Shifts in receptors during submergence of an encephalitic arbovirus

Cells and viruses

HEK 293T (human kidney epithelial, ATCC CRL-11268), Vero E6 (Cercopithecus aethiops kidney epithelial, ATCC CRL-1586) and SVG-A (human astroglial, provided by T. Kirchhausen) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) and 25 mM HEPES (Thermo Fisher Scientific). Vero 81 (C. aethiops kidney epithelial, ATCC CCL-81) cells were cultured in DMEM high glucose supplemented with 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin, 1× non-essential amino acids (NEAA, Sigma), and 1 mM sodium pyruvate. K562 (human chronic myelogenous leukaemia, ATCC CCL-243) cells were maintained in RPMI1640 (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS, 25 mM HEPES, and 1% (v/v) penicillin-streptomycin. SK-N-SH (human brain neuroblastoma, ATCC HTB-11) cells were maintained in Eagle’s minimum essential medium (EMEM, Sigma) supplemented with 10% (v/v) FBS, 25 mM HEPES, and 1% (v/v) penicillin-streptomycin. Expi293F cells (Thermo Fisher Scientific A14527) were maintained in Expi293 Expression Medium (Thermo Fisher Scientific). Cell lines were not authenticated. Absence of mycoplasma is confirmed through routine mycoplasma test using e-Myco PCR detection kit (Bulldog Bio 25234).

Full-length infectious clones of WEEV Fleming and WEEV McMillan have been previously described⁵⁵ and were provided by W. Klimstra. Plasmids were transformed into TOP10 Escherichia coli (Invitrogen) and prepared using the Plasmid Plus Midi or Maxi kits according to the manufacturer’s protocol (Qiagen). Linearization of 10 µg of plasmid was achieved with the NotI-HF restriction enzyme (NEB), followed by phenol-chloroform extraction. WEEV RNA was in vitro transcribed using the mMESSAGE mMACHINE T7 kit (Invitrogen) with 1 µg linearized plasmid. Following RNA transcription, two confluent T-150 or T-175 flasks of Vero 81 (for Fleming) or Vero E6 (for McMillan) cells were detached with 0.25% trypsin-EDTA (Gibco) and washed three times with Dulbecco’s phosphate-buffered saline (DPBS). Following the final wash, cells were resuspended in DPBS and combined with the entire volume of transcribed RNA in a 4 mm gap cuvette. The cells and RNA were subjected to three 250 V, 10 ms pulses at 1 s intervals in an ECM 830 square wave electroporation system (BTX). Cells were allowed to rest for approximately 10 min at room temperature before being transferred to a T-75 flask in the presence of medium with reduced FBS and maintained at 37 °C with 5% CO₂. Upon onset of cytopathic effect two days post-electroporation, cellular debris was pelleted by centrifugation and viral stocks were collected and stored at −80 °C.

