Structure of Venezuelan equine encephalitis virus in complex with the LDLRAD3 receptor

LDLRAD3 is a recently defined attachment and entry receptor for Venezuelan equine encephalitis virus (VEEV)1, a New World alphavirus that causes severe neurological disease in humans. Here we present near-atomic-resolution cryo-electron microscopy reconstructions of VEEV virus-like particles alone and in a complex with the ectodomains of LDLRAD3. Domain 1 of LDLRAD3 is a low-density lipoprotein receptor type-A module that binds to VEEV by wedging into a cleft created by two adjacent E2–E1 heterodimers in one trimeric spike, and engages domains A and B of E2 and the fusion loop in E1. Atomic modelling of this interface is supported by mutagenesis and anti-VEEV antibody binding competition assays. Notably, VEEV engages LDLRAD3 in a manner that is similar to the way that arthritogenic alphaviruses bind to the structurally unrelated MXRA8 receptor, but with a much smaller interface. These studies further elucidate the structural basis of alphavirus–receptor interactions, which could inform the development of therapies to mitigate infection and disease against multiple members of this family.

LDLRAD3 is a recently defined attachment and entry receptor for Venezuelan equine encephalitis virus (VEEV) 1 , a New World alphavirus that causes severe neurological disease in humans. Here we present near-atomic-resolution cryo-electron microscopy reconstructions of VEEV virus-like particles alone and in a complex with the ectodomains of LDLRAD3. Domain 1 of LDLRAD3 is a low-density lipoprotein receptor type-A module that binds to VEEV by wedging into a cleft created by two adjacent E2-E1 heterodimers in one trimeric spike, and engages domains A and B of E2 and the fusion loop in E1. Atomic modelling of this interface is supported by mutagenesis and anti-VEEV antibody binding competition assays. Notably, VEEV engages LDLRAD3 in a manner that is similar to the way that arthritogenic alphaviruses bind to the structurally unrelated MXRA8 receptor, but with a much smaller interface. These studies further elucidate the structural basis of alphavirus-receptor interactions, which could inform the development of therapies to mitigate infection and disease against multiple members of this family.
Alphaviruses enter host cells through receptor-mediated endocytosis 4 . Within the low-pH endosomal compartment, the virion envelope rearranges to enable membrane fusion and nucleocapsid penetration into the cytoplasm 5 . The 12-kilobase alphavirus RNA genome is released after capsid disassembly and is translated from two open reading frames. The structural proteins (capsid, envelope glycoprotein (E)3, E2, 6K and E1) undergo processing and modification in the endoplasmic reticulum-Golgi network. The E2 and E1 proteins facilitate binding to entry factors and subsequent membrane fusion [6][7][8][9] . The E3 protein is essential for the proper folding of p62 (a precursor to E2) and the formation of the p62-E1 heterodimer 10,11 but is cleaved by furin-like proteases during maturation 12 . Mature E2-E1 heterodimers assemble into trimeric spikes at the plasma membrane before budding and release of the virion from the host cell 13 . The 70-nm-diameter mature alphavirus virion comprises 240 E2-E1 heterodimers that are arranged into 80 trimeric spikes with T = 4 icosahedral symmetry [14][15][16] . Twenty of these trimeric spikes sit on the icosahedral three-fold (i3) symmetry axes, and the other 60 spikes sit on the quasi-three-fold (q3) axes.
Low-density lipoprotein receptor class A domain-containing 3 (LDL-RAD3) was recently identified as an attachment and entry receptor for VEEV and shown to be essential for optimal infection in cell culture and pathogenesis in mice 1 . LDLRAD3 is a conserved yet poorly characterized cell-surface protein that is expressed in neurons, epithelial cells, myeloid cells and muscle, the endogenous ligand(s) of which remain unknown. Biolayer interferometry experiments established that domain 1 (D1) of LDLRAD3 (LDLRAD3(D1)) binds directly to VEEV, and anti-LDLRAD3 antibodies and LDLRAD3(D1)-Fc fusion proteins block VEEV attachment and infection of cells. Only VEEV uses LDLRAD3 as a receptor, as EEEV, WEEV and other distantly related alphaviruses do not bind to it. How LDLRAD3 engages VEEV, and why only VEEV binds to LDLRAD3 remain unclear. We set out to address these questions using structural, genetic and biophysical approaches. within each trimeric spike (Fig. 1c). This cleft widens slightly when D1 of LDLRAD3 is bound (Supplementary Video 1). Local resolution estimation performed in RELION revealed heterogenous resolution; the capsid proteins and membrane proximal regions of the E2-E1 heterodimers were best resolved (about 4 Å) and the membrane distal regions and LDLRAD3(D1) were less-well resolved (about 5-6 Å) (Fig. 1d). To avoid under-and over-sharpening of the reconstructions by conventional global B-factor correction, post-processing was performed using Deep-EMhancer 18 . This resulted in improved continuity and reduced noise in the density (Extended Data Fig. 1c). The visibly clear tracing of the carbon backbone simplified subsequent model building.

