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Journal of Virology, September 2006, p. 8541-8553, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00830-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Inhibition by Woodchuck Hepatitis Virus of Class I Major Histocompatibility Complex Presentation on Hepatocytes Is Mediated by Virus Envelope Pre-S2 Protein and Can Be Reversed by Treatment with Gamma Interferon

Jinguo Wang1 and Tomasz I. Michalak1,2*

Molecular Virology and Hepatology Research, Division of Basic Medical Science,1 Discipline of Laboratory Medicine, Faculty of Medicine, Health Science Centre, Memorial University, St. John's, Newfoundland, Canada2

Received 21 April 2006/ Accepted 19 June 2006


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ABSTRACT
 
Presentation of class I major histocompatibility complex (MHC) is severely down-regulated on hepatocytes in chronic hepatitis caused by woodchuck hepatitis virus (WHV). To determine which of the viral proteins mediates class I MHC antigen suppression, cultured normal woodchuck hepatocytes were transfected with the complete WHV genome, sequences encoding individual virus proteins, or whole virus genomes in which transcription of selected proteins was disabled by site-specific mutagenesis. It was found that hepatocyte presentation of class I MHC antigen was significantly inhibited following transfection with complete WHV genome or with viral subgenomic fragments encoding envelope pre-S2 protein or pre-S1 protein, which naturally encompasses pre-S2 amino acid sequence. In contrast, hepatocytes transfected with WHV X gene alone demonstrated a profound enhancement in the class I antigen display, whereas those expressing virus major S protein or nucleocapsid (core) protein were not different from control hepatocytes. Analysis of the mutated WHV sequences confirmed that the envelope pre-S2 protein was responsible for inhibition of the class I MHC antigen display. Interestingly, treatment with recombinant woodchuck gamma interferon (rwIFN-{gamma}) restored the inhibited presentation of the class I antigen. Moreover, the class I antigen suppression was not associated with down-regulation of hepatocyte genes for class I MHC heavy chain, ß2-microglobulin, transporters associated with antigen processing, and proteasome subunits. These findings indicate that the defective presentation of class I MHC antigen on hepatocytes transcribing WHV is a consequence of posttranscriptional suppression exerted by virus pre-S2 protein and that this hindrance can be fully reversed by IFN-{gamma}.


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INTRODUCTION
 
Hepatitis B virus (HBV) is a member of the hepadnavirus family, which includes, among several mammalian and avian viruses, woodchuck hepatitis virus (WHV), which naturally infects eastern North American woodchucks (Marmota monax). Woodchucks infected with WHV represent a highly valuable natural model of hepatitis B in which molecular, immunological, and histological sequelae of virus-induced liver inflammation closely resemble those encountered in humans, including development of chronic hepatitis (20, 32). It is now also evident that HBV and WHV invade and replicate not only in hepatic tissue but also in the lymphatic system, although under most circumstances, less vigorously than in the liver (24-26). The host cellular immune responses directed against hepadnavirus protein epitopes displayed on infected hepatocytes, particularly those mediated by cytotoxic T lymphocytes (CTL), are regarded to be crucial in the recovery from acute hepatitis as well as in the induction of hepatocellular damage in actively progressing infection (3). In contrast to acute hepatitis, which is characterized by a strong and specific CTL response directed towards multiple virus epitopes, chronic hepatitis B is characterized by a weak and narrowly focused CTL antiviral reactivity. It is assumed that this hindrance is one of the main factors underlying the development of chronic hepadnaviral infection and protracted liver damage.

Because the triggering and strength of antiviral CTL responsiveness depend to a significant degree on the efficient presentation of viral peptides by class I major histocompatibility complex (MHC) molecules on the surfaces of infected cells, we have previously investigated characteristics of the class I MHC antigen display in woodchucks acutely and chronically infected with WHV (22). Among others, we have uncovered that chronic WHV hepatitis but not acute WHV hepatitis is associated with a profound suppression in class I antigen on the surface of infected hepatocytes. Thus, while acute hepatitis was accompanied by enhanced hepatocyte presentation of the class I antigen, inhibition of the antigen was consistently detected on these cells with chronic hepatitis. This occurred despite the fact that levels of intrahepatic expression of class I MHC-affiliated genes encoding woodchuck class I heavy and ß2-microglobulin (ß2m) chains and transporters associated with antigen processing (TAP1 and TAP2), as well as gamma interferon (IFN-{gamma}), which is one of the most powerful stimulators of the class I antigen expression (1), were found to be augmented to the same degree in both acute and chronic hepatitis. Furthermore, the hepatocyte class I antigen suppression was not linked to the histological severity of hepatocellular injury, the extent of lymphocytic infiltrations or the hepatic load of WHV. It was concluded that the deficiency in hepatocyte class I antigen display is a uniform hallmark of chronic WHV hepatitis and is caused by a virus-dependent posttranscriptional interference. However, the basis of this interference, particularly which of the WHV genome translation products might be responsible for this impediment, was not investigated.

The presentation of peptides by the class I MHC molecules on the cell surface accessible for CTL recognition is a multistep process involving the generation of peptides, their transport, loading onto class I heavy chain-ß2m complexes in the endoplasmic reticulum (ER), and trafficking the complexes to the cell surface via the Golgi apparatus (30). The generation of short peptides in the cytosol is achieved by the ubiquitin-proteasome system. Briefly, the endogenously synthesized proteins are first conjugated with polyubiquitin by ubiquitin ligase (16). Then, the ubiquitin-tagged proteins are unfolded by the 19S cap of the proteasome, which is composed of proteasome activator 28 alpha (PA28{alpha}) and proteasome activator 28 beta (PA28ß) and subsequently degraded into short peptides within the proteasome 20S core (18). The peptidase activity in the 20S proteasome resides in constitutive ß subunits, termed X, Y, and Z, which can be readily substituted by IFN-{gamma}-inducible ß subunits, such as multicatalytic endopeptidase complex-like 1 (MECL-1), low-molecular-mass protein 2 (LMP2), and low-molecular-mass protein 7 (LMP7), which display catalytic activity. The generated peptides are either further degraded into amino acids (aa) for recycling by cytosolic peptidase or transported to the ER by TAP1/TAP2 heterodimer complexes (28). An interaction between the TAP molecules and class I MHC-ß2m is required for the assembly of class I MHC-peptide complexes (29). Upon loading a peptide, the class I MHC complex is exported from the ER to the Golgi apparatus and finally transported to the cell surface, where it can be identified by the peptide-specific CTL.

In the present study, to investigate the nature of the molecular hindrance responsible for hepadnavirus-induced inhibition of the class I MHC antigen on hepatocytes observed in chronic WHV (22), we established a panel of woodchuck hepatocyte transfectants transiently or stably expressing the whole genome of WHV, subgenomic fragments encoding individual virus proteins, or complete WHV sequences in which expression of individual translation products was disabled by premature stop codons introduced through site-specific mutagenesis. The display of class I MHC antigen on these hepatocytes was measured and compared to the control naive hepatocytes and those transfected with empty vector. It was uncovered that the expression of the complete WHV genome profoundly down-regulated presentation of the class I antigen on hepatocytes in vitro, and throughout the differential analysis of the created transfected hepatocyte lines, it was determined that the virus envelope pre-S2 protein was responsible for this effect. In parallel experiments, it was also found that the inhibited presentation of the class I antigen can be fully restored by exposure of hepatocytes to IFN-{gamma}, implying that the virus-induced suppression was not tenacious but can be reversed by treatment with an appropriate exogenous agent.