Primary mouse cortical neuron culture and infection

Mouse experiments were approved under the Harvard Medical School Institutional Animal Care and Use Committee (protocol number IS00002530-3), and under the Boston Children’s Hospital Institutional Animal Care and Use Committee (protocol number 00001725). The Pcdh10-knockout mouse line was maintained on a C57BL/6J background²¹. Postnatal day 0 or day 1 pups were genotyped by genomic PCR, in which a fragment of the wild-type allele was amplified by primer P1 (5′-GCTCGCGTTTGCCAGCCGTTGATATC-3′) and primer P2 (5′- AGAGCGTCTCCAAATCGAGCCTCATT-3′), and a fragment of the mutant (null) allele was amplified by primer P1 and primer P3 (5′-ACTGGTACACGCGACTGAAAACAGTG-3′). Cortical neurons were dissected and dissociated from postnatal day 1 or 2 neonates using methods adapted from ref. ⁵⁶. In brief, pups were anaesthetized on ice and euthanized by decapitation. The cortices were then isolated in cold HBSS and dissociated in HBSS supplemented with 20 units ml⁻¹ of papain (Worthington Biochemicals) and 2000 units ml⁻¹ of DNase I (Roche). During dissociation, the cortices were first incubated at 37 °C for 5 min following trituration. Following dissociation, the papain was neutralized with 10 mg ml⁻¹ ovomucoid inhibitor (Worthington Biochemicals) in HBSS. Cells were then washed once with neurobasal medium by centrifugation at 600g for 3 min and plated at a density of 100,000 cells per well in 96-well plates (Cellvis) coated with 20 μg ml⁻¹ poly-l-lysine (Sigma) and 4 μg ml⁻¹ laminin (Thermo Fisher). The neurons were maintained in neurobasal medium supplemented with B27 (Thermo Fisher), l-glutamine and penicillin-streptomycin, unless specified otherwise. The plated neurons were treated with 3 µM cytosine arabinoside (AraC) from day 1 post-plating (day in vitro 1 (DIV 1)) to DIV 3 to reduce non-neuronal cell outgrowth. On DIV 4 we pre-incubated WEEV or SFV RVPs with 100 µg ml⁻¹ transferrin or RAP in culture medium containing 5 µg ml⁻¹ polybrene for 30 min at 37 °C. We then added the mixtures to cells. Cells were imaged every 4 h for 24 h using the Incucyte S3 Live Cell Imaging system (Sartorius) with Incucyte S3 Software version 2022B Rev2 (Sartorius) using a 20× objective. GFP-positive neurons were scored as cells with a threshold signal greater than 5 green calibrated units (GCU) above background, using a Top-hat background subtraction method. The neuronal cell body area in each image was obtained by analysing phase-contrast images using the Incucyte S3 Software. To calculate the percentage of positive cells, at the time point of 24 h post-infection, the area of GFP signal above background was divided by the total area covered by neuronal cell bodies and was multiplied by 100. We calculated relative infection as follows: Relative infection (%) for wild-type neurons = (percentage of GFP-positive wild-type cells in the presence of transferrin or RAP)/(percentage of GFP-positive wild-type cells in the absence of transferrin or RAP) × 100; relative infection (%) for Pcdh10^−/− neurons = (percentage GFP-positive Pcdh10^−/− cells in the presence or absence of transferrin or RAP)/(percentage GFP-positive wild-type cells in the absence of transferrin or RAP) × 100.

Reporter virus particle generation

RVPs were generated as previously described⁴. In brief, we transfected two plasmids into HEK 293T cells using Lipofectamine 3000 (Thermo Fisher): a modified pRR64 Ross River virus replicon⁵⁷ provided by R. Kuhn (Purdue University) (the SP6 promoter is replaced with a CMV promoter, the E3–E2–(6 K/TF)–E1 sequence is replaced with a turbo GFP or CD20 reporter preceded by a porcine teschovirus-1 2 A self-cleaving peptide), and a pCAGGS vector expressing heterologous alphavirus E3–E2–(6 K/TF)–E1 proteins. At 4–6 h post-transfection, we replaced medium with Opti-MEM (Thermo Fisher) supplemented with 5% (v/v) FBS, 25 mM HEPES, and 5 mM sodium butyrate. We collected supernatant 2 days post-transfection, centrifuged supernatant at 4,000 rpm for 5 min, filtered these using a 0.45-µm filter, and froze aliquots at −80 °C for storage.

Alphavirus E3–E2–(6 K/TF)–E1 coding sequences cloned into the pCAGGS vector include: WEEV strain 71V1658 (GenBank NC_003908.1), WEEV strain California (GenBank KJ554965.1), WEEV strain Fleming (GenBank MN477208.1), WEEV strain McMillan (GenBank GQ287640.1), WEEV strain BFS932 (GenBank KJ554966.1), WEEV strain BFS2005 (GenBank GQ287644.1), WEEV strain Y62-33 (GenBank KT844544.1), WEEV strain Montana-64 (GenBank GQ287643.1), WEEV strain CU71-CPA (GenBank KT844545.1), WEEV strain 85-452NM (GenBank GQ287647.1), WEEV strain PV012357A (GenBank KJ554987.1), WEEV strain R02PV003422B (GenBank KJ554990.1), WEEV strain Imperial 181 (GenBank GQ287641.1), SFV strain SFV4 (GenBank AKC01668.1), EEEV strain Florida 91-469 (GenBank Q4QXJ7.1), EEEV strain PE6 (GenBank AY722102.1), SINV strain Toto1101 T6P144 (GenBank AKZ17594.1), VEEV strain INH-9813 (GenBank KP282671.1) and CHIKV strain 37997 (GenBank AY726732.1).