Atomic model building and refinement
LDLRAD3(D1) was identified as an LDL receptor type A (LA) domain by the Pfam database 19 . LA domains are approximately 40 amino acids in length and contain 6 disulfide-bound cysteine residues and a cluster of conserved acidic residues that coordinate calcium ions (Fig. 2a). The LA domain architecture is well characterized with over 200 structures in the Protein Data Bank (PDB), revealing a highly conserved fold. The initial model of LDLRAD3(D1) was built from its primary amino acid sequence by threading using the SWISS-MODEL server 20 with multiple high-resolution crystal structures of related LA domains as templates. The starting coordinates of the VEEV VLP structural proteins came from a previously built model of the same VEEV strain (PDB: 3J0C; ref. 21 ). Both models were docked into the DeepEMhancer modified electron density of the asymmetric unit and underwent manual and computational real-space refinement using COOT 22 and PHENIX 23 (Methods), with LDLRAD3(D1) unambiguously oriented with the N terminus proximal to the core of the virus particle (Fig. 2b-d and Supplementary Table 2).
The resultant model shows the domains and residues of the VEEV E2-E1 heterodimers at the LDLRAD3-binding interface. The two E2-E1 heterodimers at each binding site were termed 'wrapped' and 'intraspike', as described previously for the structure of CHIKV 23 Table 3). The binding interface is around 900 Å 2 with equal contributions from the interfaces of the wrapped and intraspike E2-E1 heterodimers.   33 ). Contact residues of LDLRAD3(D1) to the wrapped and intraspike VEEV E2-E1 heterodimers are shaded dark and/or light purple, respectively. Contact residues of the cysteine-rich domain 2 of LDLR (LDLR-CR2) and LDLR-CR3 to glycoprotein G of VSV (VSV G) are shaded green and contact residues of VLDL receptor module 3 (VLDLR-V3) to viral protein 1 (VP1) of human rhinovirus 2 (HRV2) are shaded pink, as determined by PDBePISA (www.ebi.ac.uk/pdbe/ pisa/) ( Fig. 4c-e). The brackets and rectangles indicate residues that form disulfide bonds and coordinate calcium, respectively. The figure was prepared using ALINE 33 . b, Ribbon diagram of LDLRAD3(D1) and surface representation of its wrapped and intraspike E2-E1 heterodimers. LDLRAD3(D1) and VEEV E2-E1 are coloured by domain. LDLRAD3(D1) (purple); chain E1: DI (light grey), DII (medium grey), DIII (dark grey) and fusion loop (FL) (orange); chain E2: A domain (cyan), β-linker (medium blue), B domain (dark cyan) and C domain (blue). The disulfide bonds and calcium ion in the ribbon diagram are coloured yellow and green, respectively. c, d, Paired isolated views of electron density and a model of LDLRAD3(D1) and its wrapped (c) or intraspike (d) heterodimers. Wrapped refers to the E2-E1 heterodimer, the fusion loop of which is covered by LDLRAD3. Intraspike refers to the heterodimer adjacent to the wrapped heterodimer but within the same trimeric spike. The naming convention is consistent with previous alphavirus-receptor structural studies 23 . The arrows indicate the regions that are magnified in the insets, which contain views of LDLRAD3(D1). Proteins are coloured by domain as described in b. N-linked glycans are shown as balls and sticks and coloured by heteroatom. The disulfide bonds and calcium ion are coloured yellow and green, respectively.