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MATERIALS AND METHODS
 
WHV plasmids. A plasmid containing approximately 1.1 times the length (exactly 1.127) of the WHV genome was constructed by sequentially inserting into the eukaryotic expression vector pcDNA3.1 (Invitrogen, Carlsbad, Calif.) two fragments (A and B) derived from a wild-type WHV/tm3 DNA sequence (GenBank accession number AY334075) (24). Fragment A encompassed nucleotides (nt) from position 1 to 2120 (numbers denote the positions in relation to the endogenous EcoRI site in the WHV/tm3 genome), whereas fragment B encompassed nt 1698 to 3308 (Fig. 1). Fragment A was generated by PCR with sense primer 5'-TTCGAAATCAAACCTGGGCC (position 3286 to 3305), which was homologous to the sequence located upstream from the EcoRI site (position 3308 to 3305) (Fig. 1), and antisense primer 5'-CGCGGTACCCAGTGTCCACCAAAGCATTA (position 2101 to 2120). The produced amplicon had the KpnI site incorporated at the 3' end (indicated in underlined italic letters) and the EcoRI site at the 5' terminus to facilitate cloning. Fragment B was amplified with primers 5'-CGCCTCGAGTCCGGTCCGTGTTGC (position 1698 to 1712), containing an incorporated XhoI site, and 5'-CGTGGTATGTCCCGAATTCC (position, 3307 to 3318), which contained the endogenous EcoRI site (indicated in underlined letters). The PCR cycling conditions used for amplification of both fragments were as follows: denaturation at 94°C for 3 min, 30 cycles at 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 2 min at each step, and final elongation at 72°C for 3 min. The reaction was performed in a Peltier Thermal Cycler PTC-200 (MJ Research Inc., Waltham, Mass.) using a standard reagent mixture described previously (25). The resulting amplicons were purified from low-melting-point agarose using the Wizard PCR Preps DNA purification system (Promega Corp., Madison, Wis.).


Figure 1
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  		FIG. 1. Schematic presentations of p-WHV containing ~1.1 times the length of the WHV genome and expression vectors carrying WHV sequences encoding individual virus proteins which were generated and examined during the course of this study. The nucleotide 5'-end and 3'-end positions for each WHV ORF and for the p-WHV construct, as well as the location of the endogenous EcoRI site within p-WHV, are marked. Relative positions for endogenous WHV promoters (Pro) and an enhancer (Enh) are indicated along the p-WHV sketch, while the location of the pC-DNA3.1-derived cytomegalovirus promoter (CMV Pro) is indicated at the beginning of each WHV sequence insert. All numbers denote the nucleotide positions according to the WHV/tm3 genome sequence (GenBank accession number AY334075 [24]).

The construction of the plasmid containing more than full-length WHV genome was accomplished by a two-step procedure. In the first step, digestion of fragment A amplicon with EcoRI and KpnI enzymes was performed. In parallel, the pcDNA3.1 vector was linearized with the same restriction enzymes. The ligation of the excised PCR fragment and the linearized vector was done at an estimated molar ratio of 2:1 at 14°C for 16 h in a 10-µl reaction mixture containing 1 U T4 DNA ligase (Invitrogen) and 1x ligation buffer (10 mM MgCl2, 1 mM ATP, 1 mM dithiothreitol, and 5% [wt/vol] polyethylene glycol-8000 in 50 mM Tris-HCl buffer, pH 7.6). TOP10 competent Escherichia coli cells (Invitrogen) were transformed following the supplier's instructions. Screening of positive colonies was based on a standard alkaline method for minipreps of recombinant plasmid DNA, followed by digestion with EcoRI and KpnI enzymes, and agarose gel analysis of the fragments obtained. The selected positive clone was designated p-WHV(1-2120).

In the next step, WHV genome fragment B was cloned into p-WHV(1-2120) by using the same strategy as described above. Thus, the fragment B amplicon and p-WHV(0-2120) were treated with XhoI and EcoRI enzymes and the resulting products ligated. After the transformation of E. coli, a positive clone was identified by restriction enzyme mapping. The final construct, designated p-WHV, contained approximately 1.1 times the length of the WHV genome, starting from nt 1698, located upstream of the enhancer and pre-C promoter and direct repeat 1 and 2 regions and ending at nt position 2120 located downstream of the WHV polyadenylation signal sequence (Fig. 1) (7). The assessment of the correct design of the construct was confirmed by restriction digest analysis and DNA sequencing (data not shown).

In order to examine whether expression of individual WHV proteins can influence presentation of the class I MHC antigen, plasmids containing WHV genomic fragments encoding the virus envelope pre-S1 protein (WHpS1), envelope pre-S2 protein (WHpS2), envelope major S protein (WHmS), core protein (WHc), or X protein (WHx) were constructed using pcDNA3.1 as the backbone. Briefly, WHV sequences encoding the respective full-length proteins were amplified by PCR using the sequence-specific oligonucleotide primers containing appropriate restriction enzyme recognition sequences. Then, the PCR products were treated with restriction enzymes and directionally inserted into pcDNA3.1 vector. The integrity of the constructs was confirmed by DNA sequencing. The names of the plasmids encoding individual WHV proteins are delineated in Fig. 1.

