and sorting process were repeated twice. Genomic DNA from GFP-negative cells was sequenced for sgRNA abundance. b, Representative flow cytometry histogram of parental N2a (grey) and ∆B4galt7 N2a (red) cells stained for heparan sulfate surface expression using R1725, a rodent herpesvirus immune evasion protein that binds to heparan sulfate. c, Schematic of chimeric SINV–VEEV virus. The chimaera contains the non-structural genes from SINV (strain TR339), structural genes from VEEV (IAB strain TrD, IC strain INH9813 or ID strain ZPC738) and an eGFP gene (green) between the capsid and E3 protein. The insertion of GFP has minimal effects on virus infection and replication20,24. d, Sequence alignment of mouse (M. musculus), mouse ∆32 N-terminus isoform, human (Homo sapiens), rhesus macaque (Macaca mulatta), cattle (Bos taurus), horse (Equus caballus), dog (Canis lupus familiaris) and chicken (Gallus gallus) LDLRAD3 ectodomain using ESPript 3. Red boxes indicate conserved residues between orthologues. The predicted domains based on sequence similarity to other related proteins and the transmembrane domains are indicated below the sequence.

Extended Data Fig. 2 Gene editing of LDLRAD3 expression.

a, Parental and gene-edited ∆B4galt7 N2a (top) and BV2 (bottom) cells were subjected to next-generation sequencing to confirm gene editing of Ldlrad3. Sequences were aligned to the Ldlrad3 gene to identify nucleotide insertions or deletions (indels). Allele frequency is indicated next to each sequence. b, Viability of ∆B4galt7 (control, black), ΔB4galt7Ldlrad3 (red) and Ldlrad3-complemented ΔB4galt7Ldlrad3 (blue) N2a (left) and BV2 (right) cells as determined by Cell-Titer Glo assay. Mean ± s.d. of three to six experiments (N2a: n = 12; BV2: control, n = 17; ∆Ldlrad3 + vector, n = 17; ∆Ldlrad3 + Ldlrad3, n = 9). c, Anti-Flag staining of ΔB4galt7 N2a cells (control, black) and lentivirus-complemented ΔB4galt7Ldlrad3 N2a cells with empty vector (red) or Ldlrad3 cDNA (blue) containing an N-terminal Flag-tag sequence (left). Schematic of the Flag-tagged LDLRAD3 protein (bottom) indicating the signal peptide (orange), Flag tag (red), GGS linker (grey) and LDLRAD3 coding region (blue). Cells were stained with an anti-Flag monoclonal antibody and analysed by flow cytometry. Mean ± s.d. of two experiments (n = 6). Representative flow cytometry histograms (right) showing LDLRAD3 surface expression of empty vector (red) and Ldlrad3 (blue)-complemented ∆Ldlrad3 cells. d, Next-generation sequencing confirmation of Ldlrad3 gene editing in N2a (top) and BV2 (bottom) cells retaining heparan sulfate biosynthetic capacity. Allele frequency is indicated next to each sequence. e, Left, B4galt7+/+ (control, black), B4galt7+/+ ∆Ldlrad3 (red) and B4galt7+/+ ∆Ldlrad3 complemented with Ldlrad3 cDNA (blue) N2a cells were analysed for surface expression of LDLRAD3 by flow cytometry using an anti-Flag monoclonal antibody. Mean ± s.d. of two experiments (n = 6). Representative flow cytometry histograms (right) showing LDLRAD3 surface expression of empty vector (red) and Ldlrad3 (blue)-complemented ∆Ldlrad3 cells. f, Next-generation sequencing of LDLRAD3 gene editing in two independent SH-SY5Y cell lines. Allele frequency is indicated next to each sequence. g, Two clonal ∆LDLRAD3 SH-SY5Y cell populations were complemented with full-length Ldlrad3 or truncated Ldlrad3 isoform (N-terminal 32 amino acid deletion, isoform 2) cDNA containing an N-terminal Flag-tag sequence, stained with an anti-Flag monoclonal antibody and analysed by flow cytometry. Representative flow cytometry histograms are shown. h, A second clonal population of ∆LDLRAD3 SH-SY5Y (red) cells were complemented with full-length Ldlrad3 (blue) or the truncated Ldlrad3 isoform (orange), inoculated with SINV–VEEV–GFP (TrD) and infection was assessed by flow cytometry. Mean ± s.d. of three experiments (n = 9; one-way ANOVA with Dunnett’s post-test: ****P < 0.0001). i, ∆B4galt7 (control, black), ∆B4galt7Ldlrad3 (red), Ldlrad3-complemented ΔB4galt7 ∆Ldlrad3 (blue), LDLRAD3-complemented ΔB4galt7 ∆Ldlrad3 (light blue) and N-terminal Flag-tagged Ldlrad3-complemented B4galt7 ∆Ldlrad3 (teal) N2a cells were analysed for LDLRAD3 cell surface expression with anti-LDLRAD3 polyclonal serum. Mean ± s.d. of three experiments (n = 9; one-way ANOVA with Dunnett’s post-test: ****P < 0.0001). Source data