Functional assessment of the atomic model
To assess our model, non-conservative point mutations were introduced throughout D1 of LDLRAD3 and used for complementation experiments in mouse Neuro2a cells lacking Ldlrad3 (∆Ldlrad3) and glycosaminoglycan (∆B4galt7) expression 1 ; we performed these experiments in cells lacking glycosaminoglycans to minimize background infection, as some alphaviruses also attach to cells through engagement of heparan sulfate moieties 17,24,25 . Wild-type (WT) LDLRAD3 and single point mutants of LDLRAD3 were transduced into ∆B4galt7∆Ldlrad3 Neuro2a cells, which were then inoculated with a chimeric, attenuated SINV-VEEV virus that expresses the structural genes of VEEV Trinidad Donkey (TrD) such that the screen could be performed using flow cytometry at a lower biosafety containment level (BSL2) yet with VEEV structural proteins from a pathogenic subtype IAB isolate. Whereas most mutant forms of LDLRAD3 promoted SINV-VEEV infection, several (including G33D, M36T, P44R and D57V) did not support infection even though the proteins were expressed on the cell surface at similar levels compared to the WT form of LDLRAD3 (Fig. 3a, b and Extended Data Fig. 4). The residues identified as loss-of-function for infectivity all sit in a pocket of LDLRAD3 that supports direct contact with residues of E2-E1 in both the wrapped and intraspike heterodimers (Supplementary Table 3). Several other mutations in LDLRAD3(D1) that correspond to contact residues (including P32D, N39T, A46K and F56D) appear to show slight increases in infectivity with normal surface expression patterns. Although further studies are required, these changes could enhance the affinity of VEEV binding. Several years ago, a high-resolution cryo-electron microscopy (cryo-EM) structure of Fab fragments of the 3B4C-4 mouse monoclonal antibody bound to VEEV was published 26 . 3B4C-4 binds to the tip of the E2 B domain 27 and inhibits cellular attachment and entry of VEEV 28 . As the principal binding footprint (S177, V179, S180, L181, S184, T214, N216 and K223) 26 of this monoclonal antibody is proximal to the LDLRAD3-binding site, we tested whether 3B4C-4 could inhibit binding to LDLRAD3 using a competition binding enzyme-linked immunosorbent assay (ELISA). The 3B4C-4 monoclonal antibody was prebound to VEEV-VLP-coated plates before addition of the LDLRAD3(D1)-human Fc fusion protein. Notably, 3B4C-4 markedly inhibited LDLRAD3(D1) binding, whereas another anti-VEEV monoclonal antibody (TRD-14), which maps to a distinct epitope on the E2 B domain (G203, G204 and T205; N. Kafai and M. Diamond, unpublished data), did not compete for binding (Fig. 3c). A structural comparison of the monoclonal antibody epitopes on the E2 B domain revealed that 3B4C-4 binds to residues that are immediately adjacent to the LDLRAD3-binding site, probably resulting in steric hindrance (Fig. 3d). By contrast, the TRD-14 epitope is located at the distal end of the E2 B domain.