WHV mutants. To study the effect of individual WHV proteins expressed in the context of the whole WHV genome on hepatocyte transcription of the class I MHC-related genes and on the surface display of the class I antigen, a series of mutants containing premature stop codons within subgenomic sequences encoding individual WHV proteins were produced by site-directed mutagenesis using p-WHV as the backbone and an approach reported by others (14). Thus, WHV genomic regions encoding WHp-S1, WHmS, WHc, WHx, and WHV polymerase (WHp) were mutated to selectively eliminate expression of a given protein without introducing amino acid changes to other translation products of the virus genome. For the generation of each mutant (Fig. 2), a pair of specific primers was designed to introduce mutating a nucleotide(s) at the desired position(s) (indicated by underlined italic letters in the primer sequences shown below). Thus, the WHpS1 mutant ({Delta}WHpS1) was generated using 5'-CAAAATAGCAGCGTGGTAGCCTGCAGTGGGCACT (position 3027 to 3060) and 5'-AGTGCCCACTGCAGGCTACCACGCTGCTATTTTG (position 3060 to 3027), which changed TGG to TAG, i.e., from tryptophan to a stop codon at position 18 in WHpS1 amino acid sequence. The WHmS mutant ({Delta}WHmS) was created using sense primer 5'-GGTGGTGTATTTCTTGTAGACAAAAATCCTAAC (position 337 to 369) and antisense primer 5'-GTTAGGATTTTTGTCTACAAGAAATACACCACC (position 369 to 337), which changed TGG to TAG, i.e., from tryptophan to a stop codon at aa 20 of WHmS. The WHc mutant ({Delta}WHc) was produced with primers 5'-CTATTAGACAAGCTTGAGTATGCTGGGATGAA (position 2181 to 2212) and 5'-TTCATCCCAGCATACTCAAGCTTGTCTAATAG (position 2212 to 2181), which modified TTA to TGA, i.e., from leucine to a stop codon at aa position 59. The WHx mutant ({Delta}WHx) was obtained with primers 5'-GTTGCCAACTGGATCCTTAGAGGGACGTCCTTCTGC (position 1519 to 1554) and 5'-GCAGAAGGACGTCCCTCTAAGGATCCAGTTGGCAAC (position 1554 to 1519), which changed ACG to TAG, i.e., from threonine to a stop codon at aa position 12. The WHp mutant ({Delta}WHp) was created with primers 5'-CTTCCGGAACATACAGTAATTAGGAGAAGAGGAGG (position 2447 to 2481) and 5'-CCTCCTCTTCTCCTAATTACTGTATGTTCCGGAAG (position 2481 to 2447), which changed TCA to TAA, i.e., from serine to a stop codon at aa position 13. For the generation of each mutant, two rounds of PCR were performed. The first round included two reactions, one with a forward primer and a mutant reverse primer and the second with a mutant forward primer and a reverse primer. Both reactions were carried out for 15 cycles of 94°C for 20 s, 52°C for 20 s, and 72°C for 2 min by using 100 ng of p-WHV as the template. PCR products from these two reactions were gel purified, combined at an equal molar ratio, and used as the template for the second-round PCR. The primers for the second round were the same forward and reverse primers as those used in the first round. This amplification was carried out at 94°C for 20 s, 52°C for 20 s, and 72°C for 2 min for 35 cycles with a final elongation step of 72°C for 5 min. The product from the second round PCR was gel purified and cloned into an appropriate vector. The presence of the created stop codons were always verified by DNA sequencing (data not shown).


Figure 2
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  		FIG. 2. Schematic presentation of the strategy applied for the generation of p-WHV mutated sequences by introducing premature stop codons. Designated names of the constructs and the introduced nucleotide modifications are identified on the left side of the panel. Relative locations of the stop codons inserted into WHV ORFs (open arrows) are marked by crosses. Detailed information on the site-specific mutagenesis leading to creation of each mutant is shown under each ORF sketch. The bold numbers at the 5' and 3' ends of the outlined sequences show nucleotide positions in relation to the EcoRI site in the WHV/tm3 genome (see the legend to Fig. 1). Bold and blue letters in the nucleotide sequences stand for introduced nucleotides, and the underlined three-nucleotide sequences represent the stop codons created. The aa sequences of interest and their relative positions (numbers after aa) within respective viral proteins are shown above the nucleotide sequences, while the aa sequences naturally overlapping the sequence of interest are depicted under the nucleotide sequences to indicate that no aa alterations were introduced. The asterisk marks termination of amino acid translation.

Hepatocyte cultures. Woodchuck hepatocyte line WCM-260 was established from a liver biopsy specimen obtained from an adult healthy animal and was characterized in detail previously (4, 8, 19). WCM-260 cells were grown in a conditioned hepatocyte culture medium consisting of 80% (vol/vol) Hepato-STIM medium (Becton Dickinson, Bedford, Mass.) and 20% (vol/vol) culture supernatant from HepG2 cells (HB-8065; ATCC). The medium was supplemented with 10 ng/ml epithelial growth factor, 2 mM L-glutamine, 50 µg/ml penicillin, and 50 µg/ml streptomycin (all from Becton Dickinson). The conditions for WCM-260 maintenance, detachment, passage, and storage were reported previously (4, 8).

Hepatocyte transfections. The transfection of WCM-260 hepatocytes with p-WHV or plasmids carrying sequences encoding individual WHV proteins or WHV mutant proteins was done using Lipofectamine 2000 (LF2000) reagent (Gibco BRL), following the supplier's instruction. The day before the transfection, cells were seeded at 3 x 105 per well in a 12-well culture plate (Fisher Scientific Limited, Nepean, Ontario, Canada). Immediately before transfection, the attached cells were washed three times with Dulbecco's modified Eagle's medium (DMEM; Invitrogen) without supplements, and then 1 ml of the same medium was added to each well. At the same time, 2 µg of plasmid DNA and 2.5 µl of LF2000 per transfection were suspended in 100 µl of DMEM each, combined, and incubated for 20 min at an ambient temperature. The plasmid DNA-LF2000 mixture was then added to cells and incubated for 8 h at 37°C. Subsequently, the DMEM was replaced with conditioned hepatocyte culture medium and cells cultured for time periods indicated. Thus, the transient expression of a given transgene was evaluated 48 to 72 h later. For stable transfection, G418 reagent (Invitrogen) was added 3 days posttransfection at 900 µg/ml and hepatocytes maintained for at least a 4-week period, with culture medium changed every 3 to 4 days. WCM-260 cells transiently or stably transfected with pcDNA3.1 vector alone (empty vector) were used as controls.

Hepatocyte stimulation with IFN-{gamma}. To assess whether the inhibition of class I MHC presentation on hepatocytes found after expression of complete WHV genome or some of its subgenomic fragments was reversible, recombinant woodchuck IFN-{gamma} (rwIFN-{gamma}), the cytokine that is one of the most potent naturally occurring class I MHC inducers, was produced in the baculovirus expression system, as described recently (33). Briefly, the full-length IFN-{gamma} cDNA derived from woodchuck peripheral blood lymphoid cells stimulated with concanavalin A (5 µg/ml; Sigma Chemical Co., St. Louis, Mo.) was cloned and expressed in insect Sf9 cells by using the MaxBac 2.0 expression system (Invitrogen). The final concentration of rwIFN-{gamma} in Sf9 culture supernatant was 150 U/µl, as determined by inhibition of WCM-260 hepatocyte lysis caused by infection with encephalomyocarditis virus (33). For stimulation with rwIFN-{gamma} of WCM-260 hepatocytes transfected with p-WHV, plasmids encoding individual viral proteins or p-WHV mutants, 150 U/ml of rwIFN-{gamma} was added to hepatocyte culture medium 18 h prior to examination of the class I MHC expression. Naive WCM-260 cells exposed under the same conditions to culture supernatant from Sf9 cells infected with wild-type (empty) baculovirus vector were used as controls.

RNA isolation and reverse transcription. Total cellular RNA was extracted with TRIzol reagent (Invitrogen), as described previously (22, 25). Possible genomic DNA contamination was eliminated by treatment with DNase I by using a DNase-free kit (Ambion, Austin, Tex.), as per the supplier's instruction. One to 2 µg of total RNA was reverse transcribed into cDNA with random primers by using 200 U of reverse transcriptase (RT) from Moloney murine leukemia virus (Gibco BRL), as reported previously (5, 25). Each reaction was set up in parallel with a negative control that had all ingredients except RT.