Extended Data Fig. 3 Surface expression of LDLRAD3 and VEEV infection of human lymphocyte cell lines.

a, Representative flow cytometry histograms of LDLRAD3 surface expression using anti-LDLRAD3 polyclonal serum (left) and contour plots of SINV–VEEV–GFP infection (right) of Jurkat cells. b, LDLRAD3-complemented Jurkat cells were assessed for LDLRAD3 surface expression (left) and infection by SINV–VEEV–GFP (TrD) (middle and right). Representative flow cytometry histograms and contour plots are shown. Mean ± s.d. of three experiments (n = 9; Mann–Whitney test: ****P < 0.0001). c, Representative flow cytometry histograms of LDLRAD3 surface expression using anti-LDLRAD3 polyclonal serum (left) and contour plots of SINV–VEEV–GFP infection (right) of Raji cells. d. LDLRAD3-complemented Raji cells were assessed for LDLRAD3 surface expression (left) and infection by SINV–VEEV–GFP (TrD) (middle and right). Representative flow cytometry histograms are shown. Mean ± s.d. of three experiments (n = 9; Mann–Whitney test: ****P < 0.0001). Source data

Extended Data Fig. 4 Surface expression of LDLRAD3 and VEEV infection in different cell lines.

a, b, Representative flow cytometry histograms of LDLRAD3 surface expression using anti-LDLRAD3 polyclonal serum (a) and contour plots of SINV–VEEV–GFP infection (b) of 293T, 3T3, A549, HAP1, HeLa, hCMEC/D3, HT1080, Huh7.5, K562, LADMAC, MRC-5 and U2OS cells. The population of infected cells are indicated for each cell line (b). Data are representative of two or three experiments.

Extended Data Fig. 5 Assessment of LDLRAD3 surface expression and VEEV infection in gene-edited cell lines and primary cells.

a, Control and ∆LDLRAD3 or ∆Ldlrad3 293T, 3T3, HeLa and hCMEC/D3 cells were assessed for LDLRAD3 surface expression (left) and SINV–VEEV–GFP (TrD) infection via GFP expression by flow cytometry (right). Two independent Ldlrad3 or LDLRAD3 gene-edited cell lines were generated (sgRNAs no. 1 and no. 2) and evaluated. Mean ± s.d. of three experiments (LDLRAD3 surface expression, n = 6; VEEV infection, n = 9; one-way ANOVA with Dunnett’s post-test: ****P < 0.0001). b, Primary cell lines (CADMEC, HDF, HPBM and HPBT) were assessed for LDLRAD3 surface expression using anti-LDLRAD3 polyclonal serum (left) (red). Cells were inoculated with SINV–VEEV–GFP (TrD) and assessed for infection via GFP expression by flow cytometry (right) (orange). The population of infected cells are indicated for each cell line. Data are representative of two or three experiments. Source data

Extended Data Fig. 6 Expression and characterization of recombinant LDLRAD3–Fc, VEEV structural proteins and domain-truncated forms of LDLRAD3 proteins.

