Department of Immunology and Pathology, Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK
Correspondence
Shirley Ellis
shirley.ellis{at}bbsrc.ac.uk
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ABSTRACT |
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Present address: Department of Infectious Disease and Microbiology, Graduate School of Public Health, Pittsburgh, USA.
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Introduction |
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CD8+ cytotoxic T lymphocytes (CTLs) form an essential part of the immune defence against many virus infections. CTLs recognize viral peptides presented at the surface of infected cells by major histocompatibility complex (MHC) class I molecules (Townsend & Bodmer, 1989). This is the result of a complex process of antigen processing involving a number of different molecules and cellular compartments (Pamer & Cresswell, 1998
). Many viruses have evolved mechanisms to interfere with MHC class I expression as a means of evading the host immune response. This is particularly true of herpesviruses and may relate to the need to establish latency (Ploegh 1998
). Infection of appropriate host cells with herpes simplex virus (HSV), human and mouse cytomegalovirus (HCMV, MCMV), varicella-zoster virus (VZV), bovine herpesvirus-1 (BHV1) and pseudorabies virus (PrV) all result in down-regulation of MHC class I expression (Cohen, 1998
; Johnson & Hill, 1998
; Koppers-Lalic et al., 2001
; Mellencamp et al., 1991
). However the mechanisms involved are only clearly understood and well characterized in HSV and CMV infections. These mechanisms include interference with peptide transport, retention of MHC class I heavy chains in the endoplasmic reticulum, direction of mature heavy chains to the endocytic pathway and rapid degradation of MHC class I at the cell surface. A number of different viral genes are involved in these processes, but not all related viruses carry homologous genes.
In vitro assays show that EHV1-specific, MHC class I-restricted CTLs are detectable in the peripheral blood of EHV1-seropositive horses (Allen et al., 1995). Given that current vaccination strategies do not appear to elicit an effective cell-mediated response to EHV1, we have studied the effect of in vitro EHV1 infection on MHC class I expression. The data generated may shed light on the mechanisms used by EHV1 to subvert the host immune response.
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Methods |
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Equine herpesvirus-1.
The strain of EHV1 used was AB4/14 (a gift from J. Kydd). Virus was grown on EEL cells to obtain stocks with titres of 2x107 p.f.u. ml-1, which were stored in aliquots at -80 °C. For UV inactivation, 10 ml undiluted virus stock was placed in a Petri dish under a Universal UV source (Gelman-Camag) set to 254 nm at a distance of 5 cm for 15 min. UV inactivation was confirmed by the absence of cytopathology following adsorption on to NBL-6 cells and subsequent lack of expression of EHV1 glycoprotein C.
Monoclonal antibodies (mAbs).
The anti-equine MHC class I mAbs CZ3 and CZ6 were a gift from D. F. Antczak (Cornell University, USA). The anti-porcine class I mAb PT85A was a gift from W. C. Davis (Washington State University, Pullman, USA; available from VMRD, Pullman, WA). The anti-EHV1 glycoprotein C mAb, 8F8, was a gift from J. Kydd. The anti-CD44 mAb CVS18 was purchased from Serotec. The isotype control mAb (IgG2a), TRT6, recognizes turkey rhinotracheitis virus and was raised at the Institute for Animal Health, Compton. Goat anti-mouse Ig, conjugated to fluorescein isothiocyanate (FITC), was purchased from Southern Biotechnology Associates.
Infection of cells and temporal control of viral protein expression.
Subconfluent cell cultures were washed once, then infected with EHV1 at an m.o.i. of 10 for 90 min at 37 °C in a CO2 incubator. If the cells were to remain in culture for more than 90 min, they were washed with PBS and given fresh growth medium. Mock infections were carried out in parallel using cell-free supernatant (collected from EEL cell cultures) in place of the virus stock solution. To restrict viral gene expression to immediate-early (IE) and early (E) genes, cells were infected or mock-infected in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid (PAA; Sigma) at a concentration of 300 µg ml-1, followed by incubation in fresh medium also containing PAA. To distinguish between involvement of IE and E genes, cells were infected with EHV1 and incubated in the presence of the protein synthesis inhibitor cycloheximide (CX; Sigma) at a concentration of 100 µg ml-1. After 5 h, cells were washed in PBS and fresh medium was added containing the transcription inhibitor actinomycin-D (Act-D; Sigma) at 5 µg ml-1 to allow translation of accumulated IE mRNA, while preventing further transcription. Controls comprised infected cells without addition of CX/Act-D, mock-infected cells with and without CX/Act-D, and infected and mock-infected cells with CX alone, for both 5 h and 24 h.
Flow cytometry.