Detection of WHV mRNA. RNAs extracted from transiently or stably transfected hepatocytes were reverse transcribed into cDNA and examined by PCR under conditions previously established (5, 25), using primers specific for nonoverlapping regions of WHV core (C), surface (S), and X genes. Primer sequences of the X protein gene were reported before (25), whereas those for the amplification of the C and S gene sequences were as follows: for the C gene, 5'-CTAACAGGTAGGGAACATTGC (sense) and 5'-GACCTAGAAGCTCTTGCACC (antisense); for WHpS1, 5'-GCGAAGCTTAATGTATACCCAAATC (common antisense primer) and 5'-GCGCTCGAGGCAACATAATGGGCAAC (sense primer); for WHpS2, 5'-CGCCTCGAGACTATGAAAAATCAGACT (sense primer); and for WHmS, 5'-CGCCTCGAGAGATGTCACCATCAAGTC (sense primer). As controls, cDNAs prepared from WCM-260 transiently or stably transfected with pcDNA3.1 alone (empty vector) or from naïve (nontransfected) WCM-260 hepatocytes were included. In addition, samples of RNA isolated during the same extractions but not reversely transcribed were used as DNA contamination controls (5). Southern blot hybridization analysis of the PCR product with 32P-labeled complete recombinant WHV DNA (rWHV DNA) as a probe was routinely used to verify the specificity of viral sequence detected and the validity of controls, as described elsewhere (25).

Cloning of woodchuck proteasome subunit cDNAs. Since sequences of woodchuck genes encoding proteasome subunits were unknown prior to this study, we made an effort to determine their partial sequences to investigate the potential influence of WHV expression on transcriptional activity of these genes in WCM-260 hepatocytes. For this purpose, total RNA isolated from WCM-260 hepatocytes was treated with DNase and then reverse transcribed to cDNA as described above. Amplification of woodchuck LMP2, LMP7, MECL-1, PA28{alpha}, and PA28ß cDNA fragments was done using primers deduced by interspecies homology comparison of human, mouse, and rat cDNA sequences by using PC Gene software (IntelliGenetics Inc., Mountain View, Calif.). The LMP2 cDNA was amplified with primers 5'-CGGRAGAAGTCCACACCGGG and 5'-CTCATCRTAGAATTTTGGCAG and that of LMP7 with 5'-GCCTTCAARTTCCAGCATGG and 5'-CCTCCAGAATAGYTGTCTCTGTG, MECL1 with 5'-CTTCTCYTTCGAGAACTGCC and 5'-TCCACCTCCATRGCCTGCAC, PA28{alpha} with 5'-GGGAGCTATTTYCCCAAGAA and 5'-GGGCTTCTTGCGCTTCTC, and PA28ß with 5'-ATGGCCAAGCCKTGTGGG and 5'-GATGGCTTTTCTTCACCCTT. PCR amplification was performed in a PTC-200 cycler using 50 ng of template cDNA and the standard reagent mixture described before (25). Cycling conditions were 30 cycles of 94°C for 20 s, 52°C for 20 s, and 72°C for 1 min, followed by an elongation step at 72°C for 3 min. The DNA amplicons of the expected sizes were purified from low-melting-point agarose and cloned into the pCRII vector using a TOPO-TA cloning kit (Invitrogen). Plasmids with cloned fragments were examined by DNA sequencing. The nucleotide sequences obtained were compared to those of humans, mice, and rats. This comparison showed that the woodchuck sequences were more comparable with those of humans (91.5 to 98.5%) than with those of mice (87.1 to 94.2%) or rats (87.9 to 93.9%).

Quantitation of transcriptional activity of class I MHC- and proteasome-affiliated genes. The quantitation of transcription of class I MHC heavy chain, ß2m, TAP1, TAP2, LMP2, LMP7, MECL-1, PA28{alpha}, PA28ß, and ß-actin genes in WHV-transfected and control WCM-260 hepatocytes was done by real-time PCR using a Light Cycler (Roche, Laval, Quebec, Canada). cDNA samples from WCM-260 hepatocytes transfected with p-WHV or with plasmids encoding individual WHV proteins or WHV mutant proteins were prepared as described above. Real-time PCR was carried out in capillary tubes with 0.2 µl of cDNA (equivalent to 25 to 50 ng of total RNA) in a 20-µl volume using FASTstart SYBR reagent kit (Roche Diagnostics, Laval, Quebec, Canada). Primers designed to specifically amplify the sequences of interest are listed in Table 1. It is of note that primer sequences for woodchuck class I heavy chain, ß2m, TAP1, and TAP2 were designed based on the partial gene sequences previously determined in this laboratory (22). The copy number for each gene was extrapolated from a standard curve generated using 10-fold serial dilutions of a relevant cloned gene fragment, which were run in parallel in each reaction. Specificity of the products was determined by melting curve analysis. Negative controls included water instead of cDNA and samples treated exactly the same as test RNA samples but in the absence of RT. The housekeeping ß-actin gene was used to standardize the expression of the genes tested.


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  		TABLE 1. Primer sequences for real-time RT-PCR quantitation of woodchuck class I MHC- and proteasome-affiliated gene expression

Flow cytometry and confocal microscopy. To determine the presence of WHV envelope and core proteins in WCM-260 hepatocytes transfected with p-WHV or plasmids encoding WHV proteins and selected protein mutants, the cells were grown on coverslips, fixed with cold ethanol ether for 3 min, and incubated with the respective specific antibodies. The woodchuck hepatitis surface protein (WHs) antigenic reactivity was detected with guinea pig anti-pre-S antibodies raised by immunization with a recombinant WHV pre-S1/pre-S2 protein produced in an E. coli expression system (J. Wang and T. I. Michalak, unpublished data) or with guinea pig anti-WHs produced by immunization with serum-derived woodchuck hepatitis surface antigen (WHsAg) which recognized all three envelope proteins, i.e., WHpS1, WHpS2, and WHmS (23). WHV core antigen (WHcAg) was detected with rabbit anti-WHc antibodies produced by immunization with WHV core particles (27). The reactivity of guinea pig antibodies was identified with Cy5-conjugated goat anti-guinea pig immunoglobulin G (IgG) and that of rabbit antisera with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (both from Jackson ImmunoResearch Laboratories Inc., West Grove, Pa.). Cells were examined using an FluoView FV300 confocal system (Olympus America Inc., Melville, N.Y.) equipped with an Olympus BX50WI microscope. WCM-260 hepatocytes transfected with empty pcDNA3.1 vector and stained exactly the same way as test cells, as well as WCM-260 transfected with p-WHV, plasmids encoding individual WHV proteins or p-WHV mutants incubated with preimmune sera were used as controls.

To evaluate the presentation of class I MHC antigen on the transfected and control WCM-260 hepatocytes, the unfixed cells were incubated for 45 min on ice with mouse monoclonal antibody (MAb) against woodchuck class I MHC heavy chain (B1b.B9), previously prepared in this laboratory (21) or with phosphate-buffered saline (PBS), pH 7.6, a negative control. After a washing with PBS, cells were exposed to FITC-conjugated goat anti-mouse IgG (heavy plus light chain) antibodies (Jackson ImmunoResearch Laboratories Inc.) for 45 min, followed by washing with PBS. Subsequently, the cells were fixed with 4% paraformaldehyde in PBS and analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson, Palo Alto, Calif.). Flow cytometry data were interpreted with the assistance of CellQuest software (Becton Dickinson) or WinMDI software (The Scripps Research Institute, La Jolla, Calif.). Geometric mean fluorescence intensity (MFI), calculated automatically from the plots of flow cytometric histograms, was used to compare the levels of class I MHC expression. The results from three independent experiments were used to calculate the means. WCM-260 cells transfected with empty pC-DNA3.1 vector served as controls, and their MFI values were taken as 100%.