Reporter virus particle titration

Titration of GFP-expressing RVPs was performed on Vero E6 cells seeded in 96-well plates using a serial twofold or tenfold dilution of the RVP stocks. At 24 h post-infection, numbers of GFP-positive cells were counted using fluorescence microscopy and used to calculate RVP titre as infectious unit per millilitre (IU ml⁻¹), assuming that at high dilution factors, 1 GFP-positive cell = 1 infectious unit, given that RVPs can only infect cells for one cycle.

sgRNA library design, screening, analysis

Supplementary Table 2 contains genes targeted in the single guide RNA (sgRNA) library. The library, as previously described⁴, includes genes that encode proteins identified by mass spectrometry to be on the cell surface⁵⁸ and proteins either bioinformatically predicted to be on the cell surface^59,60 or annotated as associated with endosomes, lysosomes, vesicles or the cell surface by UniProt (https://www.uniprot.org). We cloned the guide RNAs into lentiGuide-Puro⁶¹ (provided by F. Zhang, Addgene #52963) and amplified the library in Endura ElectroCompetent cells (Lucigen 60242) as previously described⁶². We packaged the library into lentivirus by transfecting the plasmid library along with pMD2.G (provided by D. Trono, Addgene #12259) and psPAX2 (provided by D. Trono, Addgene #12260) into HEK 293T cells using Lipofectamine 3000 (Thermo Fisher). Supernatants were collected 1 and 2 days post-transfection, pooled, clarified by centrifugation (1,200 rpm for 5 min), filtered through a 0.45-µm membrane, and stored at −80 °C.

We used a previously described HEK 293T line that stably expresses Streptococcus pyogenes Cas9 (HEK 293T-Cas9) for the CRISPR–Cas9 screen⁴. We transduced HEK 293T-Cas9 cells with the CRISPR sgRNA lentivirus library at a MOI of 0.3. One day post-transduction, we began selection of sgRNA expressing cells by adding puromycin at 1 µg ml⁻¹. Seven to ten days post-selection, we infected cells with WEEV RVPs expressing CD20 (strain 71V1658), aiming for 80–90% infected cells compared to HEK 293T-Cas9 cells not transduced with the library, as monitored by an anti-CD20 APC-conjugated antibody (Miltenyi Biotec Clone LT20 130-113-370) used at 1:50 dilution. Three days post RVP infection, we depleted infected cells using anti-CD20 MicroBeads (Miltenyi Biotec 130-091-104). To improve the signal-to-noise ratio, we performed two additional rounds of infection with WEEV RVPs expressing CD20 following expansion of uninfected cells. We extracted genomic DNA from uninfected cells and library-transduced HEK 293T-Cas9 cells that had not been infected with RVPs. We amplified sgRNA sequences and determined sgRNA content using next-generation sequencing on an Illumina MiSeq. Tag sequences were removed and gene enrichment was analysed using MAGeCK (version 0.5.6)²⁰.

Genetic knockout and validation

We used previously described VLDLR-knockout HEK 293T cells⁴. To disrupt PCDH10 using CRISPR–Cas9, we transduced HEK 293T cells with pairs of sgRNAs targeting two sites into lentiGuide-Puro (Addgene #52963) along with lentiCas9-blast (Addgene #52962). We then isolated individual clones using clonal dilution. We confirmed the lack of cell surface expression of PCDH10 or VLDLR in knockout clones by staining cells with an anti-PCDH10 antibody (Proteintech 21859-1-AP) or an anti-VLDLR antibody (GeneTex GTX79552). Clonal PCDH10-knockout cells were genotyped by PCR.

Forward guide RNA sequences used to disrupt PCDH10 were: sgPCDH10-1, 5′-CGTGACTGACCGCGACTCAG-3′; sgPCDH10-2, 5′-TCGCATGGACTGGCGCACCG-3′. Genotyping primer sequences for clonal PCDH10-knockout HEK 293T cells are: forward, 5′-CTACACGGTACAGGAGGAGC-3′; reverse, 5′-CCAACGCGATGATGAGGATG-3′.

