Eph Receptor Tyrosine Kinases Are Functional Entry Receptors for Murine Gammaherpesvirus 68

MHV68 gH/gL interaction profile with human and murine Eph receptors.

EBV and KSHV gH/gL structures demonstrate co-folding of gH D-I and gL that provides the basis for gH/gL-Eph interactions (55, 56). To evaluate if this gL-dependent conformation is conserved in MHV68, we modelled the MHV68 gH ectodomain (gH^ecto) in complex with gL using alphafold2 multimer (Fig 1A). The predicted MHV68 gH/gL structures were found to be similar to experimental structures of gH/gL from orthologous herpesviruses (57–61). Based on the EBV gH domain structure, MHV68 gH^ecto was divided into four domains: D-I (V25-P86), D-II (L87-S369), D-III (N370-L552), and D-IV (P553-Q702). The Eph binding interface of KSHV and EBV was mapped to a co-folded region comprised of the N-terminal D-I of gH and gL, which is conserved in the MHV68 gH/gL model.

To analyze if the conserved fold of the MHV68 gH/gL complex is predictive of interactions with Eph family receptors, we performed pairwise co-precipitation analysis of MHV68 gH^ecto in the absence or presence of full-length MHV68 gL with each of the 14 full-length human Eph receptors expressed in 293T cells. MHV68 gH^ecto/gL interacted most efficiently with human (h) EphA4, with less pronounced binding to hEphB3 and hEphA6 (Fig 1B). We also observed weak signal for hEphA8, hEphA10 and hEphB1. However, this signal was consistent in the absence of MHV68 gL (Fig 1B), and observed with an Fc control (S1A Fig), indicative of non-specific binding. Among herpesviruses that interact with members of the Eph receptor family (26, 27, 29–34), KSHV exhibits the closest sequence similarity to both MHV68 gH and gL, based on sequence coverage and identity (Fig 1C). We therefore compared the interaction of hEphA4, a KSHV receptor and gH/gL interaction partner (29, 32), with MHV68 and KSHV gH/gL. Precipitation of gH^ecto/gL from MHV68 or KSHV confirmed binding of both MHV68 gH/gL and KSHV gH/gL to hEphA4. On the other hand, MHV68 gH/gL did not interact with hEphA2, a KSHV-interacting Eph receptor (Fig 1D), in line with a previous report that did not observe an effect of hEphA2 on MHV68 fusion (55). Eph receptors are an evolutionary conserved class of receptor tyrosine kinases and exhibit high amino acid identity between human and mouse homologs (Fig 1E, S1D Fig). To analyze if this high inter-species conservation is reflected in observed MHV68 gH/gL-Eph interactions, we examined co-precipitation of MHV68 gH in the presence (Fig 1F) or absence (S1E Fig) of MHV68 gL with a subset of murine (m) Eph receptors. We observed a binding pattern recapitulating observations with human Eph receptors, identifying mEphA4 and mEphB3 as the most prominent interaction partners.

MHV68 genome annotations predict two coding sequences for MHV68 gL, starting from two alternative start codons. To determine potential differences in expression, gH complex formation and Eph interaction, we compared plasmids encoding the putative 173-amino-acid-protein (GenBank accession no. U97553) and 137-amino-acid-protein (containing a predicted 20 amino acid signal peptide) (GenBank accession no. AF105037) (S1C Fig). We did not observe differences in apparent molecular weights of expressed proteins, indicating cleavage after the signal peptide for generation of mature gL. In concordance, we did not see differences in gH/gL complex incorporation or EphA4 binding. These results agree with a previous report that identified expression of transcripts that align with AF105037 (22). Subsequent gL amino acid positions are determined based on the 137-amino-acid-protein (S1C Fig).