D2 does not contribute to VEEV binding
D1 of LDLRAD3 is necessary and sufficient to support infection by VEEV 1 , but it remains unclear whether D2 also contributes to VEEV binding. To evaluate this question, we expressed soluble LDLRAD3(D1+D2) in Expi293 cells (Extended Data Fig. 5a). Electron micrographs of VEEV VLPs with or without bound LDLRAD3(D1+D2) were acquired using a 300 kV Titan Krios system equipped with a Falcon 4 detector (Supplementary Table 1). Single-particle analysis with imposed icosahedral symmetry yielded a reconstruction at 5.0 Å (Extended Data Fig. 5b). The electron density of D2 of LDLRAD3 was weak and projected away from VEEV (Extended Data Fig. 5c). Binding of purified LDLRAD3(D1+D2) to captured VLPs by surface plasmon resonance yielded a monovalent affinity of approximately 50 nM that was similar to LDLRAD3(D1) (Extended Data Fig. 5d). On the basis of this structural and biophysical analysis, and previous functional data 1 , D2 of LDLRAD3 does not appreciably contribute to VEEV binding or infection.
Cell culture infection experiments with mouse and human cells and in vivo pathogenesis studies in mice defined LDLRAD3 as a cell-surface receptor for VEEV that is required for optimal infectivity and induction of encephalitis in mice 1 . Here, our single-particle cryo-EM analyses of LDLRAD3 and VEEV VLPs provide structural insights into how VEEV engages with LDLRAD3 to facilitate interactions with target cells. We observed a network of quaternary protein-protein interactions with D1 of LDLRAD3 engaging two E2-E1 heterodimers within one trimeric spike. The specific binding determinants that we observed are supported by structure-guided mutations that we introduced into LDLRAD3, and binding competition studies with LDLRAD3(D1) and a neutralizing monoclonal antibody against VEEV that engages the top of the E2 B domain and directly blocks virus attachment. Our structures indicate that D1 of LDLRAD3 can bind with full occupancy at four distinct sites in the icosahedral asymmetric unit of the mature VEEV VLP. VEEV binds to LDLRAD3 in a manner that is notably similar to the binding of CHIKV to its receptor MXRA8, which consists of two immunoglobulin-related folds 23,29,30 (Fig. 4a, b). Although LDLRAD3 and MXRA8 have similar sites of virion engagement, LDLRAD3 forms a significantly smaller interface (about 900 Å 2 versus about 2,100 Å 2 ) even though the monovalent affinity of virus-receptor binding is similar 23 (Extended Data Fig. 5d). Inspection of the contact residues indicates that LDLRAD3 makes greater use of hydrophobic residues to bind to VEEV compared with the use of hydrophobic residues by MXRA8 when binding to CHIKV (about 40% versus about 24% of interface residues, respectively). Approximately 65% of the receptor contact positions on VEEV spikes are shared with CHIKV (Extended Data Figs. 2 and 3). Both receptors effectively shield the hydrophobic fusion loop from solvent access, and all seven of the VEEV E1 contact residues are conserved with CHIKV E1. We speculate that the common positioning of these receptors near the fusion loop might function to modulate viral fusion during endocytosis. However, the primary contact residues used by LDLRAD3 and MXRA8 are not conserved; notably, MXRA8 has a substantial number of histidine residues (7% of the ectodomain) and LDLRAD3(D1) has no histidine residues.
The distinct receptor specificities of VEEV and CHIKV can probably be explained by the low level of sequence conservation of the E2-binding residues (26% of 35 LDLRAD3 contact positions). Our structural analysis also suggests why LDLRAD3 is a receptor for VEEV but not for WEEV and EEEV-other related encephalitic alphaviruses. Other than the aforementioned conserved contact site in the E1 fusion loop (100% conservation for 7 residues), the receptor determinants in E2 of VEEV generally are not conserved in WEEV and EEEV (17% and 23% identity, respectively, for 35 contact residues; Extended Data Figs. 2 and 3). By contrast, these determinants are essentially conserved among VEEV complex members, which probably explains why LDLRAD3 supports infection of all of the VEEV strains (IAB, IC and ID) that we tested 1 .
LDL-receptor family members mediate the entry of several viruses belonging to different families. High-resolution structures have been solved for LDL receptor (LDLR) LA domains in complex with vesicular stomatitis virus (VSV) and human rhinovirus (HRV) 31,32 . Notably, an unrelated negative stranded rhabdovirus (VSV) and non-enveloped picornavirus (HRV) engage the same tryptophan residue near the calcium-binding site of the conserved cysteine-rich domain that also is a major contact for LDLRAD3 (Trp47) (Figs. 2a, Fig. 4c-e). Thus, evolutionarily distinct viruses have evolved similar structural strategies for engaging related members of a protein superfamily to enable entry into target cells. As such, it is plausible that structure-guided design of small-molecule inhibitors could prevent entry of viruses from multiple families.

Online content
Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-021-03963-9.

Recombinant LDLRAD3 protein generation and purification
Monomeric LDLRAD3 ectodomain constructs were prepared as previously described 1 34 ). Contrast transfer function parameters of the electron micrographs were estimated using Gctf 35 , and particles were auto-picked using crYOLO 36 .
Single-particle analysis, specifically reference-free 2D classification, 3D refinement, video refinement, Bayesian polishing, post-processing and local resolution estimation were performed using RELION-3.1 (ref. 37 ). Post-processing of maps for model building and figure presentation was performed using DeepEMhancer 18 . Further information for all of the samples is provided in Supplementary Table 1. Structural visualization of the electron maps was performed using ChimeraX 38 .