Expression and purification of VLPs

We transfected plasmids encoding the structural polyprotein (capsid–E3–E2–(6 K/TF)–E1) of CHIKV strain 37997⁶³, WEEV strain CBA87, which contains an nuclear-localization mutation in the gene encoding the capsid protein²⁹, or WEEV strain McMillan (GenBank GQ287640.1) containing the same mutation into Expi 293F cells using the ExpiFectamine 293 Transfection Kit (Thermo Fisher) according to the manufacturer’s protocol. Culture supernatant was collected 5 days post-transfection and cleared of cell debris by centrifugation at 3,000g for 20 min. The clarified supernatant was laid upon 5 ml 35% (w/v) sucrose cushion on top of 5 ml 70% (w/v) sucrose cushion, and ultracentrifuged at 25,000 rpm for 5 h at 4 °C. VLPs were pooled from the interface of the 35% and 70% sucrose cushion and buffer exchanged in a 100-kDa Amicon filter (Sigma) to lower the sucrose concentration to less than 20% at a volume of 1 ml. We then laid VLPs onto a 20%–70% continuous sucrose density gradient and ultracentrifuged samples at 35,000 rpm for 1.5 h at 4 °C. The VLP band was collected. VLPs were stored at 4 °C without buffer exchange and not frozen. We confirmed particle integrity and the absence of degradation products using SDS–PAGE. VLPs were always used within seven days of purification, and buffer exchanged based on the application immediately before use.

Fluorescent labelling of VLPs

Purified VLPs were buffer exchanged into 0.1 M sodium bicarbonate (pH 8.3) and diluted to a concentration of 1 mg ml⁻¹. Immediately before use, Alexa Fluor 647 (AF647) NHS ester (succinimidyl ester) (Invitrogen A37573) was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 1 mg ml⁻¹. We added 25 µg of AF647 NHS ester to 1 mg of VLP and incubated the mixture for 30 min at room temperature. We removed excess dye from the solution with a Zeba Spin Desalting Column (Thermo Fisher) and buffer exchanged labelled VLPs into PBS. Labelled VLPs were stored at 4 °C and used for confocal microscopy experiments within 12 h of labelling.

Ectopic expression construct design and generation of stable cell lines

cDNA encoding human PCDH10 (GenBank NM_032961.3), mouse PCDH10 (GenBank NM_001098170.1), human VLDLR (GenBank NP_003374.3), human MXRA8 (GenBank NM_032348.3), and human LDLR (GenBank AAP88892) were obtained from GeneScript. The coding sequences of P. domesticus PCDH10 and P. domesticus MXRA8 were obtained by aligning the coding sequences of Passer montanus PCDH10 (GenBank XM_039733439.1) and P. montanus MXRA8 (GenBank XM_039727729.1) against the genome of P. domesticus (GenBank GCA_001700915.1) and assembling aligned fragments. Gene blocks were synthesized at Integrated DNA technologies (IDT) for the following codon-optimized coding sequences: E. caballus PCDH10 (GenBank XM_023636548.1), P. domesticus PCDH10, P. domesticus MXRA8, T. sirtalis PCDH10 (GenBank XM_014072689.1), H. sapiens ApoER2 isoform 2 (GenBank NM_004631.5). A Flag tag (DYKDDDDK) was placed at the N-terminus of P. domesticus MXRA8 to monitor expression. Truncation constructs of human PCDH10 were generated as follows: PCDH10(ΔEC1) was generated by removing EC1 (Q19–F122) in the PCDH10 precursor protein sequence (numbering includes the signal peptide sequence); PCDH10 stalk–Flag was generated by removing Q19–G690 in the precursor protein sequence and adding a Flag tag between S696 and G697; PCDH10 EC1–Flag and EC2–Flag were generated by replacing Q19–G690 in the precursor protein sequence with EC1 (Q19–F122) or EC2 (P123–F250) and inserting a Flag tag between S696 and G697; PCDH10(ΔCT) was generated by removing the cytoplasmic domain (Q741–C1040 in the precursor protein sequence) of PCDH10. PCDH10–GPI and VLDLR–GPI were generated by replacing the transmembrane helices and cytoplasmic domains of PCDH10 (L716–C1040) and VLDLR (A798–A873) with a GPI anchor coding sequence (5′-CCTAATAAGGGCTCAGGCACTACTTCAGGAACCACCAGACTGCTGTCTGGCCATACCTGCTTTACACTGACCGGTCTCCTGGGGACGCTGGTCACCATGGGACTGCTGACC-3′), which encodes a GPI anchor peptide (PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT).