To quantify binding of candidate Eph receptors to gH/gL and evaluate the requirement for additional cellular factors we analyzed the binding of purified murine Eph proteins to an immobilized MHV68 gL-gH fusion protein by enzyme-linked immunosorbent assay (ELISA) (Fig 1G). We determined an EC50 of 3.6 nM and 2.3 nM for mEphA4 and mEphB3, respectively, consistent with previously observed nanomolar binding affinities of KSHV gH/gL to EphA2 (55, 56).

These co-precipitations and ELISAs reveal a direct interaction of MHV68 gH/gL with multiple Eph receptors, with strongest binding to EphA4, followed by EphB3. EphA4 and EphB3 are receptors for KSHV and RRV, respectively, demonstrating an overlapping binding profile with both human and NHP GHV pathogens.

Receptor function of MHV68 gH/gL-interacting Eph receptors.

We next analyzed the biological and functional relevance of this interaction for the entry process and infection of MHV68. First, we employed a loss-of-function approach, using soluble murine Eph receptors as decoys to test their inhibitory potential on infection of permissive target cells with a GFP-expressing MHV68 reporter virus (MHV68 ORF59-GFP (62)) (Fig 2A). NIH 3T3 murine fibroblasts, a highly susceptible cell type for MHV68 infection, exhibits broad Eph receptor expression, including Epha4 and Ephb3 (Fig 2B) in a published high throughput sequencing dataset (63). To analyze the impact of competition with soluble Eph receptors, MHV68 ORF59-GFP inocula were incubated with a concentration series of soluble mEphA4, mEphA6 and mEphB3 prior to infection of NIH 3T3 cells. PBS, and soluble mEphA2 were used as controls (Fig 2C, D). In line with MHV68 gH/gL-Eph-interaction patterns observed in precipitation or ELISA, pre-incubation with soluble mEphA4 and mEphB3 reduced MHV68 infection of NIH 3T3 cells in a dose-dependent manner, while pre-treatment with mEphA2 and mEphA6 did not block infection (Fig 2C).

While both mEphA4 and mEphB3 inhibited MHV68 infection of NIH 3T3 cells, we observed differences between Eph receptors and murine (Fig 2D–E) and human cell types (Fig 2F–G). Pre-incubation of MHV68 virions with 100 nM homodimerized mEphA4 resulted in a near complete abrogation (~93%) of MHV68 infection on NIH 3T3 murine fibroblasts (Fig 2D), whereas infection of human fibroblasts (HFF) and endothelial cells (TIME) was inhibited by approximately 50–60% (Fig 2F–G). Soluble mEphA4 exhibited the lowest blocking efficiency on the murine endothelial cell line SVEC4–10 with an approximate 25% reduction of MHV68 infection (Fig 2E). Pre-incubation with soluble mEphB3 demonstrated a similar pattern to mEphA4, leading to an approximate 40% reduction of infection on murine NIH 3T3 fibroblasts (Fig 2C–D), while inhibition on human fibroblasts and endothelial cells only reached approximately 30% (Fig 2F–G). EphB3 pre-treatment had no effect on MHV68 infection of SVEC4–10 cells (Fig 2E). In concordance with interaction assays, we observed no inhibition of MHV68 infection after pre-incubation with mEphA2 on any of the tested cell lines.

In an orthogonal approach, we performed entry experiments using ectopic Eph overexpression in a non-permissive cell type (Fig 3A). Raji cells, an EBV-positive, human B lymphoblast cell line support only low level MHV68 infection (<0.5% GFP⁺ cells under high MOI conditions), in agreement with low to absent mRNA expression of all 14 human Eph receptors (Fig 3B). To analyze contributions of single Eph receptors to MHV68 infection, Raji cells were transduced with lentiviruses encoding TwinStrep-tagged Eph receptors including human and murine EphA4 and EphB3 which bind MHV68 gH/gL (Fig 1) and inhibit infection on permissive cell lines (Fig 2). Empty vector, as well as human and murine EphA2 were used as negative controls. Immunoblot analysis of transduced and selected Raji cells confirmed expression of all analyzed Eph receptors, with lower levels of both human and mouse EphA4 observed compared to EphB3 and EphA2 (Fig 3C).

