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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ABSTRACT |
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MAIN TEXT |
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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. 1
B 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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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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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.
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ACKNOWLEDGEMENTS |
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Received 7 August 2003;
accepted 15 October 2003.