Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands1
Division of Infectious Diseases and Food Chain Quality, ID-Lelystad, PO Box 65, 8200 AB Lelystad, The Netherlands2
Laboratory for Vaccine Research, RIVM, PO Box 1, 3720 BA Bilthoven, The Netherlands3
Division of Tumor Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands4
Author for correspondence: Emmanuel Wiertz. Fax +31 71 5248148. e-mail Wiertz{at}LUMC.nl
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Abstract |
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Introduction |
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Here, we report on the construction of a vhs deletion mutant of BHV1 (BHV1vhs-). BHV1 is a member of the genus Varicellovirus and is the aetiological agent of infectious bovine rhinotracheitis and infectious pustular vulvovaginitis in cattle. Although the presence of a vhs gene in BHV1 has been documented (Schwyzer et al., 1996 ), the function of the putative BHV1 vhs gene product has not been examined. Based on the high degree of homology of the BHV1 UL41 gene with the vhs-encoding UL41 of HSV1 and HSV2, we decided to construct a UL41 deletion mutant of BHV1. Biochemical experiments described in this study indicate that, contrary to wild-type BHV1, the BHV1vhs- mutant does not interfere with host cell protein synthesis at early times post-infection. This indicates that UL41 indeed encodes the BHV1 vhs protein.
We used the BHV1vhs- mutant to examine the mechanisms by which BHV1 evades host immune responses. Like other herpesviruses, BHV1 has the capacity to establish latency and may be periodically reactivated during the lifetime of the host, despite a state of antiviral immunity (Denis et al., 1993 ; Lawman et al., 1987
; Rock, 1994
). The fact that BHV1 is capable of establishing a life-long infection in a fully immunocompetent host indicates that this virus is capable of escaping from detection by the immune system. Cytotoxic T cells (CTLs) play an important role in the elimination of virus-infected cells (Townsend & Bodmer, 1989
). CTLs recognize antigenic peptides in the context of MHC class I molecules (Townsend & Bodmer, 1989
; Yewdell & Bennink, 1992
). Highly effective strategies that specifically interfere with MHC class I-restricted activation of CTLs have been found for all herpesviruses scrutinized for such properties. Each step within the MHC class I antigen presentation pathway forms a potential target for virus evasion strategies (Brodsky et al., 1999
; Fruh et al., 1999
; Ploegh, 1998
; Wiertz et al., 1997
).
At present, viral gene products that subvert the MHC class I-restricted antigen presentation pathway have been identified for HSV1 and -2 (York et al., 1994 ), murine (MCMV; Reusch et al., 1999
) and human cytomegalovirus (HCMV; Ahn et al., 1996
, 1997
; Hengel et al., 1997
; Jones et al., 1995b
, 1996
; Wiertz et al., 1996a
, b
), human herpesvirus-8 (Coscoy & Ganem, 2000
; Ishido et al., 2000
), EpsteinBarr virus (Levitskaya et al., 1995
) and gammaherpesvirus-68 (Stevenson et al., 2000
). The varicelloviruses VZV (Cohen, 1998
), BHV1 (Hariharan et al., 1993
) and PrV (Sparks-Thissen & Enquist, 1999
) have also been found to down-regulate MHC class I expression, but the viral gene products involved remain to be identified. None of these varicelloviruses code for proteins homologous to the presently known gene products from other (herpes) viruses that interfere with MHC class I antigen presentation.
In the current study, we show that infection of MDBK cells with BHV1vhs- does not affect MHC class I synthesis during early stages of infection, which indicates that the UL41 gene product plays a role in MHC class I down-regulation. In addition to the vhs protein activity, other BHV1 gene products may interfere with MHC class I expression in a more specific fashion. Previous studies addressing this possibility have been hampered by the vhs effect, which precludes biochemical studies beyond 3 h of infection. The BHV1vhs- mutant provides us with a unique opportunity to investigate specific interference with the MHC class I pathway of antigen presentation.
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Methods |
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Construction of the BHV1vhs- mutant.