Ectopic expression of EphB3 of human and mouse origin exhibited the strongest enhancement of MHV68 ORF59-GFP infection, leading to an ~20-fold increase in GFP⁺ cells. Expression of EphA4 had a comparably less pronounced effect on MHV68 infection of ~8-fold enhancement compared to empty vector. This might be reflective of consistently lower expression levels of EphA4 constructs compared to EphB3. Nevertheless, overexpression of EphA4 led to an equivalent species-independent increase in MHV68 ORF59-GFP infection (Fig 3D–E). In summary, orthogonal soluble blockade and single receptor entry assays demonstrate that EphA4 and EphB3 act as functional entry receptors for MHV68.

The MHV68 gH/gL-Eph interaction is mediated by a conserved gammaherpesviral Eph-interaction motif.

The gH/gL-Eph interaction is dependent on a structural motif formed by D-I of gH and gL that contacts residues of Eph receptors that mediate binding of the GH loop in ephrin ligands (55, 56, 64, 65). Structure predictions of the MHV68 gH/gL heterodimer in complex with either the murine EphA4 or EphB3 ligand binding domain (LBD) were performed using alphafold2 multimer, refined using Rosetta and aligned with the previously published structure of the KSHV gH/gL-EphA2 complex (PBDid 7B7N) (Fig 4A–C). MHV68 gH/gL exhibited an Eph binding interface reminiscent of EBV and KSHV gH/gL-EphA2 complexes, with contacts observed throughout the N-terminal region of gL. As gL is co-folded with gH D-I and separated from D-II by a linker helix we sought to analyze if a complex consisting of gH D-I and full-length gL is sufficient to mediate the gH/gL-Eph interaction. We performed co-precipitation analyses of the constructs encoding the MHV68 gH ectodomain (gH^ecto) or MHV68 gH D-I and the D-I/D-II linker sequence (gH^D-I) in the absence or presence of full-length MHV68 gL with murine EphA4. MHV68 gH^D-I efficiently incorporated gL to higher levels than gH^ecto and bound EphA4 (Fig 4D). The predicted conservation of intermolecular hydrogen bonds between MHV68 gH and EphA4/ EphB3 indicated the aspartic acid at position 52 (D52^{MHV68 gH}) as a key interacting residue contacting arginine 106 on both mEphA4 and mEphB3 (R106^{EphA4/ EphB3}) (Fig 4B, C). R103^EphA2, the conserved arginine in the ephrin binding channel of EphA2, has been reported as single contact site for KSHV gH, forming a salt bridge with glutamic acid 52 (E52^{KSHV gH}) (56), the positional equivalent of D52^{MHV68 gH} (S2A Fig). Analysis of hydrogen bonds formed between MHV68 gH and gL, identified Y50^{MHV68 gH} as a potential stabilizing residue for the gH-gL interaction and K24^{MHV68 gL} as a potential contact residue for Eph receptors (Fig 4B, C). We next evaluated the impact of these residues on gH/gL-Eph complex formation. Single amino acids in the putative interaction interface were mutated to alanine and subjected to co-precipitation of wt and mutant combinations with murine EphA4 (Fig 4E, S2 Fig). Single point mutations of predicted interaction residues Y50^{MHV68 gH}, D52^{MHV68 gH} and K24^{MHV68 gL} led to comparable levels of gH expression and gL incorporation. In contrast to KSHV, in which a single point mutation in E52^{MHV68 gH} leads to complete abrogation of EphA2 interaction, single point mutations in MHV gH and gL only led to a moderate reduction in EphA4 binding in our assays. However, the combination of mutations Y50A^{MHV68 gH}, E52A^{MHV68 gH} and K24A^{MHV68 gL} reduced EphA4 binding to background levels (Fig 4E). The double point mutation in Y50-D52^{MHV68 gH} led to decreased incorporation of the low molecular weight gL. Taken together our data indicates that the MHV68 gH/gL-Eph interaction is shaped by the interaction of D52^{MHV68 gH} and R106^{EphA4/ EphB3} and a larger gL-Eph interaction interface.