The above constructs were cloned into lentiGuide-Puro (Addgene #52963). We transfected this vector along with psPAX2 (Addgene #12260) and PMD2.G (Addgene #12259) at a ratio of 3:2:1 into HEK 293T cells using Lipofectamine 3000 (Thermo Fisher). Lentiviruses were collected 2 days post-transfection and used to transduce K562 cells or clonal PCDH10-knockout HEK 293T cells. Successfully transduced K562 cells and HEK 293T cells were selected using puromycin at 2 μg ml⁻¹ and 1 μg ml⁻¹ respectively. K562 cells transduced with E. caballus PCDH10 were additionally sorted using fluorescence-activated cell sorting with anti-PCDH10 polyclonal antibodies (Proteintech 21859-1-AP) to isolate a sub-population of positive cells. Cell lines were confirmed to express the transduced constructs by cell surface antibody staining.

Cell surface antibody staining

Primary antibodies were diluted to 10 μg ml⁻¹ in binding buffer (2% (v/v) goat serum in PBS) immediately before use. Primary antibodies used include polyclonal anti-PCDH10 (Proteintech 21859-1-AP), anti-VLDLR (GeneTex GTX79552), anti-MXRA8 (MBL International W040-3), anti-LDLR (R&D Systems MAB2148), rabbit IgG isotype (Proteintech 30000-0-AP) and mouse IgG isotype (BD Biosciences BDB557351). Cells were incubated in blocking buffer (5% (v/v) goat serum in PBS) for 30 min at 4 °C followed by incubation with primary antibodies at 10 µg ml⁻¹ in binding buffer (2% (v/v) goat serum in PBS). Cells were washed three times in binding buffer and subsequently incubated with a PE-conjugated donkey anti-rabbit F(ab′)₂ fragment (Jackson ImmunoResearch 711-116-152) or a PE-conjugated donkey anti-mouse F(ab′)₂ fragment (Jackson ImmunoResearch 715-116-150) diluted 1:200 in binding buffer for 30 min at 4 °C. We washed cells twice in binding buffer and twice in PBS, fixed cells in 2% (v/v) formalin and detected cell surface receptor expression using an iQue3 Screener PLUS (Intellicyt) with ForeCyt (Sartorius) software. Antibody staining was visualized using FlowJo (version 10.6.2).

For cells expressing Flag-tagged constructs, we diluted an APC-conjugated anti-DYKDDDDK (Flag) antibody (BioLegend 637307) or an APC-conjugated control antibody (BioLegend 402306) to 5 μg ml⁻¹ in binding buffer immediately before use. Cells were blocked as described above, incubated with primary antibodies in binding buffer for 30 min at 4 °C, and washed twice in binding buffer, twice in PBS. We then detected cell surface receptor expression using an iQue3 Screener PLUS (Intellicyt) with IntelliCyt ForeCyt Standard Edition version 8.1.7524 (Sartorius) software. Antibody staining was visualized using FlowJo (version 10.6.2).

Protein purification

We cloned human PCDH10 EC1 (Q19–F122, GenBank NP_116586.1), human MXRA8 ectodomain (V20–H337, GenBank NP_001269511.1), human VLDLR ligand-binding domain (A31–C355, GenBank NP_003374.3) into a pVRC expression vector encoding the human IgG1 Fc as a fusion protein at the C-terminus, provided by A. Schmidt⁶⁴. We cloned full-length human RAP (residues 1–353, including the signal sequence) (GenBank NP_002328) into the pCAGGs vector.

To produce PCDH10_EC1–Fc and MXRA8_ect–Fc, we transfected the pVRC vectors into Expi 293F cells using the ExpiFectamine 293 Transfection Kit (Thermo Fisher) according to the manufacturer’s recommendations. At 5 days post-transfection, we purified Fc fusion proteins using the MabSelect SuRe LX protein A affinity resin (GE Healthcare) according to the manufacturer’s protocol and further by size-exclusion chromatography using a Superdex 200 increase column. Control IgG (C1A-H12 anti-SARS-CoV-2 spike antibody⁶⁵) was similarly generated. Proteins were stored in PBS. Proteins used for in vivo experiments were not subjected to size exclusion chromatography other than for a small aliquot analyzed for quality control purposes; they were also tested for the presence of endotoxin, which was measured as <0.5 endotoxin units ml^–1 using a Pierce Chromogenic Endotoxin Quantification Kit (Thermo Fisher Scientific).