Following infection or mock infection of NBL-6 or EEL, cells were trypsinized and resuspended in PBS containing 0·1 % sodium azide (PBS/azide) at selected times post-infection (p.i.). P815 transfectants were pelleted and washed once in PBS. Working on ice, the cells were dispensed into 96-well round-bottom plates at 5x105 cells per well. The cells were resuspended in 50 µl vols of the relevant mAbs diluted in PBS/azide containing 1 % BSA. Optimal dilutions for the mAbs had been determined in preliminary experiments. After 30 min incubation on ice, the cells were washed three times in PBS/azide and incubated in 50 µl vols of FITC-conjugated goat anti-mouse Ig (5 µg ml-1) for 30 min. To stain dead cells, 25 µl propidium iodide (PI, 100 µg ml-1; Sigma,) was subsequently added to each well followed by a further 5 min incubation. The cells were washed three times and either analysed immediately or fixed in 3 % paraformaldehyde in PBS and stored at 4 °C until analysis. Samples were analysed using a FACSCalibur flow cytometer (Becton Dickinson). Each sample for analysis contained 12x104 cells and dead cells were subsequently gated out according to their PI staining.
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Results |
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An EHV1 E gene(s) is responsible for MHC class I down-regulation
UV-inactivated EHV1 was used to determine whether de novo viral protein synthesis was required for MHC class I down-regulation. Fig. 5(a) shows that infection of NBL-6 cells with UV-inactivated virus at an m.o.i. of 10 resulted in no loss of class I expression. No expression of EHV1 glycoprotein C was detected (data not shown). To determine the class of viral protein involved in the down-regulation, cells were infected in the presence of PAA, which inhibits late viral gene expression. Fig. 5(b)
shows that expression of glycoprotein C (a late viral protein) was inhibited in the presence of PAA, but a similar pattern of MHC class I down-regulation was observed with or without PAA, as measured by CZ3 (Fig. 5c
). The only difference was that a very small population of cells that were still expressing MHC class I was observed in the PAA-treated sample. A similar result was seen when MHC class I was measured using CZ6 and PT85A (data not shown). These results indicate that the gene(s) responsible for MHC class I down-regulation is an IE or E EHV1 gene.
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Addition of CX to NBL-6 cells, either alone or followed by Act-D, effectively abrogated the down-regulation of MHC class I by the virus. Since removal of CX and addition of Act-D should allow translation of IE genes, this result strongly suggests that an EHV1 E gene, either alone or in conjunction with other genes, is responsible for the observed down-regulation of MHC class I.
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Discussion |
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Complete loss of MHC class I expression was seen in NBL-6 and EEL cells following EHV1 infection when monitored with the mAbs CZ6 and PT85A, whereas only partial (though significant) down-regulation was seen when monitored with mAb CZ3. There is no information available regarding the MHC class I genes/alleles expressed by NBL-6 and EEL cells; however, previous work has demonstrated that horse lymphocytes express the products of two polymorphic classical class I loci and may also transcribe a number of putative non-classical class I genes (Barbis et al., 1994; Ellis et al., 1995
). While expression of these latter genes has not been formally demonstrated, there is evidence that one of them, C1, is expressed. P815 and L cells express C1 protein at the cell surface following stable transfection with C1 cDNA in an appropriate expression vector (Holmes & Ellis, 1999
; Fig. 4b
). In addition, CTLs generated in vitro from C1-positive, EHV1-immune horses recognize and kill EHV1-infected L cells expressing C1 (S. A. Ellis & G. Rappocciolo, unpublished data). The mAb CZ3 recognizes a monomorphic epitope on equine class I molecules (including C1) that may not be dependent on correct conformation. In contrast, the mAbs CZ6 and PT85A appear to recognize a conformation-dependent polymorphic epitope(s) that is not found on the non-classical class I molecule C1 and that may also be missing from some classical class I molecules. Our results clearly demonstrate that infection with EHV1 causes only a partial loss of MHC class I molecules from NBL-6 and EEL cells; this may be the result of an allele- or locus-specific mechanism. It is less likely simply to reflect differences in the relative turnover/stability of different MHC molecules, since treatment of cells with CX or brefeldin A for 24 h did not result in complete loss of either PT85A or CZ6 recognition. A similar phenomenon has been reported in PrV infection of mouse cells, where H-2Dk and H-2Kk are differentially regulated due to the action of multiple viral genes acting in an allele-specific manner (Sparks-Thissen & Enquist, 1999
). Allele-specific binding by viral proteins is also seen in adenovirus, HCMV and HSV (Beier et al., 1994
; Hill et al., 1994
; Machold et al., 1997
). This is an important aspect of the immune system evasion mechanism of these viruses, since it has been suggested that by leaving some MHC class I alleles at the cell surface NK cell lysis of infected cells may be avoided (Sparks-Thissen & Enquist, 1999
).
