Peptide transport activity of the transporter associated with antigen processing (TAP) is inhibited by an early protein of equine herpesvirus-1

Aruna P. N. Ambagala, Raju S. Gopinath and S. Srikumaran

Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, NE 68583-0905, USA

Correspondence
S. Srikumaran
ssrikumaran1{at}unl.edu


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Equine herpesvirus-1 (EHV-1) downregulates surface expression of major histocompatibility complex (MHC) class I molecules on infected cells. The objective of this study was to investigate whether EHV-1 interferes with peptide translocation by the transporter associated with antigen processing (TAP) and to identify the proteins responsible. Using an in vitro transport assay, we showed that EHV-1 inhibited transport of peptides by TAP as early as 2 h post-infection (p.i). Complete shutdown of peptide transport was observed by 8 h p.i. Furthermore, pulse–chase experiments revealed that maturation of class I molecules in the endoplasmic reticulum (ER) was delayed in EHV-1-infected cells, which may be due to reduced availability of peptides in the ER as a result of TAP inhibition. Metabolic inhibition studies indicated that an early protein(s) of EHV-1 is responsible for this effect.


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Equine herpesvirus-1 (EHV-1), a member of the subfamily Alphaherpesvirinae of the family Herpesviridae, is a major cause of respiratory disease, reproductive failures and neurological signs in horses worldwide (Bryans & Allen, 1989). EHV-1 replicates in the epithelium of the upper respiratory tract, enters the peripheral circulation and disseminates systemically (Allen & Bryans, 1986; Gibson et al., 1992). Like other herpesviruses, EHV-1 establishes a latent infection that can become reactivated after the resolution of the primary infection (Edington et al., 1985). Natural infection, as well as vaccination, induces strong humoral immune responses (Allen et al., 1995; Bryans, 1981; Martens & Martens, 1991). However, breakthrough infections with consequent disease epidemics occur in the presence of virus-neutralizing antibodies in both infected and vaccinated horses, indicating that humoral immunity is inadequate for the control of EHV-1 infection (Burki et al., 1990; Doll & Bryans, 1963; Mumford et al., 1987). Hence, cell-mediated immunity is considered important for both protection against and recovery from EHV-1 infection.

As with other herpesviruses, immunosuppression is a characteristic feature of the pathogenesis of EHV-1 infection. Immunosuppression manifests as reduced in vitro proliferation of peripheral blood mononuclear cells (PBMCs) to both mitogens and inactivated EHV-1 antigens (Hannant et al., 1999), the absence of viral antigens on the surface of EHV-1-infected PBMCs (van der Meulen et al., 2003) and downregulation of expression of major histocompatibility complex (MHC) class I molecules on infected cells (Rappocciolo et al., 2003). MHC class I molecules are present on virtually all somatic cells. These molecules are generated by a highly intricate multi-step antigen-presentation pathway, which involves proteasomal degradation of cytosolic proteins (including viral proteins) into short peptides, ATP-dependent transport of these peptides from the cytosol into the lumen of the ER by the transporter associated with antigen processing (TAP), binding of peptides by class I molecules and egress from the ER via the Golgi apparatus for expression on the cell surface (reviewed by Williams et al., 2002). Cytotoxic T lymphocytes (CTLs), a major component of the cell-mediated immune system, recognize the peptides in the context of class I molecules on the cell surface. If class I molecules present viral peptides, CTLs lyse the virus-infected cells preventing further dissemination of the virus. Thus, interference with any of the steps in the class I antigen presentation pathway provides the virus with a means of escape from elimination by the host immune system.

Over the past decade, a number of viruses that interfere with the class I antigen presentation pathway have been identified. While some of these viruses target a single step, others encode several proteins that can interfere with multiple steps in the class I antigen presentation pathway (reviewed by Vossen et al., 2002). Recently, Rappocciolo et al. (2003) reported that the AB4/14 strain of EHV-1 also downregulates cell-surface expression of class I molecules. Using metabolic inhibitors, they demonstrated that the expression of early proteins of EHV-1 is necessary for EHV-1-mediated class I downregulation. Furthermore, their findings suggest enhanced endocytosis of class I molecules from the cell surface as one of the mechanisms of EHV-1-mediated class I downregulation. Studies in our laboratory have shown that two other animal alphaherpesviruses that belongs to the same genus (Varicellovirus) of EHV-1, bovine herpes virus 1 (BHV-1; Hinkley et al., 1998) and pseudorabies virus (PrV; Ambagala et al., 2000), interfere with the peptide transport activity of TAP as a means of downregulation of cell-surface expression of class I molecules. Hence, it was of interest to us to investigate whether EHV-1 also targets TAP activity in infected cells.