To produce VLDLR_LBD–Fc and RAP, we transfected the pVRC vector encoding VLDLR_LBD–Fc and the pCAGGS vector encoding RAP into Expi 293F cells using the ExpiFectamine 293 Transfection Kit (Thermo Fisher) at a 1:1 ratio. 5 days post-transfection, culture supernatants were subjected to protein A affinity chromatography, during which VLDLR_LBD–Fc bound by RAP was captured by the resin. We washed the resin with Tris-Buffered Saline (TBS) (20 mM Tris, 150 mM NaCl in water, pH 7.5) then eluted RAP with 300 column volumes of 10 mM EDTA in TBS overnight, then buffer exchanged RAP into TBS for storage. VLDLR_LBD–Fc was refolded on the column by washing the resin with 100 column volumes of TBS containing 2 mM CaCl₂ and subsequently eluted according to the manufacturer’s protocol. Proteins were further purified by size-exclusion chromatography using a Superdex 200 increase column. VLDLR_LBD–Fc was buffered exchange into TBS containing 2 mM CaCl₂ for storage.

Inhibition of RVP entry by recombinant proteins or antibodies

We pre-incubated GFP-expressing alphavirus RVPs in the presence of recombinant proteins or antibodies and 5 µg ml⁻¹ polybrene in culture medium for 30 min at 37 °C. The anti-PCDH10 antibodies and the control antibodies (anti-HLA-C polyclonal antibodies (Proteintech 15777-1-AP) and anti-HLA-ABC polyclonal antibodies (Proteintech 15240-1-AP) were first dialysed into PBS to remove azide preservatives. The mixtures were added to cells. 24 h post-infection, cells were washed twice in PBS and fixed in 2% (v/v) formalin. RVP entry was measured using an iQue3 Screener PLUS (Intellicyt) with IntelliCyt ForeCyt Standard Edition version 8.1.7524 (Sartorius) software. An example of the flow cytometry gating scheme used to quantify GFP expression after RVP infection is provided in Extended Data Fig. 2b. We calculated relative infection as follows: Relative infection (%) = (percentage of GFP-positive cells in the presence of recombinant proteins or antibodies)/(percentage of GFP-positive cells in the absence of recombinant proteins or antibodies) × 100.

Confocal microscopy with fluorescently labelled VLPs

A total of 10⁷ K562 cells stably expressing human PCDH10 or MXRA8 were centrifuged at 1,000 rpm for 5 min and resuspended in a mixture of heparinases (heparinase I (R&D Systems 7897-GH) at 2 units ml⁻¹, heparinase II (Sigma H8891) at 1 unit ml⁻¹, heparinase III (R&D Systems 6145-GH) at 2 units ml⁻¹). Cells were treated for 1 h at 37 °C. We then washed cells and resuspended cells to a density of 0.5 × 10⁶ ml⁻¹ in RPMI1640 supplemented with 2% (v/v) FBS and 25 mM HEPES. Twenty-five micrograms of labelled VLPs were added to 0.5 × 10⁶ cells. For cells kept at 4 °C, cells were incubated on ice following addition of VLPs for 30 min. Cells were then washed once with cold PBS and kept on ice. Immediately before imaging, cells were treated with 500 µl Alexa Fluor 488-conjugated wheat germ agglutinin (WGA-AF488) (Invitrogen W11261) at 1 µg ml⁻¹ in PBS, washed again with cold PBS, resuspended in 80 µl cold PBS, and placed in glass bottom microwell dishes (MatTek P35G-1.5-14-C) for imaging. Cells were imaged with a Yokogawa CSU-W1 single disk (50 µm pinhole size) spinning disk confocal unit attached to a Nikon Ti2 inverted microscope equipped with a Nikon linear-encoded motorized stage, an Andor Zyla 4.2 plus (6.5 µm photodiode size) sCMOS camera using a Nikon Plan Apo λS SR HP 100×C/1.45 Silicon DIC silicone immersion objective with Nikon Silicone oil. The final digital resolution of the image was 0.065 µm per pixel. Fluorescence from WGA-AF488 and VLPs conjugated to AF647 was collected by illuminating the sample with a solid state directly modulated 488 nm diode 100 mW (at the fibre tip) laser line or a solid state, directly modulated 640 nm diode 100 mW (laser tip) laser line in a Nikon LUNF XLlaser combiner, respectively. A hard-coated Semrock Di01-T405/488/568/647 multi-bandpass dichroic mirror was used for both channels. Signal from each channel was acquired sequentially with either a hard-coated Chroma ET525/36 m or Chroma ET700/75 m emission filters in a filter wheel placed within the scan unit, respectively. z-stacks were set by determining the top and bottom of the cell, using WGA-AF488 fluorescence as a reference. The approximate volume was ~20 µm, and the step size was set to 0.2 µm (approximating 80 z-slices in total), using the piezo stage insert (Mad City Labs, 500 µm). The exposure times were optimized to fill ~1/2–2/3 of the camera dynamic range and then kept consistent for one image set (individual exposure times are found in the image metadata and image legend where applicable). Fluorescence from each fluorophore was acquired sequentially at each z-step of the confocal to improve the axial precision of the measurements. Nikon NIS-Elements Advanced Research (AR) 5.02 acquisition software was used to acquire the data, and the files were exported in ND2 file format. Figures were generated using Fiji⁶⁶. A Gaussian filter of σ = 1 was applied to the image to smooth single pixel noise before adjustment of brightness and contrast. Max intensity projection (MPI) renderings were created by using the 3D projection function (Stacks>3D Project) with 10° increments and interpolation selected to smooth the 3D rendering.