Model building and refinement
The initial models of the VEEV structural proteins (E1, E2, transmembrane regions and capsid) with or without LDLRAD3 were constructed by docking the coordinates of the previously built model of VEEV strain TC-83 (PDB: 3J0C; ref. 21 ) and the model of LDLRAD3(D1) predicted by SWISS-MODEL server 20 into the electron density of the asymmetric units of the cryo-EM maps using the fitmap command in ChimeraX. N-linked glycans and coordinated calcium ions were built manually using COOT 22 . The model underwent real-space refinement in PHENIX 23 using the default parameters plus Morphing and secondary-structure, rotamer and torsion restraints with the initial model as the reference. Bond and angle restraints were also applied for the modelled N-linked glycans and calcium ions. After optimization, coordinates of the asymmetric units were checked using MolProbity. Contact residues were identified, and buried surface areas were calculated using PDBePISA (www.ebi.ac.uk/pdbe/pisa/).

Surface plasmon resonance
The binding kinetics and affinity of cleaved LDLRAD3(D1) or LDLRAD3(D1+D2) to VEEV VLPs were measured using the Biacore T200 system (GE Healthcare). Experiments were performed at 30 µl min −1 and 25 °C using HBS-P (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% (v/v) Surfactant P20) plus 1 mM CaCl 2 as running buffer. VEEV-57 monoclonal antibody (anti-VEEV E2, N. Kafai and M. Diamond, unpublished results) was immobilized onto a CM5 sensor chip (GE Healthcare) using standard amine coupling chemistry, and VEEV VLPs were captured. LDLRAD3 proteins were injected over a range of concentrations (1 µM to 16 nM) for 300 s, followed by a 600-s dissociation period. The sensor chip was regenerated after each analyte concentration with 60 s of 10 mM glycine, pH 1.7. Before the next analyte concentrated was tested, VEEV VLPs were recaptured; the response units of captured VLPs were consistent for each cycle. All sensorgrams were double-reference-subtracted using the reference flow cell (immobilized VEEV-57 monoclonal antibody, no captured VLP) and the running-buffer blank sample. The kinetic profiles and steady-state equilibrium concentration curves were fitted using a global 1:1 binding algorithm with a drifting baseline using BIAevaluation v.3.1 (GE Healthcare).

Infection assay
A comprehensive mutation library was generated using gene synthesis by mutating a single amino acid in D1 of the LDLRAD3 protein. The amino acids that are essential for maintaining the structural integrity of LDLRAD3 (the cysteines forming disulfide bonds, the amino acids coordinating the calcium and those forming the hydrophobic core) were kept intact 39 . The substitutions were determined using the BLOSUM scoring matrix 40 and a list of these is provided in Supplementary Table 4. The mutants were cloned into lentivirus vector pLV-EF1a-IRES-Hygro (Addgene, 85134) between the BamHI and MluI restriction enzyme sites (Genscript). An N-terminal Flag tag was added to each LDLRAD3 mutant to monitor protein expression. ∆B4galt7∆Ldlrad3 Neuro2a cells were transduced with each LDLRAD3 mutant and, 7 d later, were inoculated with SINV-VEEV (TrD)-GFP 1 (gift of W. Klimstra, University of Pittsburgh) infection at a multiplicity of infection of 20 for 7.5 h. Cells were stained with anti-Flag antibodies (1:2,000 dilution, Cell Signaling Technology, D6W5B) to measure the surface expression levels of the WT and mutant forms of LDLRAD3. Inoculated and stained cells were analysed using the MACSQuant Analyzer 10 (Miltenyi Biotec), and all flow cytometry data were processed using FlowJo (FlowJo).