To introduce a deletion in the coding region of the UL41 gene of the parental (Lam) strain, both an upstream region and a downstream region of the UL41 gene were sub-cloned into pUC18. Both regions were isolated from pBR322 clone p115, which contains the 10·7 kb EcoRIHindIII fragment that harbours the UL41 gene and neighbouring genes from the parent strain (Rijsewijk et al., 1999 ). The downstream fragment was the 1040 bp BstZ17ISnaBI fragment that starts in the open reading frame of UL41 at amino acid residue 336 and ends in UL40. This fragment has been cloned into the HincII site of pUC18. The upstream fragment was the 5665 bp ApoIApoI fragment that starts at the UL44 (glycoprotein C) gene and ends in the open reading frame of UL41, including the first 150 amino acids of the encoded vhs protein. This fragment has been cloned into the EcoRI site of pUC18, upstream of the downstream fragment. The resulting construct had a unique XbaI site in between the upstream and the downstream fragments. The XbaI site was made blunt and used to insert the expression cassette of pcDNA3 (Invitrogen) carrying the hGFP gene. This cassette is located on the 2·2 kb NruIPvuII fragment of pcDNA3-hGFP and contains the HCMV ie1 promoter, the green fluorescent protein gene hGFP and the bovine growth hormone terminator sequence. The resulting recombination plasmid was named pS297. It has a 562 bp deletion in the central part of the open reading frame of the UL41 gene, resulting in the removal of amino acids 151336 and the loss of expression of the amino acids downstream of residue 336 (Pak et al., 1995
; Strelow & Leib, 1996
). This deletion includes the most conserved part of the vhs protein, which shows homology to the fen-1 family of nucleases (Doherty et al., 1996
). To propagate a UL41 deletion mutant, a 6 kb XhoI fragment was liberated from plasmid pS297 (Fig. 1A
) and cotransfected with genomic DNA of the parental strain Lam into embryonic bovine trachea cells according to the method of Graham & Van der Eb (1973)
. Mutants expressing GFP in the UL41 locus were selected under the UV microscope and were plaque purified three times. Restriction enzyme analysis of the BHV1vhs- mutant showed no other gross genomic rearrangements than the intended deletion/insertion. The mutant virus can be grown to the same titres as the parent strain (107·9 TCID50/ml). The viruses were propagated on MDBK cells to obtain stocks with titres of 108 TCID50/ml, which were stored at -80 °C until use.
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Infection of cells and temporal control of viral protein expression.
Confluent MDBK cells were washed once with PBS and infected with either wild-type BHV1 or BHV1vhs- at an m.o.i. of 10 in serum-free RPMI-1640 medium for 2 h at 37 °C, followed by addition of complete RPMI-1640 medium. In all experiments, mock-infected cells were treated under the same conditions as infected cells. To restrict viral gene expression to immediate-early (IE) or early genes, the protein synthesis inhibitor cycloheximide (50 µg/ml; ICN Pharmaceuticals) was added 15 min before infection and maintained until the metabolic labelling period, when it was removed or replaced by the transcription inhibitor actinomycin D (10 µg/ml; Boehringer Mannheim), which was present throughout the chase. Late viral gene expression was prevented by infecting MDBK cells in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid (PAA; ICN Pharmaceuticals) at a concentration of 300 µg/ml.
Flow cytometry.
MDBK cells were infected with either wild-type BHV1 or BHV1vhs- at an m.o.i. of 10 for 2 h and incubated for 10 h. Cells were trypsinized and resuspended in PBS containing 1% BSA. The cells were incubated with IL-A19 or anti-glycoprotein B antibody no. 14 on ice for 1 h. After washing, the cells were incubated with phycoerythrin (PE)-conjugated anti-mouse antibody for 45 min. Stained cells were analysed by flow cytometry (FACScan, Becton Dickinson).
Biochemical experiments.
BHV1-infected and mock-infected MDBK cells were incubated for 1 h in RPMI-1640 medium lacking methionine and cysteine, followed by a 30 min pulse with 200 µCi/ml of [35S]methionine/cysteine (Redivue PRO-MIX, Amersham Life Science). Cells were lysed in NP-40 lysis mix containing 50 mM TrisHCl, pH 7·4, 5 mM MgCl2 and 0·5% NP-40, supplemented with 1 mM AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride], 1 mM leupeptin and 20 µM Cbz-L3 (carbobenzyloxy-L-leucyl-L-leucyl-L-leucinal-H; Peptides International). Samples were kept on ice throughout the experiment. The post-nuclear supernatant was precleared twice with normal mouse serum, normal rabbit serum and protein GSepharose beads (Amersham Pharmacia Biotech). Proteins of interest were extracted from the lysates by 1 h incubation with specific antibodies and 30 min with protein GSepharose beads. Immunoprecipitates were washed in 1x NET buffer (0·5% NP-40, 50 mM TrisHCl, 150 mM NaCl, 0·1% SDS, 5 mM EDTA, pH 7·4). The pellets were resuspended in SDS sample buffer and boiled at 95 °C for 5 min. Eluted proteins were separated on 10% SDSpolyacrylamide gels and were visualized by autoradiography on Kodak XAR film. Analysis and quantification of radiolabelled products were performed using Quantity One quantification software (BIO-RAD Laboratories).