3D image analysis was performed using custom pipelines built in Arivis 4DFusion 4.0 analysis software. We detected VLPs through a particle enhancement denoising filter of diameter 0.6 µm followed by a dilation morphology filter of diameter 0.13 µm (sphere shaped). We then applied a Blob Finder segmentation filter of diameter 0.52 µm, a probability threshold of 21.24%, and a split sensitivity of 50%. We segmented cellular compartments by first applying an enhance edges filter within the membrane detection operation, with a membrane width of 0.9 µm and a gap size of 0.6 µm, to enhance the AF488 signal. A discrete Gaussian denoising filter of diameter 0.2 µm was then applied. The membrane-based segmentation operation with a split sensitivity of 30% and a maximum diameter of 50 µm was executed to segment the processed image, and the whole cell masks were obtained by additional feature filters of sphericity >0.58 and volume >20 µm³. The cytoplasm mask was created by eroding the cell mask by two pixels. The membrane + cytoplasm mask was created by dilating the cell mask by three pixels, and the membrane mask was obtained by subtracting the membrane + cytoplasm mask with the cytoplasm mask. Finally, to remove segments created based on cells cut off at the edges of the imaged volume as well as cellular blebs that were segmented as independent cells, we applied a volume filter to exclude cytoplasm segments with a volume of <500 µm³ as well as their corresponding membrane segments. The number of VLPs in each compartment was then calculated by combining all masks.

Biolayer interferometry

Biolayer interferometry was performed using an Octet RED96e (Sartorius) and data were analysed using ForteBio Data Analysis HT version 12.0.1.55 software. PCDH10_EC1–Fc, VLDLR_LBD–Fc, and a control IgG (C1A-H12)⁶⁵ were loaded onto Anti-Human IgG Fc Capture (AHC) Biosensors (Sartorius 18-5063) at 100 nM in kinetic buffer (TBS containing 2 mM CaCl₂ and 0.1% (w/v) BSA) for 80 s. Coated sensor tips were dipped into kinetic buffer for a baseline measurement of 60 s, then dipped into a solution of WEEV VLPs at 100 nM for 300 s (CBA87 VLP) or 600 s (McMillan VLP), and finally in kinetic buffer for dissociation for 300 s (CBA87 VLP) or 600 s (McMillan VLP). In the case of WEEV McMillan VLP, the control IgG (C1A-H12)⁶⁵ was replaced with MXRA8_ect–Fc as a control.

Phylogenetic analysis

Sequences encoding the structural polyprotein (C–E3–E2–(6 K/TF)–E1) of 44 WEEV strains with full genome sequences available (Supplementary Table 1) were aligned in MEGA11 using the built-in MUSCLE algorithm⁶⁷. A maximum-likelihood phylogenetic tree was constructed using the aligned sequences with the Tamura 3-parameter nucleotide substitution model. The bootstrap method was used to test phylogeny with 1000 bootstrap replications.