Many different strategies have been documented that are used by alphaherpesviruses to evade the host immune system. It is clearly important to determine which one(s) is used by EHV1 and to identify the gene(s) responsible. Infection of cells in the presence of PAA clearly demonstrated that E or IE genes must be responsible for MHC down-regulation by EHV1. The small cell population that remain MHC class I-positive in the PAA-treated sample may indicate that it is difficult to achieve 100 % infection due to the PAA-induced inhibition of virus growth. It is possible that this could be overcome by using a higher m.o.i. Infection of cells in the presence of CX followed by Act-D strongly suggested that, while the IE genes are not involved, an E gene(s) is responsible for the observed MHC class I down-regulation.
Our data show that EHV1 infection may result in enhanced endocytosis of MHC class I molecules from the cell surface. This was demonstrated by the fact that EHV1 infection resulted in significantly greater loss of MHC class I molecules from the cell surface than either CX or brefeldin A treatment of uninfected cells. CD44 expression was not affected, indicating that MHC class I molecules were specifically targeted. Human herpesvirus-8 (HHV8) has been shown to down-regulate MHC class I via two genes, K3 and K5, which enhance endocytosis and direct internalized class I molecules to endolysosomal vesicles for degradation (Coscoy & Ganem, 2000). K3 and K5 act independently and each have specificity for a different set of MHC class I alleles (Lorenzo et al., 2002
). This is of interest given the apparent class I allele- or locus-specific down-regulation by EHV1 observed in this study. HHV8 is the only herpesvirus shown to mediate class I down-regulation using this mechanism, although unrelated viruses, e.g. human immunodeficiency virus, encode proteins that function in a similar manner (Piguet et al., 2000
).
There may in addition be mechanisms mediated by the same or a different EHV1 gene that result in less MHC class I reaching the cell surface. No genes have been identified in EHV1 that are homologues of those shown to be involved in MHC down-regulation in other viruses, for example ICP47 in HSV and US6 in HCMV, both of which interfere with TAP, although by completely different mechanisms (Ahn et al., 1997; Hill et al., 1995
; Telford et al., 1992
). The likelihood of an allele/locus-specific mechanism suggests that an EHV1 viral protein(s) may be interacting directly with the class I heavy chain. This could involve redirection of newly synthesized heavy chains to endolysosomes for degradation, as in MCMV infection (gp48) (Reusch et al., 1999
), or redirection to cytosolic proteasomes, as in HCMV infection (US2 and US11 proteins) (Wiertz et al., 1996
). It is also possible that a viral gene product is interfering with TAP, but that some MHC molecules are less dependent than others on TAP for loading of peptides.
Existing EHV1 vaccines that contain inactivated virus, while not leading to MHC class I down-regulation, would be unlikely to induce CTLs due to lack of endogenous processing. Attenuated EHV1 vaccines are likely to induce MHC class I down-regulation, which may also lead to inefficient CTL priming and result in incomplete protection. Understanding the mechanisms by which EHV1 exerts this effect and identification of the gene(s) responsible may ultimately lead to the development of improved vaccines.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Allen, G. P. & Bryans, J. T. (1986). Molecular epizootiology, pathogenesis and prophylaxis of equine herpesvirus1 infections. In Progress in Veterinary Microbiology and Immunology, vol. 2, pp. 74144. Edited by R. Pandey. Basel: Karger.
Allen, G. P., Yeargan, M., Costa, L. R. R. & Cross, R. (1995). Major histocompatibility complex class I-restricted cytotoxic T-lymphocytes in horses infected with equine herpesvirus 1. J Virol 69, 606612.[Abstract]
Allen, G. P., Kydd, J. S., Slater, J. S. & Smith, K. C. (1999). Advances in the Understanding of the Pathogenesis, Epidemiology and Immunological Control of Equine Herpesvirus Abortion. Equine Infectious Disease VIII, pp. 129146. Edited by U. Wernery, J. F. Wade, J. A. Mumford & O. R. Kaaden. Newmarket: R&W Publications.
Ambagala, A. P., Hinkley, S. & Srikumaran, S. (2000). An early pseudorabies virus protein down-regulates porcine MHC class I expression by inhibition of transporter associated with antigen processing (TAP). J Immunol 164, 9399.
Barbis, D. P., Maher, J. K., Stanek, J., Klaunberg, B. A. & Antczak, D. F. (1994). Horse cDNA clones encoding two MHC class I genes. Immunogenetics 40, 163.[Medline]
Beier, D. C., Cox, J. H., Vining, D. R., Cresswell, P. & Engelhardt, V. (1994). Association of human class I MHC alleles with the adenovirus E3/19K protein. J Immunol 152, 38623872.
