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Journal of Virology, April 2005, p. 4870-4876, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4870-4876.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Escape Mutations Alter Proteasome Processing of Major Histocompatibility Complex Class I-Restricted Epitopes in Persistent Hepatitis C Virus Infection

Yoichi Kimura,1 Toshifumi Gushima,1 Sharad Rawale,2 Pravin Kaumaya,2 and Christopher M. Walker1,3*

Center for Vaccines and Immunity, The Columbus Children's Research Institute,1 Departments of Obstetrics and Gynecology,2 Pediatrics, The Ohio State University, Columbus, Ohio3

Received 11 July 2004/ Accepted 30 November 2004


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ABSTRACT
 
Mutations in hepatitis C virus (HCV) genomes facilitate escape from virus-specific CD8+ T lymphocytes in persistently infected chimpanzees. Our previous studies demonstrated that many of the amino acid substitutions in HCV epitopes prevented T-cell receptor recognition or binding to class I major histocompatibility complex molecules. Here we report that mutations within HCV epitopes also cause their destruction by changing the pattern of proteasome digestion. This mechanism of immune evasion provides further evidence of the potency of CD8+ T-cell selection pressure against HCV and should be considered when evaluating the significance of mutations in viral genomes from persistently infected chimpanzees and humans.


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INTRODUCTION
 
Hepatitis C virus (HCV) infection resolves spontaneously in some humans and chimpanzees, but most develop persistent viremia. CD8+ T cells probably influence infection outcome because their expansion in blood and liver is kinetically associated with termination of viremia (4, 6, 18, 28, 29) and antibody-mediated depletion of this subset from immune chimpanzees prolongs virus replication (26). Why the CD8+ T-cell response fails in humans and chimpanzees that develop chronic infections is not fully understood. At least some CD8+ T-cell populations display defects in perforin-mediated cytotoxicity or production of gamma interferon (IFN-{gamma}) upon antigenic stimulation (1, 14, 32, 33). It is unlikely that inadequate effector function alone accounts for evasion of this response. Persistence is probably also facilitated by mutation of the positive-stranded RNA genome of HCV. Approximately 1012 new viral genomes are produced per day by a viral RNA polymerase that lacks a proofreading mechanism (19), and there is compelling evidence that this leads to selection of HCV variants that escape humoral and cellular immune responses (8, 10, 11, 16). Recent evidence that lack of CD4+ T-cell help results in accumulation of escape mutations in multiple major histocompatibility complex (MHC) class I epitopes of HCV provides additional support for this mechanism of immune evasion (13).

In a previous study of three persistently infected chimpanzees (CH-503, CBO603, and CBO609), we identified several intrahepatic HCV-specific CD8+ T-cell populations that failed to recognize HCV because of escape mutations in the Patr (Pan troglodytes) class I-restricted epitopes that they targeted (10). The mutations did not arise by chance, but instead arose by Darwinian selection pressure by the cytotoxic T lymphocytes (CTLs), because the ratio of nonsynonymous to synonymous nucleotide substitutions was significantly higher in Patr class I-restricted epitopes than in flanking regions or nonrestricted epitopes. Many of the amino acid substitutions in epitopes impaired either peptide binding to MHC class I molecules or T-cell receptor recognition (10). In this follow-up study, we report that mutations within MHC class I epitopes can also facilitate their destruction by proteasomes and impair CD8+ T-cell recognition of target cells.


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MATERIALS AND METHODS
 
Animals. Chimpanzees were enrolled in this study after 5 (CBO603, CBO609) or 7 (CH-503) years of persistent infection with the HCV-1/910 virus (10) and were housed at the New Iberia Research Center (NIRC), New Iberia, La., under a protocol approved by the NIRC Animal Care and Use Committee. MHC class I haplotypes of the animals used in this study are shown in Table 1.


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  		TABLE 1. Patr class I alleles expressed by common chimpanzeesa in this study

CTL lines. T cells recovered from liver homogenates with anti-CD8 paramagnetic beads were cloned by limiting dilution culture with 5 x 104 irradiated human peripheral blood mononuclear cells (PBMCs) as feeder cells, 50 U of interleukin-2 (IL-2) per ml, and anti-CD3 monoclonal antibodies (MAbs) (10). Panels of clones were screened for cytotoxicity against target cells infected with recombinant vaccinia viruses expressing different regions of the HCV-/910 polyprotein, and minimum epitopes were identified with synthetic overlapping peptides (10).