The NVSL laboratory strain 7/3/72 of EHV-1 (obtained from the Veterinary Diagnostic Center, University of Nebraska-Lincoln) was propagated and titrated on equine kidney (EK) primary fibroblasts (McGuire et al., 1994). First, we wanted to confirm whether the NVSL strain 7/3/72 of EHV-1 downregulated expression of class I molecules on EK cells. Therefore, EK cells were either mock-infected or infected with EHV-1 at an m.o.i. of 5. At 12 h p.i, cells were trypsinized and suspended in FACS buffer (PBS containing 3 % horse serum and 0·01 % sodium azide). Cells then were dispensed in duplicate into wells of a 96-well U-bottomed microtitre plate and incubated with 50 µl of primary antibody for 1 h at 4 °C, followed by 50 µl of appropriate secondary antibody for 30 min at 4 °C. Anti-MHC class I mAb PT85A (IgG2a), anti-EHV-1 polyclonal goat serum (all from VMRD, Inc.) and anti-EHV-1 gC mAb 56B4 were used as primary antibodies. PT85A, which detects peptide-bound porcine class I, cross-reacts with class I molecules of a wide range of species including equine. MM605 (IgG2a) specific for leukotoxin secreted by Mannheimia haemolytica (Gentry & Srikumaran, 1991) and pre-immune goat serum were used as isotype-matched controls. FITC-conjugated goat anti-mouse IgG (Amersham) and FITC-conjugated rabbit anti-goat IgG (Biomeda) were used as secondary antibodies. To exclude dead cells, propidium iodide (1 µg ml-1) was added and samples were analysed using a FACScan flow cytometer (Becton Dickinson). By 12 h p.i, approximately 75 % of cells were infected with EHV-1 (Fig. 1B and C). Compared with mock-infected cells, surface expression of class I molecules in infected cells was reduced by >90 % (Fig. 1A).



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Fig. 1. Downregulation of class I expression on EHV-1-infected EK cells. EK cells, either mock-infected or infected with EHV-1 at an m.o.i. of 5, were subjected to flow cytometric analysis at 12 h p.i. Histogram overlays of MHC class I expression detected by the mAb PT85A (A), EHV-1 gC expression detected by mAb 56B4 (B) and EHV-1 protein expression detected by goat anti-EHV-1 serum (C) are shown. The mAb MM605 and normal goat serum were used as isotype-matched control antibodies. Dead cells were gated using propidium iodide and 10 000 viable cells were analysed for each sample. Results of one representative experiment of three are shown.

 
To determine whether EHV-1 interfered with peptide transport activity of TAP in EK cells, we employed an in vitro transport assay that allowed direct assessment of the transport activity of TAP (Ambagala et al., 2000; Hill et al., 1995; Hinkley et al., 1998; Neefjes et al., 1993). This assay takes advantage of the fact that peptides, once transported into the lumen of the ER, are N-glycosylated. In this assay, cells, either mock-infected or infected with EHV-1 at an m.o.i. of 5, were washed twice in propagation medium, twice in ice-cold transport buffer (130 mM KCl, 10 mM NaCl, 1 mM CaCl2, 2 mM EGTA, 2 mM MgCl2, 5 mM HEPES, pH 7·3 with KOH) and permeabilized using Streptolysin O (1–2 U ml-1; Murex Diagnostics). To the permeabilized cells resuspended in transport buffer supplemented with 1 mM ATP (Sigma), a library of peptides containing a glycosylation consensus motif and labelled with 125I was added. Samples were incubated at 37 °C for 10 min and lysed in lysis buffer (50 mM Tris containing 5 mM MgCl2, 0·5 % Igepal and 1 mM PMSF). The lysate was then incubated with concanavalin A-coupled Sepharose beads (Amersham) for 1 h at 4 °C and the bound radioactivity was measured using a gamma counter.