For pulsechase analysis, cells were labelled for 30 min with 200 µCi/ml of [35S]methionine/cysteine at 37 °C and were either lysed (chase time 0) or incubated in complete medium, supplemented with cold methionine and cysteine at a final concentration of 1 mM for the chase times indicated.
Endoglycosidase H (EndoH) digestion was performed following the instructions of the manufacturer (New England Biolabs). Mock-treated samples served as reaction controls.
Peptide transport assay.
BHV1-infected and mock-infected MDBK cells were washed twice with incubation buffer (130 mM KCl, 10 mM NaCl, 1 mM CaCl2, 2 mM EGTA, 2 mM MgCl2, 5 mM HEPES, pH 7·3) at 4 °C. The cells were permeabilized in incubation buffer containing 2 IU/ml of Streptolysin O (Murex Diagnostics) for 10 min at 37 °C. Permeabilization was assessed using trypan blue staining. Permeabilized cells (3x106 cells per sample) were incubated with 10 µl (100 ng) of a radioiodinated model peptide 417 (TVNKTERAY) in the presence or absence of ATP (10 mM final concentration) in a total volume of 100 µl at 37 °C for 10 min (Neefjes et al., 1993
). Peptide translocation was terminated by adding 1 ml of ice-cold lysis buffer (1% Triton X-100, 500 mM NaCl, 5 mM MgCl2, 50 mM TrisHCl, pH 7·5) and samples were left on ice for 20 min. After centrifugation at 12000 g, supernatants were collected and incubated with 100 µl of concanavalin ASepharose to isolate the glycosylated peptides (Amersham Pharmacia Biotech) at 4 °C for 1 h. The beads were washed four times with lysis buffer and the amount of radioiodinated peptide associated was quantified by gamma counting.
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Results |
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In contrast, BHV1vhs- infection of MDBK cells did not influence the synthesis of host cell proteins at early stages of infection. At 3 and 5 h after infection with BHV1vhs-, cellular proteins were synthesized at levels comparable to those in uninfected cells (Fig. 1B, compare lanes 1 and 3 with lanes 4 and 6). These results indicate that the BHV1 vhs protein is both necessary and sufficient to reduce de novo synthesis of host cell proteins at these time-points. Therefore, the BHV1vhs- mutant allows us to perform biochemical studies at time-points at which the wild-type virus blocks protein synthesis. We have used the new BHV1vhs- mutant to investigate the mechanisms underlying the down-regulation of MHC class I expression in BHV1-infected cells.
BHV1vhs- affects intracellular trafficking of newly synthesized MHC class I molecules
Using flow cytometry, BHV1 has been shown to down-regulate cell surface expression of MHC class I molecules (Nataraj et al., 1997 ). From the experiments performed, however, it cannot be concluded whether this down-regulation is the result of a general effect of BHV1 infection, e.g. is caused by the vhs function, or whether the virus specifically interferes with the surface expression of MHC class I molecules. The BHV1vhs- mutant allows us for the first time to distinguish between these possibilities.
Cell surface expression of MHC class I molecules on wild-type BHV1- and BHV1vhs--infected cells was examined by flow cytometry (Fig. 2AD). All cells were infected, as shown by expression of the viral glycoprotein gB (Fig. 2B
, D
). This experiment indicates that even when the vhs gene is deleted, MHC class I surface expression is still impaired (Fig. 2A
, C
). It is unlikely that the observed down-regulation is caused by expression of the GFP gene carried within BHV1vhs-, since uninfected, GFP-transfected MDBK cells show no reduction in MHC class I cell surface expression (data not shown). For control proteins, such as the transferrin receptor or MHC class II molecules, no down-regulation was observed (data not shown). Thus, in addition to the effect of the vhs protein, post-translational mechanism(s) are responsible for down-regulation of MHC class I surface expression by BHV1.
