Equus caballus Major Histocompatibility Complex Class I Is an Entry Receptor for Equine Herpesvirus Type 1

Cells, viruses, antibodies, plasmids, and cDNA library.B78H1 murine melanoma cells were generously provided by Gary Cohen and Roselyn Eisenberg (University of Pennsylvania). B78H1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). The construction and packaging of the equine macrophage cDNA library into the pFB murine retrovirus (7, 48) were described earlier, and this library was kindly provided by Ron Montelaro (University of Pittsburgh). The EHV-1 lacZ reporter virus, L11ΔgIΔgE was described previously (16). The monoclonal anti-equine MHC-I antibody CZ3 was provided by Douglas Antczak (Cornell University). The polyclonal anti-MHC-I antibody HLA B27 (ab93150) was purchased from Abcam (Cambridge, MA). Secondary goat anti-rabbit IgG-horseradish peroxidase (HRP) (sc-2030) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary goat anti-mouse Alexa Fluor 594 antibody was purchased from Invitrogen (Carlsbad, CA). The PCR-amplified equine cDNA (MHC-I clone B9) obtained from the screening process was subcloned into the pCDNA2.1 Topo Vector (Invitrogen) to generate pCDNA2.1TOPO-B9MHC-I. This vector was then digested with MluI and BglII to release the B9 MHC-I gene. The MHC-I gene was ligated into the pCDNA3.1-hygro plasmid (Invitrogen) to generate the MHC-I expression plasmid, pCDNA3.1-B9.

Transduction of the equine cDNA library into B78H1 cells and screening for EHV-1 infectivity.Two 35-mm dishes of B78H1 cells (1 × 10⁵) were seeded. One dish was infected with the pFB murine retrovirus containing the equine macrophage cDNA library (48) at a multiplicity of infection (MOI) of 5, and the other dish was not infected. Forty-eight hours postinfection (p.i.), medium was replaced with DMEM containing 5% FBS. Cells from each dish were harvested, resuspended, and seeded into 96-well plates 24 h after medium replacement, and the cells were incubated in 5% CO₂ at 37°C for 48 h. Duplicate 96-well plates were prepared for each original 96-well plate, and the cells were allowed to divide for an additional 48 h. Cells in each original 96-well plate (pFB infected and mock infected) were infected with EHV-1 L11ΔgIΔgE at an MOI of 10 for 16 to 20 h and then fixed with 0.5% glutaraldehyde in phosphate-buffered saline (PBS). X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) was added to the plate, and the wells were screened for the appearance of blue cells. Wells with the most blue cells were identified, and cells from the corresponding well of the duplicate plate were trypsinized and subdivided into a 96-well plate (one 96-well plate for each individual well that was selected for further characterization). This screening process was repeated for a total of five rounds. After the final round, cells from individual positive wells were harvested, counted, and then subdivided into numerous 96-well plates such that each well of the 96-well plate received approximately a single cell from the harvested cell population. Cells in these plates were grown for 7 days until they reached confluence. Cells were then trypsinized and split equally into duplicate plates. After a further 48 h of cell growth, cells were infected with L11ΔgIΔgE at an MOI of 3 for 20 h. Cells were then fixed and stained as described above, and wells that contained 100% blue cells were isolated from their corresponding duplicate well and used for genomic DNA isolation.

Genomic DNA isolation and PCR amplification of equine cDNA.Genomic DNA (1 × 10⁶ cells/sample) was collected from the B78H1 clones (B78H1R+) that exhibited 100% infection using a Promega Wizard Genomic DNA purification kit as per the manufacturer's directions (Promega, Madison, WI). Total DNA isolated from each sample was quantitated using a NanoVue spectrophotometer (GE Healthcare, Piscataway, NJ), and PCR was performed on each sample using the following primers: forward, 5′-CCCGGGGGTGGACCATCCTCT-3′, and reverse, 5′-GCCGGCCTTTATCGAACCCCAGAGAT-3′. DNA was denatured for 5 min at 95°C, and then 35 cycles were run as follows: 15 s at 94°C, 30 s at 55°C, and 2 min at 72°C, followed by a final extension of 7 min at 72°C. PCR products were run on a 1% agarose gel and isolated using a QuickClean 5 M gel extraction kit (Genscript Corporation, Piscataway, NJ) following the manufacturer's protocol.

DNA sequencing of PCR products.The PCR products were sequenced by Macrogen, Inc. (Seoul, South Korea), using the forward PCR primer listed above. All sequence data were analyzed using Sequencher, version 4.8, software (Gene Codes Corporation, Ann Arbor, MI).