MHV68 infected mice generate neutralizing antibodies targeting D-I of gH.

The gH/gL complex has been identified as a major target for GHV neutralizing antibodies in vivo (19–23). To analyze the effect of depletion of gH/gL-directed antibodies on serum neutralization capacity, we inoculated C57BL/6 mice with 1000 PFU wild-type (WT) MHV68 by the restrictive intranasal route of infection that models oropharyngeal GHV infection of people (Fig 5A). At 28 dpi, serum was collected from two independent groups of WT infected mice and analyzed by ELISA using plates coated with purified gL-gH fusion protein. WT infection elicited robust gH/gL-targeting antibody responses (Fig 5B). We next evaluated the serum neutralizing capacity on MHV68 infection of NIH 3T3 fibroblasts by analyzing reduction in GFP⁺ cells after pre-incubation of MHV68 ORF59-GFP with pooled, heat-inactivated sera (Fig 5C). We observed similar neutralizing capacity of serum from both MHV68 WT infected groups when compared to pre-incubation with serum from naïve mice. To evaluate the relative contribution of gH/gL-targeting nAbs to MHV68 neutralization, we depleted gH/gL-specific antibodies from serum of naïve or MHV68 WT infected mice using three rounds of depletion with either gH^ecto/gL or gH^D–I /gL complexes pre-coupled to magnetic beads. Adsorption using gH^ecto/gL or gH^D–I/gL decreased gH/gL-specific antibodies as measured by ELISA, but we observed slightly higher residual antibody levels in gH^D–I/gL-depleted samples (S3A Fig). To measure the neutralizing capacity of sera following adsorption, serum samples were pre-incubated with MHV68 ORF59-GFP prior to infection of NIH 3T3 cells. Depletion of gH/gL-targeting antibodies reduced neutralizing activity by approx. 50% with no pronounced differences between gH^ecto/gL or gH^D-I/gL (Fig 5D, E, S3B Fig). These findings indicate that the gH/gL interface is a primary target for gH/gL nAbs.

Cell culture.

Human embryonic kidney (HEK) 293T cells (RRID:CVCL_0063), human foreskin fibroblasts (HFF) (RRID:CVCL_XB54k) and SVEC4–10 (RRID:CVCL_4393) murine endothelial cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM), high glucose (Thermo Fisher Scientific, Waltham, MA; Corning, Corning, NY) supplemented with 10% FBS (R&D Systems, Minneapolis, MN), 100 U/mL penicillin, 100 μg/mL streptomycin (Corning), and 2 mM L-glutamine (Corning) (D10). NIH 3T3 (RRID:CVCL_0594) and NIH 3T12 (ATCC CCL-164, Manassas, VA) murine fibroblasts were maintained in DMEM supplemented with 8% FCS, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine (D8). TIME (RRID:CVCL_0047) human telomerase-immortalized human microvascular endothelial cells were cultured in Vascular Cell Basal Medium (ATCC PCS-100–030) supplemented with Microvascular Endothelial Cell Growth Kit-VEGF (ATCC PCS-110–041) and blasticidin (Invivogen, San Diego, CA) at 12.5 μg/ml. Raji cells (RRID:CVCL_0511) were cultured in RPMI (Thermo Fisher Scientific, Corning) supplemented with 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine (R10). All cells were cultured at 37°C in 5% CO₂.