To ascertain whether the transport assay could be used to measure peptide transport activity of TAP in EK cells, mock-infected cells were subjected to this assay in the absence or presence of ATP. As shown in Fig. 2(A), addition of exogenous ATP increased the transport of peptides, as indicated by an increased recovery of glycosylated peptides. These data confirmed that the transport assay measured TAP-mediated peptide transport in EK cells. In EHV-1-infected EK cells, peptide transport activity of TAP was inhibited as early as 2 h p.i. The reduction was greater than 50 % by 4 h p.i., and an almost complete shutdown of peptide transport (93 % inhibition) was observed by 8 h p.i (Fig. 2B). To confirm the specificity of TAP inhibition, a dose titration was performed at 4 h p.i. As shown in Fig. 2(C), peptide transport activity of TAP was down-regulated by EHV-1 in a dose-dependent manner. It could be argued that the inhibition of TAP activity observed in EHV-1-infected cells might be due to the inhibition of synthesis of TAP proteins as a result of virus-encoded virion host shut-off activity. However, this scenario is highly unlikely because EHV-1, in contrast to BHV-1 and HSV, does not exhibit early shut-off of protein synthesis (Feng et al., 1996).



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Fig. 2. Inhibition of peptide transport activity in EHV-1-infected EK cells. (A) The peptide transport observed in EK cells was TAP-mediated. To confirm that the transport assay measured peptide transport activity of TAP, permeabilized EK cells were either mock-treated (-) or treated with ATP (+) before the addition of radiolabelled peptides. Then the samples were incubated for 10 min at 37 °C, lysed, incubated with concanavalin A–Sepharose beads and the radioactivity recovered was measured using a gamma counter. (B) Kinetics of TAP inhibition by EHV-1. EK cells, either mock-infected or infected with EHV-1 at an m.o.i. of 5, were subjected to an in vitro transport assay at the times indicated p.i. (C) EHV-1-mediated TAP inhibition is dose-dependent. EK cells, either mock-infected or infected with EHV-1 at the indicated m.o.i.s, were subjected to an in vitro transport assay at 4 h p.i. (D) An early protein(s) of EHV-1 is responsible for inhibition of TAP activity in EK cells. EK cells, mock-infected (M) or infected (I) with EHV-1 at an m.o.i. of 5, in the presence or absence of the metabolic inhibitors cycloheximide (c), phosphonoacetic acid (p), or cycloheximide followed by actinomycin D (c/a), were subjected to an in vitro transport assay.

 
Reduced availability of peptides due to inhibition of TAP activity in EHV-1-infected cells would result in delayed maturation of class I molecules in the ER. To compare the rate of maturation of class I molecules in EHV-infected EK cells with that of mock-infected cells, a pulse–chase experiment was performed. EK cells, either mock-infected or infected with EHV-1 at an m.o.i. of 5 for 2 h, were incubated with cysteine- and methionine-free medium for 30 min and pulsed with 500 µCi [35S]methionine/cysteine (Dupoint NEN) for 30 min. After a chase period of 180 min in regular growth medium, cells were lysed and post-nuclear supernatant fluid was incubated with mAb H58A (VMRD) for 1 h. H58A, an anti-equine class I antibody, recognizes equine class I molecules irrespective of peptide binding. Immune complexes recovered on protein A-coupled Sepharose beads were eluted. Immunoprecipitated proteins were subjected to Endo-H (NEB) digestion, separated by SDS-PAGE and subjected to autoradiography. In mock-infected cells, virtually all class I molecules became Endo-H resistant within 180 min (Fig. 3), indicating that class I molecules had completed their transit from the ER to the medial Golgi by this time. In contrast, in EHV-1-infected cells, only about 50 % of class I molecules attained Endo-H resistance, indicating that EHV-1 infection results in delayed maturation of class I molecules.