Generation of MHC-I receptor lines in B78H1 cells.B78H1 cells were transfected with the pCDNA3.1-B9 MHC-I expression plasmid using Lipofectamine 2000 (Invitrogen) per the manufacturer's instructions. Transfected cells were grown in the presence of 100 μg/ml hygromycin B. Hygromycin-resistant cells were maintained in hygromycin B and then split into a 96-well plate such that each well was seeded with approximately one cell. After cells reached confluence in the 96-well plate, they were trypsinized and placed into wells of a 24-well plate. Cells were grown until confluence and split into a duplicate 24-well plate, and then one of the plates was infected with L11ΔgIΔgE at an MOI of 10 for 24 h. The cells were then fixed with 0.5% glutaraldehyde in PBS, stained with X-Gal, and screened for the appearance of blue cells. Cells that showed 100% infection by X-Gal staining were selected for further growth in six-well plates.

Immunofluorescence analysis of MHC-I receptor expression.A total of 5 × 10⁵ parental EHV-1-resistant B78H1 cells and the cDNA-transduced B78H1R+ cells selected for their susceptibility to EHV-1 infection were seeded onto number 1.5 coverglass, 35-mm MatTek dishes (MatTek Corporation, Ashland, MA) and incubated overnight at 37°C in 5% CO₂. Cells were washed once with cold PBS and then fixed with 4% paraformaldehyde (PFA) for 30 min at 4°C. PFA was removed, and cells were washed with PBS two times for 15 min per wash. Cells were blocked with 0.1 mg/ml bovine serum albumin (BSA) for 1 h at room temperature with gentle agitation on a rocking platform. Cells were washed once with PBS, and then mouse anti-equine MHC-I antibody (CZ3) was added to each plate at a concentration of 1:200 for 24 h at 4°C with gentle agitation on a rocking platform. Cells were washed three times for 10 min per wash with cold PBS. Goat anti-mouse Alexa Fluor 594 (Invitrogen) was added to the cells at a concentration of 1:400, the dishes of cells were covered with aluminum foil, and the cells were rocked for 1 h at room temperature on a rocking platform. Cells were washed three times for 10 min per wash with PBS, and then images were obtained using an Olympus Fluo-View 1000 confocal microscope (Olympus America, Inc., Center Valley, PA). Dishes of B78H1 and the cDNA-transduced B78H1R+ cells were also incubated with secondary antibody alone as a further control. All images were captured at a magnification of ×60.

Western blot assay.A total of 1 × 10⁶ B78H1 or B78H1 MHC-I-expressing (B78H1-MHC-I) cells were seeded in 60-mm dishes. Twenty-four hours postseeding, cells were harvested in radioimmunoprecipitation assay (RIPA) lysis buffer, and total protein lysates were quantitated using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Twenty micrograms of B78H1 and B78H1-MHC-I protein lysates were run on a 10% SDS-PAGE gel at 100 V, and then the separated proteins were transferred to an Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) overnight at 30 V. The membrane was incubated in 10% nonfat dry milk in Tris-buffered saline-Tween (TBST) for 1 h at room temperature, and then the rabbit polyclonal primary anti-MHC-I antibody, HLA B27 (ab93150; Abcam), was added at a concentration of 1:250 for 24 h at 4°C in 5% nonfat dry milk in TBST. The membrane was washed three times for 10 min each in TBST at room temperature, and then the secondary antibody, goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology), was incubated on the membrane for 1 h at RT in TBST at a concentration of 1:1,000. The membrane was washed three times for 10 min each in TBST at room temperature, and then SuperSignal West Pico chemiluminescent reagent (Pierce) was added to the membrane for 5 min. The membrane was exposed to X-ray film and processed.

Semiquantitative reverse transcription-PCR (RT-PCR).A total of 1 × 10⁶ B78H1 or B78H1-MHC-I cells were seeded in 60-mm dishes. At 24 h postseeding, total RNA was extracted using an EZNA kit (Omega Bio-Tek, Norcross, GA) as per the manufacturer's instructions. mRNA was reverse-transcribed into cDNA using the avian myeloblastosis virus (AMV) reverse transcriptase and a first-strand cDNA synthesis kit (Invitrogen). MHC-I cDNA was amplified using equine MHC-I-specific forward (5′-ATTGGGAGCGGAACACGCGG-3′) and reverse (5′-CCCTCATGCTGCACACGGCA-3′) primers. PCR was run as follows: initial denaturation for 5 min at 95°C and then 35 cycles consisting of 15 s at 94°C, 30 s at 55°C, and 2 min at 72°C, followed by a final extension of 7 min at 72°C. PCR products were run on a 1% agarose gel.