Moreover, WCM-260 hepatocytes transfected with p-WHV, plasmids encoding individual WHV proteins, or mutated p-WHV sequences with disabled expression of the selected viral proteins, as well as naive WCM-260 hepatocytes and those stably transfected with empty pC-DNA3.1 vector, were examined for class I antigen display after treatment with exogenous rwIFN-{gamma}, as described above, and compared to the cells not treated with this cytokine.

Statistical analysis. The statistical analysis was carried out using computer software (GraphPad Software Inc., San Diego, Calif.). P values were determined by an unpaired t test, and P values of <0.05 were considered significant.

Nucleotide sequences accession numbers. The accession numbers for the woodchuck nucleotide sequences established in this study submitted to GenBank were as follows: LMP2 gene, AY726002; LMP7 gene, AY7260023; MECL-1 gene, AY726004; PA28{alpha} gene, AY726005; and PA28ß gene, AY726006.


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RESULTS
 
Validation of p-WHV expression in hepatocytes. Expression of p-WHV, carrying more than the full-length WHV genome, was driven by the cytomegalovirus promoter contained in the pC-DNA3.1 vector and most likely by WHV genome intrinsic promoter/enhancer sequences, as illustrated in Fig. 1. After transient transfection of WCM-260 hepatocytes with p-WHV, mRNA transcripts were identified by reverse transcriptase PCR (RT-PCR) using primer pairs specific for nonoverlapping regions of the C, S, and X open reading frames (ORFs) (Fig. 3A). The specificity of the mRNA detections was ascertained through the absence of DNA signals when the RT step was omitted from RT-PCR and by Southern blot hybridization analysis of the amplified products. In addition, total RNA isolated from the liver of a woodchuck with chronic WHV yielded RT-PCR amplicons with molecular sizes were identical to sizes of amplicons detected in the transfected WCM-260 cells when amplified with the same primer pairs (Fig. 3A). WCM-260 cells stably transfected with p-WHV gave the same results (Fig. 3B).


Figure 3
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  		FIG. 3. Expression of WHV genes in WCM-260 hepatocytes transfected with p-WHV. WCM-260 hepatocytes transiently transfected (after 48 to 72 h) (A) or stably transfected (after 2 to 3 months in the presence of G418) (B) with p-WHV were analyzed for expression of viral mRNAs by RT-PCR using primer pairs specific for S, C, and X genes. The amplified products were identified by Southern blot hybridization by probing with complete rWHV DNA. Total RNA isolated from the liver of a woodchuck with chronic WHV hepatitis was examined each time as a positive control. RNA samples not transcribed to cDNA (RT –) were analyzed in parallel with those subjected to RT reaction (RT +) to exclude possible contamination with DNA. The molecular sizes (bp) of the detected PCR products are marked on the right side of the panel. In panel C, WHV pre-S1/pre-S2 and core proteins were visualized by immunostaining in WCM-260 hepatocytes transiently transfected with p-WHV. Following exposure of the cells to anti-WHV pre-S and anti-WHc antibodies, secondary antibodies conjugated with Cy5 or FITC were applied to detect WHV pre-S (left) and WHcAg (center), respectively. The overlaid image (right) is shown to illustrate pre-S1/pre-S2 and WHcAg colocalization. Bars, 20 µM.

The synthesis of WHsAg and WHcAg in the transfected cells was examined with specific antibodies by confocal microscopy. As shown in Fig. 3C, both antigens were colocalized in the cytoplasm of WCM-260 cells transiently transfected with p-WHV. The cells transfected with empty pC-DNA3.1 plasmid remained entirely negative (data not shown), confirming specificity of the antigen detections. The efficiency of the transfection, estimated based on the number of WHsAg- or WHcAg-positive cells identifiable by immunostaining, was between 10 and 20%. A comparable result was obtained when WCM-260 hepatocytes were transiently transfected with a plasmid encoding green fluorescent protein (pEGFP; Clontech, Mountain View, Calif.) (data not shown).

Further, to determine whether WHV virions were produced by WCM-260 hepatocytes transfected with p-WHV, culture supernatants from these cells transiently transfected with this plasmid were concentrated by ultracentrifugation, as described elsewhere (6), and injected intravenously into a healthy WHV-naive woodchuck. The animal developed WHV infection characterized by the persistent presence of WHV DNA in serum, peripheral lymphoid cells, and liver tissue when tested at week 57 postinoculation. The infection progressed in the absence of serological markers of WHV infection, i.e., WHsAg and anti-WHc, and its pattern was comparable to that of the primary occult infection, which, with time, spread to the liver, as described previously (5, 24). Taken together, these findings showed not only that p-WHV was transcriptionally active in WCM-260 hepatocytes but also that infectious virions were produced. However, due to the relatively small number of hepatocytes displaying WHV antigens after transient transfection (≤20% by immunofluorescence staining), it was concluded that the use of the stably transfected hepatocytes, which all a priori express viral genome, would be more advantageous for an examination of WHV influence on class I MHC expression.

Validation of plasmids encoding individual WHV proteins. To assess which of the translation products of the WHV genome may modify class I MHC expression, WCM-260 hepatocytes were transfected with plasmids carrying WHV sequences encoding WHpS1, WHpS2, WHmS, WHc, or WHx protein. As illustrated in Fig. 4A, when total RNA extracted from WCM-260 hepatocytes transiently transfected with the plasmids described above was analyzed with appropriate specific primer pairs, the resulting RT-PCR amplicons had the same molecular sizes as those obtained by amplifying cDNA derived from RNA extracted from the liver of a woodchuck with chronic WHV hepatitis (Fig. 4A). The signals were detected only in RNA samples subjected to RT but not in those in which the RT step was omitted, as confirmed by Southern blot hybridization analysis (Fig. 4A). These results showed that the genes encoding individual WHV proteins were successfully transcribed in WCM-260 hepatocytes. The same was found to be true for WCM-260 hepatocytes stably transfected with the plasmids listed above (Fig. 4B).


Figure 4
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  		FIG. 4. Validation of the expression of the plasmid constructs carrying WHV subgenomic sequences encoding individual virus proteins. WCM-260 hepatocytes transiently transfected (A) or stably transfected (B) with p-WHpS1, p-WHpS2, p-WHmS, p-WHc, or p-WHx were analyzed for the presence of relevant mRNA by RT-PCR using primer pairs specific for the pre-S1 (S1), pre-S2 (S2), or mS region of the S gene or for the C or X gene. The amplicons were identified by Southern blot hybridization by probing with rWHV DNA. For other details, see the legend to Fig. 3.