Replication kinetics assay with authentic WEEV

K562 cells (2.5 × 10⁶) transduced with an empty lentiGuide-Puro vector or transduced to overexpress MXRA8, PCDH10, VLDLR, or ApoER2 were pelleted by centrifugation at 450g for 2 min. The medium was discarded and the cell pellets were gently resuspended in 1 ml of solution containing WEEV (strain McMillan or Fleming) diluted to 2.5 × 10⁴ PFU/ml in maintenance medium (RPMI1640 supplemented with 2% (v/v) FBS, 25 mM HEPES, 1% (v/v) penicillin-streptomycin) for a MOI of 0.01. The infection was allowed to proceed for 1 h at 37 °C with 5% CO₂. Following the infection, the cells were washed three times with 5 ml DPBS (Sigma) by centrifugation as above. Finally, the cells were resuspended in 5 ml maintenance medium. Immediately following this final resuspension, and again at 6, 12, 24 and 48 h post-infection, 500 µl supernatant was collected from each sample and stored at −80 °C. The removed volume was replaced with 500 µl fresh maintenance medium, and the samples were returned to the incubator. Sample titres were determined by plaque assay on Vero E6 cells.

Plaque assay

Culture supernatants containing WEEV (strain McMillan or Fleming) were serially diluted tenfold in DPBS supplemented with 2% (v/v) FBS. Diluted samples were allowed to infect confluent monolayers of Vero E6 cells in 12-well plates for 1 h at 37 °C with 5% CO₂. Following the infection, monolayers were overlaid with minimum essential medium (MEM) (Gibco) supplemented with 2% (v/v) FBS, 1% (v/v) GlutaMax (Gibco), 1% sodium bicarbonate (7.5% solution) (Gibco), 1% penicillin-streptomycin, and 0.4% LE agarose (Promega). Infected, overlaid plates were incubated for two days at 37 °C with 5% CO₂ prior to fixation with 10% buffered formalin (Thermo Fisher). Monolayers were stained with 1% (v/v) crystal violet (Sigma) and plaques were visualized with the aid of a light box.

Plaque reduction neutralization assay

For plaque neutralization assays with infectious WEEV (strain McMillan), PCDH10_EC1–Fc or MXRA8_ect–Fc fusion proteins were serially diluted in PBS supplemented with 2% (v/v) FBS to concentrations of 150–0.073 µg ml⁻¹. Diluted proteins (or diluent alone as a control) were combined with an equal volume of virus containing approximately 30 PFU of WEEV McMillan. The mixture of protein and virus was incubated at 37 °C with 5% CO₂ for one hour. Following this incubation, samples were processed as described for the plaque assay. Per cent neutralization was calculated as follows: per cent neutralization = (1 – (number of plaques in an experimental well/average number of plaques in diluent-only control wells)) × 100%.

In vivo protection study

Mouse studies were performed as approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee (protocol number 1708051) in accordance with the NIH Guidance for the Care and Use of Laboratory Animals. Mice were fed a 19% protein diet (Teklad, 2919, Irradiated), had 12 h light:dark cycle (06:00–18:00), and were housed in a facility maintained at a temperature range of 20 to 26 °C with a humidity range of 30 to 70%. Food and water were provided ad libitum. Sample sizes for mouse studies were determined based on previously published results for similar in vivo experiments⁴. Randomly assigned mixed-sex cohorts (n = 5 female and n = 5 male) of 6-week-old CD1 IGS mice (Charles River) received a 50 mg kg⁻¹ dose of either PCDH10_EC1–Fc, control IgG (C1A-H12 anti-SARS-CoV-2 spike antibody)⁶⁵, or PBS via the intraperitoneal route. Six hours later, all mice were infected with 1,000 PFU WEEV McMillan via the subcutaneous route in the left rear footpad. Weights were recorded and health checks were performed daily up to 14 days post-infection. We were not blinded to the treatment or infection status of the mice, also for safety reasons, since WEEV can cause severe disease in humans.

Statistical analysis

Data were deemed statistically significant when P values were <0.05 using version 10 of GraphPad Prism. Experiments were analysed by one-way or two-way ANOVA with multiple comparison correction, or by log-rank (Mantel–Cox) test in GraphPad Prism. P values are indicated in each of the figure legends. Statistical methods were not used to predetermine sample sizes.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.