Antibody blocking of B78H1-MHC-I cells.A total of 2.5 × 10⁴ B78H1-MHC-I cells were seeded in a 96-well plate. Twofold dilutions were made of mouse anti-equine MHC-I antibody (CZ3). Before antibody was added to the cells, the maintenance medium was removed, 100 μl of cold DMEM with 10% FBS was added to each well, and the plate was placed on ice for 5 min. The DMEM and 25 μl of each antibody dilution were added to the cells in triplicate. The cells were kept on ice for 1 h. The cells were then infected with L11ΔgIΔgE at an MOI of 3 for 24 h. The cells were washed with PBS, and 150 μl of ONPG (o-nitrophenyl-β-D-galactopyranoside) was added to each well. The plate was incubated at 37° for 30 min. The plate was then read on a plate reader at 405 nm.

Transduction of an equine macrophage cDNA library into EHV-1-resistant cells and screening for cells that become susceptible to EHV-1.To identify an EHV-1 entry receptor, EHV-1-resistant B78H1 murine melanoma cells (1 × 10⁵ cells) were infected with a pFB murine retrovirus that contains an equine macrophage cDNA library (48). The infected cells were grown for 3 days and then collected and seeded into two 96-well plates. Once the cells reached confluence, they were trypsinized and split evenly into two plates each in order to generate duplicate 96-well plates. Cells in each of the duplicate plates were infected with the EHV-1 lacZ reporter virus, L11ΔgIΔgE (16), at an MOI of 10. In addition, B78H1 cells that were not transduced with the cDNA library were also infected and served as a negative control.

As shown in Fig. 1, a subset of B78H1 cells that were transduced with the cDNA library stained positive for infection, while very few blue cells were observed in the nontransduced, B78H1 control cells (Fig. 1). Eight wells that contained the highest number of blue cells were selected for further characterization. Cells from the duplicate wells were trypsinized and split into two 96-well plates, grown for 48 h, and then infected with L11ΔgIΔgE. Four wells were selected from this round of screening, and cells from the corresponding duplicate wells were isolated and seeded into two more 96-well plates for additional screening. This process was continued for four more rounds, and, in the final screening round, three wells were selected, and the cells were counted and subdivided into 96-well plates so that each well contained approximately one cell. The cells were then allowed to divide and grow for 7 days, at which time they were trypsinized and split evenly into duplicate plates. After an additional 48 h, the cells were infected with L11ΔgIΔgE at an MOI of 3 and then screened for lacZ expression the following day. After this infection, some wells exhibited 100% staining for X-Gal (Fig. 2), indicating that the entire clonal population of cells had become susceptible to EHV-1 infection.

• Open in new tab
• Download powerpoint

FIG. 1.

B78H1 cells were mock infected (A) or infected with the retrovirus cDNA library (B), and then each group of cells was subdivided into 96-well plates and infected with EHV-1 L11ΔgIΔgE at an MOI of 3. At 16 to 20 h postinfection, cells were stained with X-Gal to allow for identification of EHV-1-infected cells.

• Open in new tab
• Download powerpoint

FIG. 2.

L11ΔgIΔgE infection (MOI of 3) of a clonal population of cDNA-transduced B78H1 (A) and parental B78H1 (B) cells. Cells were infected for 20 h and then stained with X-Gal.

Genomic DNA isolation, PCR amplification, and sequencing of cDNAs from the positive cells.Cells from wells that exhibited 100% X-Gal staining were selected for further characterization. Cells from each selected well were expanded into six-well plates, and then genomic DNA was isolated from each population as described in Materials and Methods. PCR was performed on each genomic DNA isolate using primers that are specific for the flanking regions of the cDNA insert in the retrovirus construct. As shown in Fig. 3, PCR products were obtained from the genomic DNA of the selected EHV-1-susceptible B78H1 cells but not from the parental EHV-1-resistant B78H1 control cells (Fig. 3). The sizes of the PCR products were identical in each sample, with a strong band at approximately 1.5 kb. The 1.5-kb band was isolated and purified from four of the samples, A4, B9, C2, and D4, as described in Materials and Methods. The purified PCR products were sequenced, and the sequence data for each isolated PCR product revealed an exact match for the E. caballus MHC-I antigen (NCBI reference sequence, XM_001915759.1) which was derived from shotgun sequencing of the equine genome (NCBI, EquCab2 chrUn0268). The complete nucleotide and amino acid sequences for this MHC-I receptor are shown in Fig. 4.