Plasmids and recombinant proteins.

pcDNA4 vectors expressing full-length human Eph constructs were previously described (33). pCMV3-C-Myc vectors containing full-length constructs for murine EphA2, EphA4, EphA6, EphA7, EphB1 and EphB3 were purchased from Sino Biological (S1 Fig for accession numbers). Sequence similarity with orthologs was determined using the BLAST algorithm (81). The expression construct for the soluble, ectodomain of MHV68 gH (gH^ecto), endcoded by orf22 fused to the Fc fragment of human IgG (gH-FcStrep) contains the codon-optimized sequence (see S1 Table) coding for the predicted extracellular domain of MHV68 gH (amino acids 25 to 702, as predicted by DeepTMHMM) (analogously to (30, 82)) fused to the carboxy terminus of the 21-amino-acid murine immunoglobulin G kappa subunit ([IgG(κ)] signal peptide (PIR locus KVMS32) and the amino terminus of the Fc part from human IgG1 (GenBank accession no. S72664, amino acids 146 to 374) followed by a Twin-Strep-tag and 6xHis-tag. MHV68 gH ectodomain single point mutants and a truncated construct encoding D-I and the D-I/D-II linker sequence (gH^D-I, amino acids 25 to 106) were generated using site-directed mutagenesis. The expression construct for MHV68 gL, encoded by orf47 contains the codon-optimized sequence coding for full-length MHV68 gL (NP_044884.3, see S1 Table) followed by a single C-terminal Flag tag. An N-terminally truncated MHV68 gL expression construct lacking the first 36 amino acids (gL^ΔN36) and single point mutants were generated using site-directed mutagenesis. Mutations were verified by Sanger Sequencing of the targeted region. See S1 Table for a complete list of primers.

Recombinant soluble Eph receptors were purchased as Fc fusion proteins from R&D Systems (#639-A2, #641-A4, #607-A6, #432-B3). Cloning, protein expression and purification of the MHV68 gL-gH fusion protein was performed at the CCR Protein Expression Laboratory (PEL). Synthesized DNA, gene-optimized for mammalian expression, was used to create Entry clones containing the DNA sequences for gL-Linker-gH-Linker-His6. An Entry clone containing the CMV51 promoter sequence was created by PCR using pDest-720 (Sabine Geisse, Novartis) as template DNA and pDonr-233 (Esposito lab) as backbone vector via Gateway recombination (83). Final Expression clones were created by assembling promoter and gene sequence Entry clones by Gateway multisite reactions as described before (84) using pDest-303 (Addgene #159678) as destination vector. Expi293F (Thermo Fisher Scientific) culture supernatant was harvested 96 h after transfection and stored at −80°C prior to protein purification. The culture supernatant was thawed, dialyzed against 20 mM HEPES, pH 7.4, 300 mM NaCl (Buffer A) and filtered through a bottle top 0.45 μm filter. Imidazole was added to a final concentration of 25 mM. All purification steps were performed on a Bio-Rad NGC chromatography system at room temperature. A 2 × 1-ml HisTrap FF column (Cytiva, Marlborough, MA) was equilibrated in 10 column volumes (CV) 98% Buffer A and 2% Buffer B (20 mM HEPES, pH 7.4, 300 mM NaCl, 500 mM imidazole). Filtered culture supernatant was loaded onto the column followed by 10 CV wash in 2% Buffer B. Protein was eluted from the column with bump to 100% Buffer B. Fractions were collected across the gradient and analyzed by SDS-PAGE/Coomassie Blue staining. Elution fractions containing target protein were pooled.

Virus stock production and titration.