Similarly to what was found for the cells transfected with p-WHV (see Fig. 3C), WCM-260 cells transiently transfected with p-WHc displayed cytoplamic localization of WHcAg in 10 to 20% of the cells. WHsAg was detected in similar proportions of the cells after transfection with p-WHpS1, p-WHpS2, or p-WHmS (data not shown). The display of WHx protein was not examined, due to the lack of an appropriate antibody. The WCM-260 cells stably transfected with the same plasmid constructs were seemingly WHcAg and WHsAg nonreactive by immunostaining, although they were positive by Western blotting (data not shown). This suggested that the antigens were expressed but at levels which were below the detection limit of the immunofluorescence method applied. In subsequent experiments, it was clearly established that these expression levels were sufficient to exert a consistent modifying effect on class I MHC antigen presentation.

Expression of complete WHV genome and subgenomic sequences encoding pre-S1 and pre-S2 proteins inhibits hepatocyte class I MHC antigen display. Stable transfection of WCM-260 cells with p-WHV resulted in a very significant (20 to 35%; P = 0.004) suppression of the surface class I MHC antigen display, as determined by flow cytometry, compared to the control WCM-260 cells transfected with empty pC-DNA3.1 vector (Fig. 5A) or to naive WCM-260 cells (data not shown). Similarly, WCM-260 cells transfected with WHV sequences encoding WHpS1 or WHpS2 protein showed a meaningful inhibition of the surface display of class I antigen (22 to 36%; P = 0.001 and P = 0.0003, respectively). In contrast, WCM-260 cells transfected with p-WHmS or p-WHc did not show a noticeable change in the class I antigen expression level (Fig. 5A). Interestingly, transfection with p-WHx led to a dramatic up-regulation (>50%; P = 0.0001) of the class I antigen presentation (Fig. 5A). Figure 5B illustrates the detected shifts in the class I MHC heavy chain fluorescence on WCM-260 cells transfected with p-WHV, p-WHpS1 p-WHpS2, or p-WHx, as well as after transfection with other plasmids in comparison to the cells transfected with the empty vector. Overall, the data revealed that while the expression of whole WHV genome down-regulated class I MHC antigen display on WCM-260 hepatocytes, expression of its individual proteins had different modifying effects from the inhibition exerted by WHpS1 and WHpS2, through the lack of a noticeable effect in the case of WHmS and WHc, to the considerable augmentation in the class I antigen presentation when the cells were transfected with p-WHx.


Figure 5
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  		FIG. 5. Modulation of the class I MHC antigen display on WCM-260 hepatocytes stably transfected with p-WHV or with plasmid constructs encoding individual WHV proteins. (A) The class I antigen presentation on WCM-260 hepatocytes stably transfected with p-WHV, p-WHpS1, p-WHpS2, p-WHmS, p-WHc, or p-WHx was assessed by flow cytometry by comparing the means (horizontal short lines) of MFI values obtained from three independent experiments. The data are presented as the percentages of MFI where the MFI values given by WCM-260 hepatocytes stably transfected with empty pC-DNA3.1 vector (e) were taken as 100%. P values were determined as indicated in Materials and Methods. (B) A representative example of flow cytometry histograms illustrating the display of the class I antigen on WCM-260 hepatocytes from one of three experiments shown in panel A. The levels of the class I antigen on the hepatocytes were determined prior to (IFN-{gamma} –) and after (IFN-{gamma} +) treatment with rwIFN-{gamma}, as described in Materials and Methods. The data are shown as overlaid histograms representing the cells transfected with plasmids carrying WHV sequences (open symbols) and the cells transfected with empty vector (filled symbols).

Effects of WHV genome site-specific mutations on hepatocyte class I MHC antigen presentation. To determine whether individual WHV translation products which were found to be capable of modifying hepatocyte class I MHC antigen display when expressed alone would yield a similar effect when expressed in the context of the complete WHV genome, WCM-260 hepatocytes were transfected with p-WHV mutants in which transcription of selected genes was blocked by premature stop codons (Fig. 2). As far as the availability of specific antibodies allowed, transcription of individual WHV proteins or their lack and the fact that the introduced mutations did not influence the expression of virus envelope and core proteins were confirmed by immunofluorescence staining (Fig. 6). Thus, WCM-260 cells transfected with p-{Delta}WHc and stained with anti-WHc were WHcAg negative, while those transfected with plasmids carrying mutated ORFs other than the C ORF were WHcAg reactive (Fig. 6; hepatocytes transfected with p-WHc are shown in Fig. 3C). Similarly, pre-S1/pre-S2 antigenic reactivity was detected after staining with anti-pre-S antibodies in WCM-260 cells transfected with p-{Delta}WHc, p-{Delta}WHpS1, p-{Delta}WHx, and p-{Delta}WHp but not in those transfected with p-{Delta}WHmS (Fig. 6). It is important to emphasize that anti-pre-S antibodies recognize both WHpS1 and WHVpS2 proteins but not WHmS (see Materials and Methods). Therefore, WCM-260 cells transfected with p-{Delta}WHpS1 should be reactive, as they were, since expression of WHpS2 was not affected. These findings not only verified that WHcAg and WHsAg synthesis was abrogated in WCM-260 cells transfected with p-{Delta}WHc and p-{Delta}WHmS but also implied that the absence of a single protein in the context of expression of the whole WHV genome in our system was without a major effect on the expression of other virus proteins.


Figure 6
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  		FIG. 6. Determination of the competence of p-WHV mutants with the disabled expression of selected structural or nonstructural proteins for the encoding of virus envelope and core proteins. WCM-260 hepatocytes transiently transfected with p-{Delta}WHpS1, p-{Delta}WHmS, p-{Delta}WHc, p-{Delta}WHx, or p-{Delta}WHp were probed with anti-WHV pre-S or with anti-WHc by immunostaining, as described in Materials and Methods and in the legend to Fig. 3. Bars, 20 µM.

As shown in Fig. 7A, the introduction of the premature stop codon in the WHmS-encoding region, which abrogated expression of not only WHmS but also WHpS1 and WHpS2 proteins (Fig. 2), resulted in a significant enhancement (P < 0.05) in the class I MHC surface presentation over that on hepatocytes transfected with p-WHV. On the other hand, blocking of the expression of WHx led to a profound suppression in the class I antigen display when compared with the control cells transfected with the empty vector (P = 0.0024). Overall, the results given above were in good agreement with the findings from WCM-260 hepatocytes transfected with plasmids encoding WHpS1, WHpS2, and WHx (Fig. 5A). Furthermore, when WCM-260 cells transfected with p-{Delta}WHpS1, which contained the stop codon disabling transcription of WHpS1 but still carried functional intrinsic start codons for WHpS2 and WHmS, were analyzed, it was found there was no change in the class I antigen display in comparison to the cells transfected with p-WHV (Fig. 7A). This finding strengthened the notion that WHpS2 protein was mainly, if not entirely, responsible for the observed inhibition of the class I antigen on WCM-260 hepatocytes transfected with p-WHV. Further, transfection with p-{Delta}WHp was without any statistically significant effect on the class I antigen staining. Finally, although the mean expression of class I antigen on WCM-260 hepatocytes transfected with p-{Delta}WHc was somehow enhanced compared to the cells transfected with p-WHV, the difference was not statistically significant, and it was likely related to a variation in one of the experiments. Figure 7B shows examples of the changes in the class I antigen surface presentation detected on WCM-260 hepatocytes transfected with different p-WHV mutants.


