Equine major histocompatibility complex class I molecules act as entry receptors that bind to equine herpesvirus-1 glycoprotein D

Abstract

The endotheliotropism of equine herpesvirus-1 (EHV-1) leads to encephalomyelitis secondary to vasculitis and thrombosis in the infected horse central nervous system (CNS). To identify the host factors involved in EHV-1 infection of CNS endothelial cells, we performed functional cloning using an equine brain microvascular endothelial cell cDNA library. Exogenous expression of equine major histocompatibility complex (MHC) class I heavy chain genes conferred susceptibility to EHV-1 infection in mouse NIH3T3 cells, which are not naturally susceptible to EHV-1 infection. Equine MHC class I molecules bound to EHV-1 glycoprotein D (gD), and both anti-gD antibodies and a soluble form of gD blocked viral entry into NIH3T3 cells stably expressing the equine MHC class I heavy chain gene (3T3-A68 cells). Treatment with an anti-equine MHC class I monoclonal antibody blocked EHV-1 entry into 3T3-A68 cells, equine dermis (E. Derm) cells and equine brain microvascular endothelial cells. In addition, inhibition of cell surface expression of MHC class I molecules in E. Derm cells drastically reduced their susceptibility to EHV-1 infection. These results suggest that equine MHC class I is a functional gD receptor that plays a pivotal role in EHV-1 entry into equine cells.

Figure 1

Equine herpesvirus-1 (EHV-1) infection of NIH3T3 cells expressing equine major histocompatibility complex (MHC) class I. (A) Flow cytometric detection of EHV-1 virions bound to the cell surface of equine brain microvascular endothelial cells (EBMECs) (upper panel) and NIH3T3 cells (lower panel). EHV-1-treated (solid line) and mock-treated (dashed line) cells were stained with an anti-EHV-1 antibody. (B) EBMECs and NIH3T3 cells were inoculated with Ab4-GFP at an MOI of 5 for 12 h. EHV-1 infected cells were identified by observing green fluorescent protein (GFP) signals under a fluorescence microscope. Scale bars: 100 μm. (C) NIH3T3 cells transiently transfected with pA68-DsRed, pB118-DsRed or pluc-DsRed were infected with EHV-1 Ab4-GFP at an MOI of 5. The number of GFP- and DsRed-positive cells was analyzed simultaneously by flow cytometry. Scale bars: 100 μm. (D) The expression of equine MHC class I was analyzed by flow cytometry using an anti-equine MHC class I antibody PT85A. Numbers on plots indicate the percentage of cells in the designated gate.

Figure 2

Effect of equine major histocompatibility complex (MHC) class I expression on NIH3T3 cells. (A) Flow cytometric detection of equine MHC class I expression on 3T3-A68 (upper panel) and NIH3T3 cells (lower panel) stained with anti-MHC class I antibody PT85A (red), H58A (blue), B5C (orange) or isotype controls IgG2a and IgG2b (black and gray, respectively). (B and C) Infectivity neutralizing assay. 3T3-A68 cells (B) and equine brain microvascular endothelial cells (EBMECs) (C) were preincubated with 50 μg/mL of anti-MHC class I or control antibodies for 30 min and then infected with equine herpesvirus-1 (EHV-1) Ab4-GFP at an MOI of 5 in the presence of the antibody for 2 h at 37 °C. After treatment with a low-pH citrate buffer to inactivate extracellular virions, incubation was continued for a further 12 h. Green fluorescent protein (GFP)-positive cells were counted by flow cytometry. Bars represent the means from three samples, and error bars show standard deviations. Statistical significance was determined by Student's t-tests and is indicated by asterisks (**P < 0.01). (D) Effect of cellular ATP depletion on EHV-1 entry into cells. RK13, 3T3-A68, CHO-K1 and E. Derm cells were treated with ATP depletion media during or postviral entry. These cells were infected with Ab4-GFP, and GFP-positive cells were counted by flow cytometry. The number of infected cells treated with ATP depletion media postviral entry was defined as one. The graphs show the mean and standard deviation of three independent experiments. (E) Expression of viral RNAs in 3T3-A68, NIH3T3 and E. Derm cells. Cells were infected with EHV-1 Ab4-GFP, and total RNA was extracted at 0 and 4 h postinfection (p.i.) Immediate early (IE), early (ICP0) and late (gB, gD, gK) transcripts were detected by RT-PCR. GAPDH was used as an internal control. (F) Time course of viral growth in E. Derm (white bars), 3T3-A68 (gray bars) and NIH3T3 (black bars) cells. Cells were infected to Ab4-GFP at an MOI of 5 for 1h and washed with PBS to remove uninfected virus. The viral titer of each supernatant was determined by a plaque formation assay on RK13 cells. Each bar represents the amount of cell-free virus as the mean of three independent samples. Error bars show standard deviations.

Figure 3

Involvement of equine herpesvirus-1 (EHV-1) glycoprotein D (gD) in equine major histocompatibility complex (MHC) class I-mediated viral entry. (A) Inhibition of EHV-1 entry by anti-gD polyclonal antibody. EHV-1 Ab4-GFP at a final MOI of 5 was incubated with anti-gD polyclonal antibody (open circles) or control rabbit IgG (solid squares) for 30 min, and the virus–antibody mixture was added to 3T3-A68 cells. After incubation for 2 h, extracellular virus was inactivated by treatment with citrate buffer. Green fluorescent protein (GFP)-positive cells were counted by flow cytometry. (B) Inhibition of EHV-1 entry by gD-Ig fusion protein. 3T3-A68 cells were incubated with gD-Ig (open circles) or control-Ig (solid squares) for 30 min and infected with the EHV-1 Ab4-GFP at an MOI of 5. After incubation for 2 h, extracellular virus was inactivated by treatment with citrate buffer. GFP-positive cells were counted by flow cytometry. Error bars represent standard deviations of three independent samples.