a, b, Coomassie-stained SDS–PAGE under non-reducing (NR) and reducing (R) conditions of mouse LDLRAD3 domain variants (D1, D1-HRV, D2 and D1+D2) fused to mouse IgG2b Fc domain (a) and LDLRAD3(D1) fused to human IgG1 Fc domain (b). Data are representative of two experiments. c, Binding of human LDLRAD3(D1)–Fc, CHIKV positive control (humanized CHK-152 (CHK-152)), or negative control (humanized E16 (E16)) to VEEV (top) or CHIKV (bottom) VLPs by ELISA. Mean ± s.d. of two experiments (n = 8). d, Silver-stained SDS–PAGE of LDLRAD3(D1-HRV)–Fc ((–)HRV protease) and HRV 3C protease-digested LDLRAD3(D1) ((+)HRV protease) under non-reducing conditions. Data are representative of three experiments. e, Coomassie-stained SDS–PAGE of baculovirus-generated VEEV p62–E1 under non-reducing and reducing conditions. Data are representative of two experiments. f, Binding of LDLRAD3(D1) (2,000 nM starting concentration, twofold dilutions) to CHIKV p62–E1 by surface plasmon resonance. LDLRAD3(D1) does not bind appreciably to CHIKV p62–E1. Cartoon diagram (inset) and sensograms of HRV-cleaved monovalent LDLRAD3(D1) (purple) binding to CHIKV p62–E1 (E3, yellow; E2, cyan; E1, grey). Data are representative of three experiments. g, h, ∆Ldlrad3B4galt7 N2a cells were complemented with either full-length Ldlrad3 (black), Ldlrad3 domain truncations D1+D2 (cyan), D2+D3 (purple) or an Ldlrad3 isoform that lacks 32 N-terminal residues (orange). Cells were assessed for LDLRAD3 surface expression by N-terminal Flag-tag staining (g) and SINV–VEEV–GFP (TrD) infection (h) by flow cytometry analysis. The population of infected cells are indicated for each cell line. Data are representative of three experiments. i, ∆B4galt7Ldlrad3 N2a cells were complemented with either empty vector (red) or LDLRAD3 D1 truncation (blue), inoculated with SINV–VEEV–GFP, and infection was assessed by flow cytometry (left). A representative flow cytometry plot of LDLRAD3(D1)-complemented ∆B4galt7Ldlrad3 N2a cells infected with SINV–VEEV–GFP (TrD) infection is shown. Mean ± s.d. of three experiments (n = 9; one-way ANOVA with Dunnett’s post-test: ****P < 0.0001). Flow cytometry histogram of LDLRAD3(D1) surface expression as assessed by N-terminal Flag-tag staining and flow cytometry analysis (middle). Data are representative of two experiments. SINV–VEEV–GFP (TrD) infection of ∆B4galt7 (control, black), ∆B4galt7Ldlrad3 (red) and Ldlrad3(D1)-complemented ∆B4galt7Ldlrad3 (blue) cells was normalized for Flag-positive cells (right). Mean ± s.d. of three experiments (n = 9; one-way ANOVA with Dunnett’s post-test: ****P < 0.0001). For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 7 Weight change and clinical assessment of C57BL/6J and CD-1 mice treated with LDLRAD3(D1)–Fc.

a, b, Four-week-old C57BL/6J mice were administered 750 μg of anti-IFNAR1 monoclonal antibody via intraperitoneal (i.p.) route 24 h before virus inoculation. Two hundred and fifty μg of LDLRAD3(D1)–Fc or isotype control monoclonal antibody JEV-13 was given 6 h before (a) or 24 h after (b) i.p. inoculation with 105 FFU of SINV–VEEV TrD. Mice were monitored for weight change. Mean ± s.d. from two or three experiments (a: n = 15; b: n = 10; two-way ANOVA with Dunnett’s post-test: *P < 0.05; **P < 0.01, ****P < 0.0001; n.s., not significant). a, One day post infection (dpi), P = 0.0271; b, 1 dpi, P = 0.9978; 2 dpi, P = 0.9940; 3 dpi, P = 0.0082. c, Six-week-old C57BL/6J mice were administered 250 μg of LDLRAD3(D1)–Fc or isotype control monoclonal antibody JEV-13 via i.p. route 6 h before subcutaneous inoculation with 102 FFU of VEEV ZPC738. Mice were monitored for weight change. Data are mean ± s.d. from two experiments (n = 10; two-way ANOVA with Dunnett’s post-test for weight change: *P < 0.05, ***P < 0.001, ****P < 0.0001; n.s., not significant). One dpi, P > 0.9999; 2 dpi, P = 0.05; 8 dpi, P = 0.0001. df, Six-week-old CD-1 mice were administered 200 μg of LDLRAD3(D1)–Fc or isotype control monoclonal antibody JEV-13 via i.p. route 6 h before subcutaneous (d) or intracranial (e, f) inoculation with 103 PFU of VEEV TrD. Mice were monitored for weight change (left) and clinical disease (right) was assessed over time (healthy, ruffled fur, hunched posture, seizures, ataxia, moribund or death). Mean ± s.d. from two experiments (two-way ANOVA with Dunnett’s post-test for weight change: *P < 0.05, ****P < 0.0001; n.s., not significant; d, JEV-13, n = 7; LDLRAD3(D1)–Fc, n = 8; e, n = 10). d, One dpi, P = 0.8267; 2 dpi, P = 0.0531; 3 dpi, P = 0.032; e, 1 dpi, P > 0.9999; 2 dpi, P = 0.2961; 3 dpi, P = 0.0482. At 4.5, 5.5, 8 and 14 dpi, IVIS imaging was used to visual VEEV TrD luciferase infection in CD-1 mice that received LDLRAD3(D1)–Fc or isotype control monoclonal antibody JEV-13 prophylactic treatment and were challenged via intracranial inoculation (f). Isotype control treated mice became moribund at 4.5 dpi. The total flux (photons s−1) in the head region of each mouse was quantified. IVIS images shown are representative images from two experiments (n = 10). Source data