Figure 7
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  		FIG. 7. The class I MHC antigen display on WCM-260 hepatocytes transfected with p-WHV mutants in which expression of selected viral proteins was disabled by the introduction of appropriate premature stop codons. (A) Hepatocytes stably transfected with p-{Delta}WHpS1, p-{Delta}WHmS, p-{Delta}WHc, p-{Delta}WHx, or p-{Delta}WHp or with p-WHV or empty vector (e) as controls were assessed for class I antigen surface presentation by flow cytometry, as described in the legend to Fig. 5 and in Materials and Methods. (B) A representative example of flow cytometry histograms demonstrating the class I antigen display on WCM-260 hepatocytes transfected with different p-WHV mutants from one of three experiments depicted in panel A prior to (IFN-{gamma} –) and after (IFN-{gamma} +) treatment with rwIFN-{gamma}. For further details, see the legend to Fig. 5 and Materials and Methods.

Inhibition of class I MHC antigen presentation by WHV can be fully restored by treatment with IFN-{gamma}. Exposure of WCM-260 hepatocytes transfected with p-WHV, p-WHpS1, or p-WHpS2 to exogenous rwIFN-{gamma} led to up-regulation in the class I MHC antigen to the level comparable with that detected on WCM-260 hepatocytes transfected with empty pC-DNA1.3 or on naive WCM-260 cells treated with rwIFN-{gamma} (Fig. 5C and 8A). It is of note that the observed augmentation in the class I antigen presentation induced by rwIFN-{gamma} was significantly greater than that mounted by transfection of hepatocytes with p-WHx ([11.7 ± 1.6]-fold versus [1.6 ± 0.06]-fold; P = 0.01). Also, treatment with rwIFN-{gamma} of WCM-260 hepatocytes expressing mutated p-WHV sequences, including p-{Delta}WHx, led to a comparable increase in the class I MHC antigen surface display (Fig. 7C and 8B). Taken together, the experiments detailed above clearly showed that the inhibition of class I MHC antigen on cultured hepatocytes expressing whole WHV genome or its fragments encoding particular proteins was not irreversible but can be restored by treatment with exogenous IFN-{gamma}.


Figure 8
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  		FIG. 8. The display of class I MHC antigen on the surface of WCM-260 hepatocytes transfected with different WHV expression constructs following treatment of the cells with IFN-{gamma}. Hepatocytes stably transfected with p-WHV, p-WHpS1, p-WHpS2, p-WHmS, p-WHc, or p-WHx (A) or with p-{Delta}WHpS1, p-{Delta}WHmS, p-{Delta}WHc, p-{Delta}WHx, or p-{Delta}WHp and control cells transfected with empty pC-DNA3.1 vector (e) or with p-WHV (B) were treated with rwIFN-{gamma} prior to staining for class I antigen with B1b.B9 MAb, as outlined in Materials and Methods. It is of note that the treatment with rwIFN-{gamma} led to the unified augmentation in the class I antigen display to approximately the same level on hepatocytes transfected with different WHV DNA constructs and on the control cells. The data should be interpreted in the context of those shown in Fig. 5 and 7.

WHV-induced inhibition of class I antigen presentation is not due to suppressed transcription of class I MHC- or proteasome-linked genes. Quantification of the mRNA levels of the class I MHC-related genes (i.e., class I MHC heavy chain, ß2m, TAP1, and TAP2 genes) and the genes encoding subunits of proteasome (i.e., LMP2, LMP7, MECL-1, PA28{alpha}, and PA28ß genes) revealed no meaningful differences between WCM-260 hepatocytes transfected with p-WHV or with sequences encoding individual virus proteins of the virus and naive WCM-260 cells or those transfected with empty vector (Fig. 9). Similarly, there were no noticeable differences between the transcription levels of these genes between WCM-260 cells transfected with p-WHV or p-WHV mutants and the control cells (data not shown). Taken together, the results revealed that the inhibited or augmented presentation of the class I MHC antigen on WCM-260 cells transfected with WHV sequences identified as those capable of modifying the class I antigen display was not connected to the transcriptional activity of the genes examined. In a supplementary experiment, analysis of the class I MHC- and proteasome-linked gene mRNA levels in hepatocytes transfected with p-WHV, its mutants, or WHV subgenomic fragments and in control hepatocytes following treatment with rwIFN-{gamma} showed that the genes' transcriptional activity was up-regulated to approximately the same levels in all hepatocytes tested (Fig. 9 and data not shown).


Figure 9
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  		FIG. 9. Quantitation of the class I MHC- and proteasome-linked gene expression in hepatocytes transfected with p-WHV and with constructs encoding individual WHV proteins and either left untreated or subjected to treatment with IFN-{gamma}. The data were generated in three independent experiments by real-time RT-PCR. They are presented as the mean gene copy numbers per reaction, which were calculated using serial 10-fold dilutions of internal plasmid standards and normalized against housekeeping woodchuck ß-actin gene cDNA level. The open bars represent hepatocytes not treated with rwIFN-{gamma}, while the stratified bars represent those treated with the cytokine. h.c., heavy chain.


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DISCUSSION
 
To investigate the impact of WHV expression on the class I antigen presentation on hepatocyte surface, we generated a panel of woodchuck hepatocyte lines transcribing complete WHV genome, subgenomic fragments encoding individual viral proteins, or virus genome mutants in which transcription of selected products in the context of expression of an otherwise complete genome was disabled by site-specific mutagenesis. The transcriptional activity of viral DNA sequences was confirmed by detecting relevant mRNAs and, when the availability of specific antibodies allowed, expression of viral proteins. The data acquired showed that the stable transfection with complete WHV genome consistently resulted in a significant suppression of the class I MHC antigen on the hepatocyte surface, although virus antigens were displayed at relatively low levels and small amounts of virions were apparently assembled when hepatocyte supernatants were examined for infectivity by injecting into a naive woodchuck.

Importantly, the level of class I antigen inhibition on hepatocytes transfected with the whole WHV genome was comparable to that identified on the cells expressing either WHpS1 or WHpS2 alone. In contrast, transfection with p-WHmS did not alter the antigen display in comparison to the intact hepatocytes or those stably transfected with empty plasmid vector. In consideration with the facts that all three envelope proteins are encoded within the single S protein ORF, which has three in-frame translation initiation codons (Fig. 1), and that all of them share the same C-terminal portion while differing in the N-terminal amino acid sequences, the finding described above implied that the sequence shared by both WHpS1 and WHpS2 (hence, most likely located within WHpS2) was responsible for the inhibitory effect observed. The analysis of hepatocytes transfected with the complete WHV genome in which expression of WHpS1 was selectively disabled, provided supporting evidence which helped to resolve this issue. Thus, transfection with p-{Delta}WHpS1, which was unable to translate WHpS1 due to the stop codon introduced into the extreme 5'-terminal portion of the S gene (Fig. 2) but was still able to translate WHpS2 from the downstream initiation site, did not restore suppressed class I antigen display. This indicated again that the pre-S2 segment of the WHpS1 protein plays a critical role in class I antigen suppression. Furthermore, the data from the transfection experiments with p-{Delta}WHmS, which was unable to initiate translation of WHmS, showed that the hepatocyte class I antigen display was restored almost to the same level as that displayed by hepatocytes transfected with empty vector. Because the C-terminal portion of both WHpS1 and WHpS2 proteins is constituted by WHmS, this suggests that the complete intact sequence of WHpS2 protein is required to act as a functional class I antigen inhibitor. Taken together, this differential analysis provided cumulative evidence that suppression of the class I antigen on WCM-260 hepatocytes was mediated by WHpS2 protein.