Figure 4

Interaction of equine major histocompatibility complex (MHC) class I with equine herpesvirus-1 (EHV-1) glycoprotein D (gD). (A, B) Immunoprecipitation assay. (A) Protein A beads coupled with gD-Ig or control-Ig were incubated with a lysate of 293T cells co-transfected with HA-tagged A68 and equine β2m expression plasmids. (B) Protein A beads coupled with A68-Ig or control-Ig were incubated with a lysate of 293T cells transfected with the EHV-1 gD expression plasmid. Precipitated proteins were separated by SDS-PAGE and subjected to immunoblotting (IB) using the antibodies indicated. Solid arrowhead, open arrowhead and arrow indicate gD-Ig, A68-Ig and control Ig, respectively. (C, D) Binding assay of gD and A68. (C) A68 and equine β2m co-transfected 293T cells (solid line) or mock vector transfected 293T cells (dashed lines) were incubated with anti-equine MHC class I monoclonal antibody (left), gD-Ig (middle) or control-Ig (right). (D) 293T cells transfected with EHV-1 gD (dashed lines) or mock vector (dashed lines) were incubated with anti-gD polyclonal antibody (left), A68-Ig (middle) or control-Ig (right). Bound Ig fusion proteins were stained with phycoerythrin-conjugated anti-human IgG and analyzed by flow cytometry.

Figure 5

Inhibition of equine herpesvirus-1 (EHV-1) entry into equine cells with an anti-major histocompatibility complex (MHC) class I antibody. (A) E. Derm and (B) equine peripheral blood mononuclear cells were preincubated with 50 μg/mL of anti-MHC class I or control antibodies for 30 min and then infected with EHV-1 Ab4-GFP at an MOI of 5 in the presence of the antibody for 2 h at 37 °C. After removing virus–antibody mixtures, cells were treated with a low-pH citrate buffer to inactivate extracellular virions. Incubation was continued for a further 12 h. Green fluorescent protein (GFP)-positive cells were counted by flow cytometry. Bars represent the means from three samples, and error bars show standard deviations. Statistical significance was determined by Student's t-tests and is indicated by asterisks (*P < 0.05, **P < 0.01).

Figure 6

Knock-down of the cell surface expression of major histocompatibility complex (MHC) class I. E. Derm cells were infected with recombinant lentiviruses carrying both shRNA and mRFP1 expression cassettes. E. Derm cells were transduced with equine β2m-specific shRNA (shβ2m1 and shβ2m2) or luciferase-specific shRNA (shluc). (A) Flow cytometric analysis of the cell surface expression of MHC class I. Cells transduced with shRNAs (solid lines) or nontreated E. Derm cells (dashed line) were stained with anti-MHC class I antibody PT85A. (B–M) Effect of shRNA transduction on the susceptibility of E. Derm cells to equine herpesvirus-1 (EHV-1). Cells were infected with Ab4-GFP at an MOI of 1 for 24 h. Transduction of shRNA lentiviral vectors was confirmed by mRFP1 expression and infection of Ab4-GFP was confirmed by green fluorescent protein (GFP) expression under a fluorescence microscope. Scale bars: 100 μm. (N) After infection with Ab4-GFP, GFP-positive cells were counted by flow cytometry. The bars represent the means from three samples, and error bars show standard deviations. Statistical significance was analyzed by Student's t-test and is indicated by an asterisk (**P < 0.01).

Figure 7

Major histocompatibility complex (MHC) class I-independent infection of CHO-K1 cells by equine herpesvirus-1 (EHV-1). (A) Flow cytometric detection of hamster MHC class I expression on CHO-K1 cells stained with anti-MHC class I antibody PT85A (red), H58A (blue), B5C (orange) or isotype controls IgG2a and IgG2b (black and gray, respectively). (B) CHO-K1 cells were preincubated with 50 μg/mL of anti-MHC class I or control antibodies for 30 min and then infected with the EHV-1 Ab4-GFP at an MOI of 5 in the presence of antibody for 2 h at 37 °C. After removing virus–antibody mixtures, cells were treated with a low-pH citrate buffer to inactivate extracellular virions. Incubation was continued for a further 12 h. Green fluorescent protein (GFP)-positive cells were counted by flow cytometry. Bars represent means from three samples, and error bars show standard deviations. (C, D) Knock-down of the cell surface expression of MHC class I by transduction of Chinese hamster's β2m-specific shRNA (shβ2m3 and shβ2m4). (C) Flow cytometric analysis of the cell surface expression of MHC class I. Cells expressing shβ2m4 (solid line) and no shRNA (dashed line) were stained with anti-MHC class I antibody H58A. (D) Cells expressing shRNA were infected with the EHV-1 Ab4-GFP at an MOI of 1 for 24 h. GFP-positive cells were counted by flow cytometry. Bars represent means from three samples, and error bars show standard deviations.