Extended Data Fig. 8 RNA in situ hybridization and histopathological analysis of VEEV infection in LDLRAD3(D1)–Fc- or isotype-control-treated mice.

ad. Six-week-old C57BL/6J mice were administered 250 μg of isotype control monoclonal antibody JEV-13 (a, b) or LDLRAD3(D1)–Fc (c, d) via intraperitoneal route 6 h before subcutaneous inoculation of 102 FFU of VEEV ZPC738. Six days post-infection, brain tissues were collected, fixed, paraffin-embedded and subjected to RNA in situ hybridization using VEEV ZPC738-specific probes (a, c) and haematoxylin and eosin staining (b, d). Scale bars, 2 mm. Representative high-power (10×) magnification insets of the olfactory bulb (1), cortex/midbrain (2), thalamus (3), cerebellum (4) and hippocampus (5) are shown for isotype control (a, top) or LDLRAD3(D1)–Fc (c, bottom) treated mice. Scale bars, 100 μm. Haematoxylin and eosin staining of brain sections from isotype control- (b) or LDLRAD3(D1)–Fc (d)-treated mice. Scale bars, 2 mm. Representative high-power (10×) magnification insets of the cerebral cortex (6), thalamus (7), cerebellum (8) and hippocampus (9) are shown for isotype control- (b, top) or LDLRAD3(D1)–Fc (d, bottom)-treated mice. Scale bars, 100 μm. Representative images from one experiment (n = 5 per group) are shown. Source data

Extended Data Fig. 9 Generation and clinical assessment of C57BL/6 mice with deletions in Ldlrad3 by CRISPR–Cas9 gene targeting.

a, Scheme of Ldlrad3 gene locus with two sgRNA targeting guides for a site in exon 2 of both isoforms. The full-length and truncated ∆32 N terminus residue Ldlrad3 isoforms are coloured red (top) and orange (bottom), respectively. b, Sequencing and alignment of Ldlrad3 sgRNA targeting region in exon 2 (11- and 14-nucleotide frameshift deletions) in gene-edited Ldlrad3 mice. The amino acid residues and the two sgRNA guides used for gene-editing (blue and orange arrows) are indicated above. c, d, Seven-week-old male and female mice with deletions in Ldlrad3 (Δ11 or Δ14 nucleotides; homozygous or compound heterozygous) or wild-type C57BL/6 mice were inoculated subcutaneously with 103 PFU of VEEV TrD (c, left) or 102 FFU of VEEV ZPC738 (d). Mice were monitored for weight change. Data are from two experiments (VEEV TrD: WT, n = 12; ∆Ldlrad3, n = 10; VEEV ZPC738: WT, n = 9; ∆Ldlrad3, n = 8; two-way ANOVA with Dunnett’s post-test: **P < 0.01, ****P < 0.0001; n.s., not significant). c, One dpi, P > 0.999; 2 dpi, P = 0.2136; 3 dpi, P = 0.5489; 4 dpi, P = 0.0065; 8 dpi, P = 0.0014. d, One dpi, P = 0.8383; 2 dpi, P = 0.001. Clinical disease (right) was assessed over time (healthy, ruffled fur, hunched posture, seizures, ataxia, moribund or death) in mice inoculated with VEEV TrD (c, right).

Extended Data Fig. 10 Ldlrad3 mRNA expression in tissues from mice.

a, Generation of a TaqMan primer/ probe set against the Ldlrad3 gene targeting exons 2 and 3. b, c, Profile of Ldlrad3 mRNA expression in different mice tissues (b) and the brains of wild-type and Ldlrad3-deficient mice (c). Data are the mean ± s.d. of one experiment (b, spinal cord, kidney, superior cervical lymph node, heart, brain, lung, colon, liver, muscle, jejunum, spleen, inguinal lymph node, ileum and pancreas, n = 5; testis and ovary, n = 3; c, n = 3). d, In situ hybridization (brown) of Ldlrad3 (olfactory bulb, cortex, thalamus, and hippocampus) from wild-type mice (left). Ldlrad3 RNA puncta are indicated by left-pointing red arrows. A Zika virus (ZIKV) RNA in situ hybridization probe was used as a negative control (right). Slides were counterstained with Gill’s haematoxylin. Representative high-power (63×) magnification images from n = 5 per group are shown. Scale bar, 10 μm. Source data

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Supplementary Figure 1. Uncropped gels for indicated Extended Data Figures.

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Supplementary Table 1. List of genes and scores after MAGeCK analysis.

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Ma, H., Kim, A.S., Kafai, N.M. et al. LDLRAD3 is a receptor for Venezuelan equine encephalitis virus. Nature 588, 308–314 (2020). https://doi.org/10.1038/s41586-020-2915-3

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