Viruses utilized in this study include WT MHV68 (WUMS strain) for infection of C57BL/6 mice and bacterial artificial chromosome (BAC)-derived MHV68 ORF59-GFP. The MHV68 DsRed bacmid carrying a C-terminal GFP fusion to vPPF (ORF59) was previously described (62). Virus passage and titer determination on NIH 3T12 fibroblasts were performed as previously described (85). In short, 3T12 cells were infected at low MOI conditions (MOI 0.01 – 0.05) for virus stock production. Cells were harvested 8 dpi and homogenized using dounce homogenizers on ice. Supernatant was clarified by two sequential centrifugation steps at 1,600 rpm for 7 min and 4,000 × g for 15 min. Virus was concentrated from clarified supernatant by high-speed centrifugation (14,000 × g, 2 h) and resuspended in 1:80 of original cell culture volume. Aliquots were stored at −80°C. Titers were determined by quantification of plaque formation on NIH 3T12 cells. NIH 3T12 cells were plated at a density of 1 × 10⁵ cells per ml. One day after plating, cells were incubated with serial dilutions of viral inocula for 1 hr at 37°C, with rocking every 15  min, followed by an overlay of 1.5% (w/v) methylcellulose (Sigma-Aldrich, Saint Louis, MO) in DMEM supplemented with 5% FCS, 100 IU/mL penicillin, 100 μg/mL streptomycin. After 7–8 d incubation at 37°C, cells were fixed with 100% methanol (Sigma- Aldrich), stained with 0.1% crystal violet (Sigma- Aldrich) in 20% methanol, and plaques were scored.

Animal studies.

All animal procedures reported in this study that were performed by NCI-CCR affiliated staff were approved by the NCI Animal Care and Use Committee (ACUC) and in accordance with federal regulatory requirements and standards. All components of the intramural NIH ACUC program are accredited by AAALAC International. Female C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA). Seven-week-old mice were anesthetized using 1–4% isoflurane and inoculated intranasally (IN) with 1,000 PFU WT MHV68 diluted in 20 μl D10. On day 28 post infection, blood was collected following humane euthanasia using isoflurane by post-mortem cardiac puncture, transferred to serum separator tubes (BD, Franklin Lakes, NJ) and centrifuged (21,100 × g, 20 min). Serum was carefully collected, aliquoted and stored at −80°C.

Lentivirus production and transduction.

For production of lentiviral particles, 10 cm cell culture grade petri dishes of approximately 80% confluent 293T cells were transfected with 3 μg each of pLP1 (HIV-1 gag and pol), pLP2 (HIV-1 rev), pLP/VSVG (VSV G glycoprotein (VSV-G)) and lentiviral expression constructs (pLenti CMV Blast DEST (706–1), pLenti-CMV-Blast-hEphA2-Strep, pLenti-CMV-Blast-hEphA4-Strep, pLenti-CMV-Blast-hEphB3-Strep, pLenti-CMV-Blast-mEphA2-Strep, pLenti-CMV-Blast-mEphA4-Strep, pLenti-CMV-Blast-mEphB3-Strep) using Transporter 5 Transfection Reagent (Polysciences, Warrington, PA) as per manufacturer instructions. The supernatant containing the pseudo-typed lentiviral particles was harvested 2–3 d after transfection and filtered through 0.45 μm membranes (Merck Millipore, Burlington, MA). For transduction, lentivirus stocks were added at a 1:5 dilution in the presence of 8 μg/ml polybrene, followed by centrifugation (1,500 × g, 1 h, RT). After 48 h, the selection antibiotic blasticidin (Invivogen) was added to a final concentration of 10 μg/ml.

Immunoprecipitation and immunoblot analysis.