• Open in new tab
• Download powerpoint

FIG. 3.

PCR amplification of cDNA inserts from genomic DNA of clonal cDNA-transduced B78H1 cells that are susceptible to EHV-1 infection. Lane 1, DNA ladder, lane 2, PCR product from control ELR1 plasmid; lane 3, PCR on parental B78H1 cells; lanes 4 to 9, PCR products from clonal B78H1-EHV-1 susceptible cells.

• Open in new tab
• Download powerpoint

FIG. 4.

Nucleotide and amino acid sequences of MHC-I encoded from amplified equine cDNA.

Detection of equine MHC-I on B78H1 receptor-positive cells (B78H1R+).The cell surface expression of MHC-I on the cDNA-transduced B78H1 cells (B78H1R+) was assessed by confocal immunofluorescence microscopy. Parental B78H1 and B78H1R+ cells were stained with the anti-MHC-I antibody, CZ3 (9), and then imaged with a confocal microscope, as described in Materials and Methods. As shown in Fig. 5, strong cell surface staining for the MHC-I receptor was observed on the B78H1R+ cells but not on the parental B78H1 cells (Fig. 5). The intense staining was located almost exclusively on the plasma membrane and was observed on all B78H1R+ cells in the monolayer. These results show that the MHC-I receptor is highly expressed on the surface of B78H1R+ cells but not on the parent B78H1 cells.

• Open in new tab
• Download powerpoint

FIG. 5.

Cell surface expression of MHC-I. Anti-equine MHC-I antibody CZ3 was incubated on monolayers of B78H1 parental cells (A) and clonally derived B78H1 EHV-1-susceptible cells, B78H1R+ (B). Images were captured using a confocal microscope, as described in Materials and Methods.

EHV-1 infection of B78H1 cells stably transfected with MHC-I receptor cDNA and antibody blocking of infection.The B9 MHC-I receptor cDNA was cloned into the pCDNA3.1-hygro plasmid, and stable cell lines expressing the MHC-I receptor (B78H1-MHC-I cells) were generated as described in Materials and Methods. The expression of the equine MHC-I receptor in these cells was assessed by both semiquantitative RT-PCR and Western blotting (Fig. 6). MHC-I mRNA and protein were readily detected in the B78H1-MHC-I cells but not in the parental B78H1 cells. The expression of the MHC-I receptor in the B78H1-MHC-I cells correlated with robust EHV-1 infection, as shown in Fig. 7. The B78H1 parental cells remained resistant to EHV-1 infection.

• Open in new tab
• Download powerpoint

FIG. 6.

Detection of equine MHC-I expression in stably transfected B78H1 cells. (A) MHC-I mRNA was detected by semiquantitative RT-PCR as described in Materials and Methods. Lane 1, 1-kb DNA ladder; lane 2, B78H1; lane 3, B78H1-MHC-I; lane 4, distilled H₂O. (B) Detection of MHC-I by Western blotting.

• Open in new tab
• Download powerpoint

FIG. 7.

EHV-1 infection of B78H1-MHC-I cell lines. B78H1 (A) and B78H1-MHC-I (B) cells were infected with EHV-1 L11ΔgIΔgE at an MOI of 5 for 20 h and then stained with X-Gal.

In order to further test the specificity of the EHV-1-MHC-I interaction, an antibody blocking assay was performed using an antibody specific for equine MHC-I (CZ3). As shown in Fig. 8, the CZ3 antibody blocked EHV-1 infection on the B78H1-MHC-I cells in a dose-dependent manner. Infection was completely inhibited with the anti-MHC-I antibody out to a dilution of 1/32, and infection was inhibited over 50% at a dilution of 1/512. These results confirm that EHV-1 entry into the B78H1-MHC-I cells is mediated by MHC-I.

• Open in new tab
• Download powerpoint

FIG. 8.

The MHC-I antibody CZ3 inhibits EHV-1 infection of B78H1-MHC-I cells in a dose-dependent manner. Cells were incubated for 1 h at 4°C with CZ3 antibody (Ab) at the dilutions indicated on the x axis and then infected with EHV-1 (L11ΔgIΔ gE) at an MOI of 3 for 20 h. ONPG substrate was added to the cells, and β-galactosidase activity was assessed by measuring the absorbance at 405 nm.