Cohen, J. I. (1998). Infection of cells with varicella-zoster virus down-regulates surface expression of class I MHC antigens. J Infect Dis 177, 13901393.[Medline]
Coscoy, L. & Ganem, D. (2000). Kaposi's sarcoma-associated herpesvirus encodes two proteins that block cell surface display of MHC class I chains by enhancing their endocytosis. Proc Natl Acad Sci U S A 97, 80518056.
Ellis, S. A., Martin, A. J., Holmes, E. C. & Morrison, W. I. (1995). At least 4 MHC class I genes are transcribed in the horse phylogenetic analysis suggests an unusual evolutionary history for the MHC in this species. Eur J Immunogenet 22, 249260.[Medline]
Hill, A. B., Barnett, B. C., McMichael, A. J. & McGeogh, D. J. (1994). HLA class I molecules are not transported to the cell surface in cells infected with herpes simplex virus types 1 and 2. J Immunol 152, 27362741.
Hill, A. B., Jugovic, P., York, I., Russ, G., Bennink, J., Yewdell, J., Ploegh, H. L. & Johnson, D. (1995). Herpes simplex virus turns off TAP to evade host immunity. Nature 375, 411415.[CrossRef][Medline]
Holmes, E. C. & Ellis, S. A. (1999). Evolutionary history of MHC class I genes in the mammalian order Perissodactyla. J Mol Evol 49, 316324.[Medline]
Johnson, D. C. & Hill, A. B. (1998). Herpesvirus evasion of the immune system. Curr Top Microbiol Immunol 232, 150165.
Koppers-Lalic, D., Rijsewijk, F. A. M., Verschuren, S. B. E., van Gaans-van den Brink, J. A. M., Neisig, A., Ressing, M. E., Neefjes, J. & Wiertz, E. J. (2001). The UL41-encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of MHC class I molecules by bovine herpesvirus 1. J Gen Virol 82, 20712081.
Lorenzo, M. E., Jung, J. U. & Ploegh, H. L. (2002). Kaposi's sarcoma-associated herpesvirus K3 utilizes the ubiquitin-proteasome system in routing class I MHC to late endocytic compartments. J Virol 76, 55225531.
Machold, R. P., Wiertz, E. J., Jones, T. R. & Ploegh, H. L. (1997). The HCMV gene products US11 and US2 differ in their ability to attack allelic forms of murine MHC class I heavy chains. J Exp Med 185, 363366.
Mellencamp, M. W., O'Brien, P. C. & Stevenson, J. R. (1991). Pseudorabies virus-induced suppression of MHC class I antigen expression. J Virol 65, 33653370.[Medline]
Mumford, J. A., Rossdale, P. D., Jesset, D. M., Gamm, S. J., Ousey, J. & Cook, R. (1987). Serological and virological investigations of an equine herpesvirus-1 (EHV-1) abortion storm at a stud farm in 1985. J Reprod Fertil 35 (Suppl.), 509518.
Pamer, E. & Cresswell, P. (1998). Mechanisms of MHC class I-restricted antigen processing. Annu Rev Immunol 16, 323340.[CrossRef][Medline]
Patel, T. R., Edington, N. & Mumford, J. A. (1982). Variation in cellular tropism between isolates of equine herpesvirus-1 in foals. Arch Virol 74, 4151.[Medline]
Piguet, V., Wan, L., Borel, C., Mangasarian, A., Demaurex, N., Thomas, G. & Trono, D. (2000). HIV-1 Nef protein binds to the cellular protein PACS-1 to downregulate class I MHC. Nature Cell Biol 2, 163167.[CrossRef][Medline]
Ploegh, H. L. (1998). Viral strategies of immune evasion. Science 280, 248253.
Reusch, U., Muranyi, W., Lucin, P., Burgert, H. G., Hengel, H. & Koszinowski, U. H. (1999). A CMV glycoprotein re-routes MHC class I complexes to lysosomes for degradation. EMBO J 18, 10811091.
Sparks-Thissen, R. L. & Enquist, L. W. (1999). Differential regulation of Dk and Kk MHC class I proteins on the cell surface after infection of murine cells by pseudorabies virus. J Virol 73, 57485756.
Su, R. & Miller, R. G. (2001). Stability of surface H-2Kb, H-2Db, and peptide-receptive H-2Kb on splenocytes. J Immunol 167, 48694877.
Telford, E. A., Watson, M. S., McBride, K. & Davison, A. J. (1992). The DNA sequence of equine herpesvirus1. Virology 189, 304316.[Medline]
Townsend, A. & Bodmer, H. (1989). Antigen recognition by class I-restricted T lymphocytes. Annu Rev Immunol 7, 601624.[CrossRef][Medline]
Wiertz, E. J., Tortorella, D., Bogyo, M., Geuze, H. J. & Ploegh, H. L. (1996). The human CMV US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 84, 769779.[Medline]
Received 29 May 2002;
accepted 4 October 2002.