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TAP activity is inhibited in MDBK cells infected with BHV1vhs-
In addition to being retained in an ER/cis-Golgi compartment, MHC class I molecules isolated from BHV1vhs--infected cells are unstable at 37 °C (data not shown). This is indicative of a lack of peptides that stabilize MHC class I heavy chain2m complexes (Ljunggren et al., 1990
). The absence of peptide within MHC class I complexes may be the consequence of inhibited peptide translocation from the cytosol into the ER by the transporter associated with antigen presentation (TAP). HSV1, HSV2 and HCMV encode proteins which act as efficient inhibitors of TAP (Fruh et al., 1995
; Hill et al., 1995
; Tomazin et al., 1996
; York et al., 1994
; Ahn et al., 1997
; Hengel et al., 1997
; Lehner et al., 1997
). PrV (Ambagala et al., 2000
) and BHV1 (Hinkley et al., 1998
) also inhibit transport of peptides by TAP. However, no homologues of viral proteins known to interfere with TAP transport have been found in these viruses. It is possible that the inhibition of TAP in BHV1-infected cells is due to an indirect effect of the vhs protein. To investigate this possibility, we compared TAP activity in mock-infected MDBK cells and in cells infected with either wild-type BHV1 or BHV1vhs-.
At 4 h post-infection, peptide translocation was inhibited in cells infected with wild-type BHV1 (Fig. 3). Similar results were obtained with the BHV1vhs- mutant, which indicates that the vhs protein is not responsible for inhibition of TAP. This also implies that BHV1 encodes a protein that directly interferes with peptide transport by TAP.
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Maturation of bovine transferrin receptor was not influenced by BHV1 infection or PAA treatment, as this molecule acquired resistance to EndoH within 30 min of chase in BHV1vhs--infected, PAA-treated cells (Fig. 4A, top panel).
In the following series of experiments, viral gene expression was limited to IE genes. Cells were infected with BHV1vhs- in the presence of the protein synthesis inhibitor cycloheximide, which prevents synthesis of viral transactivators of early viral genes. The protocol used in this experiment is summarized in Fig. 5. Cycloheximide treatment not only inhibits protein synthesis but also enhances transcription of IE genes. Prior to metabolic labelling, cycloheximide was replaced by actinomycin D, which inhibits RNA transcription. IE mRNAs are then translated, whereas transcription of early genes is blocked, which leads to enhanced and selective synthesis of IE proteins.
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To prove that BHV1 early genes are responsible for the observed ER retention of MHC class I molecules, actinomycin D was not added immediately after removing cycloheximide, but 75 min later. Thus, a time window was created in which the IE proteins that were synthesized could transactivate transcription of early genes. The duration of early gene transcription is defined by the time at which actinomycin D is added. Maturation and intracellular trafficking of MHC class I molecules were monitored by pulsechase analysis and EndoH treatment of immunoprecipitates (Fig. 5B). In mock-infected cells, the majority of the MHC class I heavy chains acquired EndoH resistance during 120 min of chase. Sequential presence of the transcriptional and translational inhibitors had no effect on expression of MHC class I molecules in control cells. However, more than half of the MHC class I molecules isolated from BHV1vhs--infected cells still exhibited sensitivity to EndoH treatment after 120 min of chase. The ratio of EndoH-sensitive versus EndoH-resistant material was 25/75 in mock-infected cells and 73/27 in BHV1vhs--infected cells. In conclusion, chemical control of the virus replication cycle strongly suggests that BHV1 early protein(s) specifically interfere with the maturation of MHC class I molecules.
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Discussion |
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Whereas the vhs protein of wild-type BHV1 precludes biochemical experiments beyond 3 h after infection, the BHV1vhs- mutant allowed us to monitor the biosynthesis and intracellular trafficking of MHC class I molecules at later stages of infection. Interestingly, the behaviour of MHC class I molecules still remains abnormal in cells infected with the BHV1vhs- mutant. A temporal analysis of intracellular transport of MHC class I molecules by pulsechase analysis revealed that in BHV1vhs--infected cells the conversion of mannose-rich glycans of MHC class I molecules into the complex form is inhibited, most likely due to retention of newly synthesized MHC class I proteins in an ER/cis-Golgi compartment (Fig. 2E). BHV1vhs- does not affect intracellular trafficking of other cellular proteins, such as the transferrin receptor (Fig. 4
).
The observed ER retention of MHC class I molecules may be related to a lack of peptides, which may be due to inhibition of TAP-mediated peptide transport by BHV1. The experiments shown in Fig. 3 indicate that in cells infected with BHV1vhs-, TAP-dependent peptide translocation is inhibited by 76%. Similar results were obtained with wild-type BHV1, which is in accordance with previous reports (Hinkley et al., 1998
). Based on our findings with the BHV1vhs- mutant, we can now conclude that there must be one or more BHV1 gene products that specifically interfere with the function of TAP. Ultimately, MHC class I molecules that have not been loaded with peptides will be degraded in the cytosol (Hughes et al., 1997
; Wiertz et al., 1996b
).