It is of note that it has been reported that HBV pre-S1/2 protein may play a role in regulating host gene transcription via interaction with cellular signaling pathways (12, 13) which, indirectly, may influence expression of class I MHC antigen. Furthermore, it has been suggested that interference with the host gene transcription may result in the development of HCC, a common consequence of chronic HBV infection (17). One of the strategies shared by both malignant tumors and viral infections to avoid recognition by host CD8+ CTL is suppression of cell surface class I MHC display to abrogate class I MHC-T-cell receptor interactions. Thus, inhibition of expression of class I MHC antigen by hepadnavirus pre-S2 protein shown in our study may contribute not only to persistence of virus in infected hepatocytes but also to escape of hepadnavirus-transformed hepatocytes from immune recognition.

Remarkably, but not unexpectedly, transfection with p-WHx induced a dramatic increase in hepatocyte class I antigen display. The X protein of hepadnaviruses is known to be a promiscuous transactivator capable of promoting expression of a variety of viral and cellular genes, including those involved in control of cell growth, and it can interact with cellular organelles, such as mitochondria and proteasomes, and modify their functions (2, 9, 10, 15, 34). In regard to HBV X protein and class I MHC expression, it has been reported that transfection of the same cells with the HBV X gene together with a reporter plasmid driven by class I MHC promoter led to augmented expression of luciferase reporter, suggesting that X protein was responsible for this event (35). This result seems to corroborate the findings in our study, although significantly different experimental approaches were used. It has been also shown, by employing a two-hybrid system, that HBV X protein can interact with PSMA7 and PSMA1 of the proteasome complex (10, 15, 34). It was postulated that this interaction modifies the function of both X protein and proteasome. However, the effect of these potential modifications of the class I MHC antigen presentation was not characterized in these studies.

The observed diametrically opposed effects on class I MHC presentation following transfection with p-WHpS1, p-WHpS2, or p-WHx and the fact that the expression of the whole WHV genome strongly inhibited the antigen display suggest that a highly complex interplay between individual virus products is involved in suppression of the class I antigen. Thus, if we assume that, in the context of expression of the complete virus genome, both WHpS2 and WHx exerted their respective inhibitory or stimulatory action to the same degrees as those identified on hepatocytes transfected with the sequences encoding WHpS2 or WHx alone, this may suggest that WHpS2 is a very potent class I antigen inhibitor which is able not only to counteract the stimulating effect of WHx but also to suppress the antigen significantly below the level displayed on intact hepatocytes. This intriguing possibility will require further examination, for example by cotransfecting cells with p-WHpS2 and p-WHx. It also remains to be established whether the same event occurs in naturally infected hepatocytes.

Exposure to rwIFN-{gamma} completely restored the class I antigen presentation on hepatocytes transfected with the whole WHV genome, with gene sequences encoding WHpS1 and WHpS2, or with WHV DNA with an introduced stop codon disabling transcription of the pre-S1 region. These findings demonstrated that the inhibition of class I antigen was reversible in all hepatocyte lines in which defective class I antigen display was detected. This supports the notion that intrahepatic induction of IFN-{gamma} could not only suppress virus replication, as it has been shown for HBV in a transgenic mouse model (11), but also enhance class I MHC expression on chronically infected hepatocytes, promoting their recognition by virus-specific CTL. This is further supported by the results from the present study, showing that treatment with IFN-{gamma} was highly effective in augmenting expression of the genes encoding different components of the endocytic antigen presentation pathway and proteasome subunits in hepatocytes with suppressed class I antigen display. Therefore, treatment with this cytokine or with functionally equivalent agents may enable hepatocytes in chronic hepadnaviral infection to process and present viral peptides more efficiently in the context of class I antigen and, in consequence, increase their accessibility for recognition by specific CTL. Furthermore, as a result of the increased expression of IFN-{gamma}-inducible proteasome subunits, the assembly of immunoproteasomes might also be enhanced; enhanced immunoproteasome assembly has been shown to be required for the generation of certain HBV-specific CTL epitopes (31).

The precise mechanism of WHpS2-mediated suppression of the class I MHC antigen remains to be determined. We did not find a noticeable entrapment of the antigen within hepatocyte cytoplasm using class I heavy chain-specific MAb and confocal microscopy (data not shown). However, we cannot exclude the possibility that the increased recycling or release of the antigen was responsible for its depletion from the hepatocyte surface. As the quantitative analysis of the mRNA levels of several class I MHC- and proteasome-affiliated genes revealed, the class I antigen inhibition was not related to hepatocyte-down-regulated transcription of any of the genes examined. This suggests that the suppression has to be due to WHpS2-dependent posttranscriptional interference. This conclusion is consistent with the findings from the previous study, in which, based on the analysis of hepatocytes naturally infected with WHV derived from woodchucks with chronic hepatitis, a virus-dependent posttranscriptional event was suspected as a reason behind the impaired hepatocyte presentation of class I molecules (22).

The present study provides experimental evidence that the envelope pre-S2 protein of WHV functions as a suppressor of class I MHC antigen in hepatocytes actively transcribing virus genome. The identification of the translation product responsible for a defect in class I antigen presentation on hepatocytes, which are the main targets of hepadnavirus invasion and the fount of clinical symptoms, advances our understanding of how hepadnaviruses may subvert the host immune system, persist, and uphold protracted liver damage. Moreover, this study demonstrates that IFN-{gamma} can completely reverse the virus-induced defect in the class I antigen presentation. This provides an indication of how the host immune system, either intrinsically or due to exogenous stimulation, may potentially overcome this deficiency in the course of chronic hepadnaviral infection.


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ACKNOWLEDGMENTS
 
We thank Norma D. Churchill and Colleen L. Trelegan for their expert technical assistance and Clifford S. Guy and Sherri L. Rankin for immunoblot analyses.

This research was supported by grant MOP-14818 from the Canadian Institutes of Health Research (to T.I.M.). T.I.M. is the Canada Research Chair (Tier 1) in Viral Hepatitis/Immunology, supported by the Canadian Institutes of Health Research and the Canada Foundation for Innovation.


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FOOTNOTES
 
* Corresponding author. Mailing address: Molecular Virology and Hepatology Research, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John's, NL, Canada A1B 3V6. Phone: (709) 777-7301. Fax: (709) 777-8279. E-mail: timich{at}mun.ca. Back


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Journal of Virology, September 2006, p. 8541-8553, Vol. 80, No. 17
0022-538X/06/$08.00+0     doi:10.1128/JVI.00830-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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