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Fig. 3. EHV-1 infection delays maturation of MHC class I in the ER. EK cells were either mock-infected [EHV-1 (-)] or infected with EHV-1 [EHV-1 (+)] for 120 min, starved of methionine and cysteine for 30 min, then labelled with [35S]methionine and [35S]cysteine for 30 min and lysed (pulse) or chased for 180 min before lysis (chase). The class I molecules were immunoprecipitated using mAb H58A and subjected to SDS-PAGE without further treatment [Endo H (-)] or after digestion with Endo-H [Endo H (+)]. The relative locations of Endo-H-sensitive (S) and -resistant (R) forms of class I molecules are marked.

 
The next set of experiments was designed to identify the EHV-1 protein(s) responsible for TAP inhibition. First, to determine whether de novo viral protein synthesis is necessary for TAP inhibition, EK cells were infected in the presence of cycloheximide (CHX, 100 µg ml-1; Sigma), a protein synthesis inhibitor, and cells were subjected to the transport assay. By 4 h p.i., CHX treatment had completely abrogated EHV-1-mediated TAP inhibition, indicating that EHV-1 protein synthesis is required for TAP inhibition (Fig. 2D, bars 3 and 4). Next, to determine the role of late proteins in TAP inhibition, the transport assay was conducted with cells infected in the presence of phosphonoacetic acid (PAA, 300 µg ml-1 for 4 h), a DNA replication inhibitor and hence a late protein synthesis inhibitor. PAA could not prevent EHV-1-mediated inhibition of TAP activity, indicating that EHV-1 late proteins are not responsible for this effect (Fig. 2D, bars 5 and 6). To determine the role of immediate-early (IE) proteins in TAP inhibition, the transport assay was conducted with EK cells under conditions that allowed accumulation of only IE proteins. CHX was added 0·5 h before cells were infected and infection was allowed to proceed for 5 h to allow accumulation of mRNAs for IE proteins. Then cells were extensively washed with sterile PBS and incubated with fresh growth medium containing actinomycin D (2·5 µg ml-1), which inhibits transcription but allows translation of already transcribed IE mRNAs. At 20 h p.i, cells were subjected to an in vitro transport assay. As shown in Fig. 2(D, bars 7 and 8), even when large amounts of IE proteins of EHV-1 were expressed, TAP activity was not inhibited, suggesting that IE proteins of EHV-1 are not responsible for TAP inhibition. Taken together, these results indicate that an EHV-1 early protein(s) is responsible for TAP inhibition by EHV-1.

Four other herpesviruses, herpes simplex virus (HSV), human cytomegalovirus (HCMV), BHV-1 and PrV, interfere with peptide transport activity of TAP. Of these, TAP inhibition by HSV and HCMV has been well-characterized. ICP47, the 9 kDa IE protein of HSV, competes with peptides for the cytosolic peptide-binding site of TAP (Tomazin et al., 1996). US6, an early protein of HCMV, interferes with TAP function by binding to the ER luminal domain of the TAP molecule (Hengel et al., 1997; Lehner et al., 1997). US6 does not show any significant sequence homology to ICP47. We could not find a US6 or ICP47 homologue in the BHV-1, PrV or EHV-1 genomes. Results of this study and those of others (Ambagala et al., 2000; Koppers-Lalic et al., 2001) indicate that an early protein(s) of EHV-1, BHV-1 and PrV is responsible for TAP inhibition. Therefore, it is likely that EHV-1, BHV-1 and PrV, all of which belong to the genus Varicellovirus, encode a distinct protein(s) to interfere with TAP. Identification of the protein(s) responsible for TAP inhibition is currently underway in our laboratory. This novel viral inhibitor(s) of TAP should help to elucidate the molecular mechanisms involved in TAP inhibition.


   ACKNOWLEDGEMENTS
 
We thank Dr T. McGuire, Washington State University, Pullman, WA, for providing EK cells; Dr G. Allen, University of Kentucky, Lexington, KY, for the mAb 56B4; and Dr F. A. Osorio, Veterinary Diagnostic Laboratory, University of Nebraska-Lincoln, NE, for EHV-1. This article is published as Agricultural Research Division Journal Series No. 14164 with the approval of the University of Nebraska Agriculture Research Division.


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Received 7 August 2003; accepted 15 October 2003.