For interaction analysis of gH/gL complexes with Eph receptors, 293T cells were transfected using Transporter 5 Transfection Reagent (Polysciences), per manufacturer instructions. Lysates of 293T cells transfected with expression constructs for human or mouse Eph receptors were prepared 2 days after transfections in NP40 lysis buffer (1% Nonidet P40 Substitute (Sigma-Aldrich), 150 mM NaCl (Quality Biological, Gaithersburg, MD), 50 mM HEPES (Quality Biological), 1 mM EDTA (Crystalgen) with freshly added cOmplete Mini Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche). For input immunoblot, 2 μl of each lysate was denatured in 1x NuPAGE LDS sample buffer (Thermo Fisher Scientific ) at 70°C for 10 min, separated by polyacrylamide gel electrophoresis (PAGE) using 8–16% Tris-Glycine polyacrylamide gradient gels (Thermo Fisher Scientific) with Tris-Glycine SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) and transferred to Polyvinylidendifluorid (PVDF) membranes (iBlot2). The membranes were blocked in 1x Fluorescent blocking buffer (Thermo Fisher Scientific) for 1 h, at room temperature, washed once in TBS-T and incubated with the respective antibodies for 2 h at room temperature or overnight at 4°C. After three washes with TBS-T, the membranes were incubated with the respective HRP-conjugated secondary antibody in TBS-T for 1 h at room temperature, washed three times in TBS-T, and imaged on an iBright Imaging system (Thermo Fisher Scientific) using Immobilon Forte Western HRP substrate (Merck Millipore). Recombinant FcStrep, gH-FcStrep or gH-FcStrep/gL-Flag complexes were precipitated from the supernatant of 293T cells transfected with the respective expression constructs. Supernatant was collected 2 d after transfection, filtered through 0.45 μm membranes (Millipore) and incubated with magnetic StrepTactinXT beads (IBA Lifesciences, Saint Louis, MO) overnight at 4°C with agitation. After three washes in NP40 lysis buffer, StrepTactinXT beads with pre-coupled FcStrep, gH-FcStrep or gH-FcStrep/gL-Flag complexes were incubated overnight at 4°C with agitation with myc-tagged Eph receptor containing lysates normalized for Eph receptor expression levels as determined by immunoblot. Volumes were adjusted with lysate of untransfected 293T cells. After overnight incubation, magnetic StrepTactinXT beads were washed three times in NP40 lysis buffer on Dynamag magnetic racks. Precipitates were heated in 1x LDS sample buffer (70°C, 10 min) and immunoblot was performed as described above (see S1 Table for a complete list of antibodies).

Enzyme-linked immunosorbent assay.

To measure binding of soluble Eph proteins or MHV68 gH/gL-specific antibodies in mouse serum, clear flat-bottom 96-well Maxisorp plates (Thermo Fisher Scientific, #439454) were coated with recombinant MHV68 gL-gH fusion protein at 2.5 μg/ml in PBS overnight at room temperature. After three washes in 0.05% Tween20 in PBS (PBS-T), wells were blocked with 1% BSA in PBS (R&D Systems #DY995) for 1 h at room temperature. Incubation with soluble Eph proteins at the indicated concentration or serum at the indicated dilutions was performed for 2 h at room temperature in 1% BSA in PBS. After three washes in PBS-T, bound protein was detected using horseradish peroxidase-conjugated ProteinG (Merck Milipore #18–161) at 12.5 ng/ml (soluble Eph proteins) or goat-anti-mouse IgG (Invitrogen #31430) (serum) at 10 ng/ml in in 1% BSA in PBS. After three washes, a 1:1 mixture of Color Reagent A (H₂O₂) and Color Reagent B (Tetramethylbenzidine) (R&D Systems) was added. The reaction was stopped by adding 2 N H₂SO (R&D Systems). Absorbance at 450 nm and 540 nm (reference wavelength) was read on a Biotek Synergy 2 plate reader (Agilent, Santa Clara, CA). Correction for non-specific absorbance was per performed by subtracting absorbance at 540 nm and blank controls from absorbance at 450 nm.

Infection assays and flow cytometry.

For infection assays of adherent cell lines, cells were plated at 12,500 cells per well in 96 well plates. One day after plating, cells were infected with the indicated amounts of virus. Block of MHV68 infection with soluble decoy receptor was assayed by infection with MHV68 ORF59-GFP inocula that were pre-incubated with the indicated concentrations of soluble mEphA2-Fc, mEphA4-Fc, mEphA6-Fc or mEphB3-Fc at room temperature for 30 min. 16 h post infection (hpi) cells were harvested by brief trypsinization, followed by addition of 5% FCS in PBS to inhibit trypsin activity, spun down (1,500 rpm, 5 min), washed once with PBS, re-pelleted, fixed in PBS supplemented with 2% formaldehyde (ThermoFisher Scientific) and analyzed for GFP expression on a Cytoflex flow cytometer (Beckman Coulter, Brea, CA). Transduced Raji cells were plated at 25,000 cells per well in 96 well plates, virus was added at MOI 10–30, followed by centrifugation (1,500 × g, 1 h, RT). Micrographs of GFP⁺ Raji cells were taken at 24 hpi and cells were harvested for flow cytometry. Data analysis was performed in FlowJo (v10). Raw data was log-transformed and normalized to controls prior to statistical analysis, and back transformed for visualization.