Competition binding ELISA
Nunc MaxiSorp plates (Thermo Fisher Scientific) were coated with 2 µg ml −1 of capture monoclonal antibody (mouse anti-VEEV-1A4A) 41 in 100 µl of sodium bicarbonate coating buffer (0.1 M Na 2 CO 3 , pH 9.3) and incubated overnight at 4 °C. Plates were washed four times with PBS and incubated with 150 µl of blocking buffer (PBS, 4% BSA) for 1 h at room temperature. VEEV VLPs were diluted to 1 µg ml −1 in PBS containing 2% BSA and added (100 µl per well) to plates for 1 h at room temperature. After four additional PBS washes, 50 µl of mouse anti-VEEV monoclonal antibody (3B4C-4 or TRD-14) at 20 µg ml −1 in PBS with 2% BSA was added to plates for 30 min at room temperature to allow for binding to VEEV VLPs. Then, 50 µl of human LDLRAD3(D1)-Fc at 20 µg ml −1 was added directly, with no additional washes. One hour later, the plates were washed four times with PBS and incubated with 100 µl per well 1:5,000 horseradish-peroxidase-conjugated goat anti-human IgG (H+L; Jackson ImmunoResearch) diluted in PBS with 2% BSA for 1 h at room temperature for detection of LDLRAD3(D1)-Fc binding. The plates were washed four times with PBS and then incubated with 100 µl of 3,3′,5,5′-tetramethylbenzidine substrate (Thermo Fisher Scientific) for 3 min at room temperature before quenching by addition of 50 µl of 2 N H 2 SO 4 . Absorbance was read at an optical density of 450 nm using the TriStar Microplate Reader (Berthold Technologies).

Statistical analysis
Statistical significance was assigned when P < 0.05 using Prism (v.8, GraphPad) and is indicated in each of the figure legends. Cell culture or ELISA experiments were analysed using one-way ANOVA.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Data availability
All data supporting the findings of this study are available within the paper and its Supplementary Information. All structures have been deposited in the PDB and Electron Microscopy Data Bank databases (PDB: 7N1I, 7N1H; EMDB: 24117, 24116, 24394). Source data are provided with this paper.  (IAB strain TC-83, AAB02517; IAB strain TrD,  AAC19322; IC strain INH9813, AJP13627; ID strain ZPC738, AUV65225) and other  alphaviruses (EEEV strain FL93-939, ABL84687; WEEV strain CBA87, ABD98014;  SINV strain Girdwood, AUV65223; CHIKV strain 37997, ABX40011). Structurebased sequence alignments were performed between alphaviruses that do (group 1, left margin) or do not (groups 2 and 3, left margin) use LDLRAD3 as a receptor for infection using PROMALS3D with VEEV numbering. The figure was prepared using ESPript 3.0. Domains are coloured (A (light cyan), B (medium cyan), C (blue) and β linker (medium cyan)) and indicated above the sequence, along with the secondary structure features and nomenclature (PDB: 3J0C; ref. 21 ). Red boxes indicate residues that are 100% conserved; white boxes and red letters indicate homologous residues within the specific group; white boxes and black letters indicate non-conserved residues. Determinants of receptor binding to the individual E2-E1 heterodimers are indicated by stars below the alignment and are coloured magenta if specific to LDLRAD3, cyan if specific to MXRA8, or yellow if shared between the two receptors. Wrapped denotes contacts to the wrapped E2-E1 heterodimer, the fusion loop of which is covered by LDLRAD3(D1) or MXRA8. Intraspike refers to the intraspike heterodimer, which is adjacent to the wrapped heterodimer but within the same trimeric spike. Contact residues were determined using PDBePISA. and carboxy (c) termini. D1 is coloured in a rainbow spectrum of blue to red. b, Fourier shell correlation plots for VEEV VLP with LDLRAD3(D1+D2). c, Electron density of one asymmetric unit of VEEV-LDLRAD3(D1+D2) complex coloured by protein: E1 (grey), E2 (cyan), capsid (forest green) and LDLRAD3(D1+D2) (purple). Density map viewed at a low contour level to show weak density for D2 of LDLRAD3. A ribbon diagram of docked LDLRAD3(D1) model is shown with the amino to carboxy termini in a rainbow spectrum of blue to red. The cysteine residues and the acidic residues responsible for calcium ion coordination are shown as balls and sticks. The disulfide bonds and calcium ion are coloured yellow and green, respectively. Inset: magnified view of LDLRAD3 with the weak density for D2 circled. d, Representative surface plasmon resonance sensograms with the binding parameters of LDLRAD3(D1+D2) (left) and LDLRAD3(D1) (right) to VEEV VLPs. n = 4 experiments. Data are mean + s.e.m. A 1:1 binding model (red traces) was used to fit the experimental curves (black traces).

Corresponding author(s): Daved Fremont
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