Inhibition of TAP-dependent peptide transport represents an immune evasion strategy that is often employed by herpesviruses. Three other members of the alphaherpesvirus subfamily, HSV1 (Hill et al., 1995 ), HSV2 and PrV (Ambagala et al., 2000
) also interfere with peptide transport by TAP. In the case of HSV1 and HSV2, inhibition of TAP relies on a cytosolic protein, ICP47. For PrV, the molecule(s) involved in TAP inhibition remain to be identified. Interestingly, HCMV also interferes with TAP-dependent peptide transport, but does so in a completely different fashion. HCMV encodes an ER-resident type 1 membrane glycoprotein, US6, that interacts with lumenal domains of the TAP transporter (Ahn et al., 1997
; Hengel et al., 1997
; Lehner et al., 1997
).
Despite the high degree of genomic and biological similarity, searches of genomic and protein databases have not yielded obvious homologues of the closely related HSV1/2 ICP47 or the more distantly related HCMV US6 within the BHV1 genome. Based on our experiments, we cannot conclude whether the BHV1-associated inhibition of TAP involves a soluble or a membrane protein. Permeabilization of BHV1-infected cells and repeated washes prior to the peptide translocation assay did not abrogate the inhibitory effect. A putative cytosolic inhibitor should therefore possess a high binding affinity for TAP, like ICP47 (Tomazin et al., 1996 ). Alternatively, the TAP inhibitor could be a membrane protein like US6 (Ahn et al., 1997
; Hengel et al., 1997
), and would thus be resistant to cytosol depletion by washing the permeabilized cells.
The BHV1 genome encodes about 70 proteins, which are expressed during IE, early and late stages of virus infection. To investigate to which sub-group the BHV1 genes that interfere with MHC class I expression belong, we have controlled the viral gene expression cascade using transcription/translation inhibitors and inhibitors of viral DNA synthesis. These experiments indicate that early gene product(s) of BHV1 are responsible for altered intracellular trafficking of MHC class I molecules. In this respect, BHV1 differs from HSV1 and HSV2, which block MHC class I cell surface expression via an IE gene product, ICP47. A recent report on immunoevasive strategies used by PrV also suggests the involvement of an early gene product in down-regulation of MHC class I surface expression (Ambagala et al., 2000 ).
At present, it is unclear whether the ER retention of MHC class I molecules in BHV1-infected cells is solely due to a lack of antigenic peptides. The observed retention of MHC class I molecules could involve additional viral protein(s) specifically binding to MHC class I molecules and retaining them in the ER, analogous to the adenovirus E3/19K protein (Burgert & Kvist, 1985 ), MCMV gp40 (Ziegler et al., 1997
, 2000
) or HCMV gpUS3 (Jones et al., 1996
; Lee et al., 2000
), which all prevent egress of MHC class I molecules from the ER. In our biochemical experiments, we did not observe any co-precipitation of viral protein(s) with MHC class I molecules. The association with the viral protein, however, could be weak or transient, as is the case for the MCMV-encoded gp40 (Ziegler et al., 2000
).
The use of multiple strategies to subvert antigen presentation is advantageous for viruses. The vhs protein efficiently reduces the synthesis of new MHC class I molecules but has no effect on the existing pool of MHC class I molecules that will continue to present antigenic peptides to CTLs. The effectiveness of multiple independent evasion mechanisms has been clearly illustrated for HSV1 (Tigges et al., 1996 ). The presence of either the HSV1 vhs protein or ICP47 partially inhibits the lysis of infected fibroblasts by CTLs. When both proteins are present, they act synergistically and almost completely inhibit lysis by specific CTL clones.
In conclusion, we have shown that BHV1 uses at least two different strategies to interfere with the expression of antigen-loaded MHC class I molecules at the cell surface. The BHV1vhs- mutant allowed a biochemical analysis of the integrity of the MHC class I-restricted antigen presentation pathway at time-points at which wild-type BHV1 shuts off host protein synthesis. The BHV1vhs- mutant will facilitate the identification and characterization of the gene product(s) involved in the BHV1-mediated inhibition of MHC class I-restricted antigen presentation.
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Acknowledgments |
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Received 26 March 2001;
accepted 28 May 2001.