MHV68 Neutralization and antibody depletion assays.

For neutralization assays, murine serum was serially diluted and incubated with MHV68 ORF59-GFP for 30 min at room temperature. Subsequently, serum/virus inocula were added to cells plated at 12,500 cells in 96 well plates one day prior to infection. 16 h post infection cells were harvested by brief trypsinization, followed by addition of 5% FCS in PBS to inhibit trypsin activity, spun down (1,500 rpm, 5 min), washed once with PBS, re-pelleted and fixed in PBS supplemented with 2% formaldehyde and analyzed for GFP expression on a Cytoflex flow cytometer. Data was analyzed using FlowJo (v10). For depletion of MHV68 gH/gL-specific antibodies, recombinant FcStrep, gH^ecto-FcStrep/gL-Flag or gH^D-I-FcStrep/gL-Flag complexes were pre-coupled to magnetic StrepTactinXT beads overnight at 4°C with rotation as described above. After three washes in PBS/ 1% FCS/ 2 mM EDTA on a Dynamag magnetic rack StrepTactinXT beads with pre-coupled FcStrep, gH^ecto-FcStrep/gL-Flag or gH^D-I-FcStrep/gL-Flag complexes were incubated with murine serum to adsorp gH/gL-binding antibodies for at least 5 h at 4°C with rotation. A total of three adsorption steps were performed. Serum pre- and post-depletion was stored at −80°C. Evaluation of MHV68 neutralizing potential of depleted sera was performed as described above.

Structure prediction and analysis.

The MHV68 gH ectodomain (NP_044860, V25-Q702) was predicted in complex with MHV68 gL (AAF19311, C21-W137) using ColabFold v1.5.5, followed by relaxing of the top ranked structure using amber force fields (pLDDT=84.1 pTM=0.865 ipTM=0.912) (86). N- and C-terminal residues with pLDDT < 70 were excluded in the visualization. Truncated chains for MHV68 gH (NP_044860.1, residues 1–95) and gL (NP_044884, residues 1–137) were predicted in complex with either murine EphA4 (NP_031962) or murine EphB3 (NP_034273) using ColabFold (NP_031962: mean pLDDT=60.9, median pLDDT = 61.5; NP_044884: mean pLDDT = 73.3, median pLDDT = 84.6). The resulting models were aligned against known structures (PDBIDs 3PHF, 7CZE, 7B7N, 7CZF) and closest matching MHV68 gH/gL-mEphA4 model (PBDid 7B7N, rmsd = 1.1 Å, DALI Z = 25.5) was refined using one round of Rosetta fast_relax to minimize steric clashes and improve the hydrogen bonding network (87). Further analysis and visualization was performed in ChimeraX (88).

Mathematical and statistical analysis.

Curve fitting of binding of Eph receptors and gH/gL-directed antibodies from mouse serum to gL-gH fusion protein was performed using a built-in A 4-parameter logistic (4PL) curve in GraphPad Prism version 10 (GraphPad Software, La Jolla, CA). Statistical analysis of multiple groups was performed using ordinary one-way analysis of variance (ANOVA) followed by Holm-Šídák’s multiple comparisons test (Fig 2D–G, Fig 3D) or ordinary two-way ANOVA followed by Tukey’s multiple comparisons test (Fig 5E, S3A Fig). All Statistical analyses were performed with GraphPad Prism version 10. For all statistics, *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001, ****: p-value < 0.0001.