Peptides. All peptides were produced by fMoc solid-phase chemistry with free amine NH2 and free acid COOH termini. Two polypeptides spanning 30 amino acids (aa) of the HCV-1/910 NS4B protein containing either wild type (1712-SQHLPYIEQGMMLAEQFKQKALGLLQTASR-1741) or position 1 (P1) M1723L substitution mutant NS4B1723 epitopes were also synthesized and purified by semipreparative reverse-phase high-performance liquid chromatography (RP-HPLC). The identity of the peptides was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry as described below.

Cloning and sequencing of HCV genomes. RNA was extracted from chimpanzee plasma (100 µl) (RNAEasy; QIAGEN), and cDNA was reverse transcribed. cDNA was amplified with nested sets of PCR primers that flanked the class I MHC epitopes (10). PCR products were subcloned by thymidine/adenosine (T/A) ligation into a DNA plasmid (Invitrogen), and multiple molecular clones were sequenced in both directions by dye terminator chemistry with an ABI 3100 automated DNA sequencer.

Proteasomes and peptide digestion. Human constitutive and immunoproteasomes purifed from LCL-721 and LCL-721.174 cells were purchased from Immatics Biotechnologies (Tubingen, Germany). Wild-type or mutant NS4B1723 peptides (5 µg) were incubated with 3 µg of either proteasome in 150 µl of digestion buffer (20 mM HEPES-KOH, pH 7.6, 2 mM Mg-acetate, 0.01% sodium dodecyl sulfate) for 16 h and then frozen at –20°C. Both peptides were also treated with digestion buffer alone as a control. Mock- and proteasome-digested peptides were then analyzed by mass spectrometry and tested for T-cell recognition.

MALDI-TOF mass spectrometry. MALDI-TOF analysis of mock and proteasome-digested NS4B peptides was performed on a Bruker Reflex III (Bruker, Breman, Germany) mass spectrometer operated in the reflectron, positive-ion mode with an N2 laser. Laser power was used at the threshold level required to generate signal. Accelerating voltage was set to 28 kV. The instrument was calibrated with protein standards bracketing the molecular weights of the protein samples (typically mixtures of bradykinin fragments 1 to 5 and adrenocorticotropin fragments 18 to 39 as appropriate). Salt buffers from the protein samples were cleaned with ZipTips (Millipore, Bedford, Mass.) according to the manufacturer's directions. Cyano-4-hydroxy cinnamic acid was used as the matrix and prepared as a saturated solution in 50% acetylnitrile-0.1% trifluoroacetic acid (in water). Allotments of 1 µl of matrix and 1 µl of sample were thoroughly mixed together; 0.5 µl of this was applied as spots to the target plate and dried.

Cytotoxicity assays. Autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines (BLCL), fibroblasts, or Patr class I-transfected 721.221 cells were used as targets. Targets (T) were labeled with 51Cr and various concentrations of peptide epitopes, washed, and incubated with effector (E) CTL lines at various E:T ratios for 4 h. Supernatants (50 µl) were harvested into Packard Lumaplates, and radioactivity was counted with a Wallac Microbeta 1420 scintillation instrument. In some experiments, we determined the stability of the peptide-target cell binding by pulsing Patr class I-transfected target cells with a 10 µM concentration of the peptide epitopes for 1 h before washing and further culture in RPMI-1640 medium supplemented with 10% fetal calf serum (R-10) for periods up to 96 h. Targets were then 51Cr labeled and tested for lysis by CTL lines.

Intracellular cytokine staining. Mock- or proteasome-digested NS4B peptides were incubated with the Patr-A0101-transfected targets for 1 h, washed once, and incubated with a NS4B1723-specific CTL line in the presence of brefeldin A (5 µg/ml) for 16 h. After fixation, cells were permeabilized and intracellular cytokine staining was performed with a phycoerythrin (PE)-conjugated anti-IFN-{gamma} MAb. PE-conjugated rat immunoglobulin G1 (IgG1) MAb served as a control. Stained cells were analyzed with a BD FacsCalibur instrument.

HCV plasmids and T7-driven gene expression in target cells. In some experiments, HCV genes were expressed transiently in chimpanzee fibroblast target cells by an infection/transfection procedure described elsewhere (9). The pTM1(C-E2) plasmid expressing the HCV-1 core, E1, and E2 structural proteins (aa 1 to 746) and the pTM1(NS2-5) plasmid expressing all nonstructural proteins (aa 1027 to 3033), including NS4, were kind gifts from the Chiron Corporation. They were derived from the consensus sequence of the HCV-1/910 genotype 1A virus used to infect the animals. A bacterial T7 promoter regulates transcription of HCV genes in these plasmids. Site-directed mutagenesis (QuikChange; Stratagene, La Jolla, Calif.) was used to introduce the core and NS4 mutations into these plasmids. Plasmid pTM1(C-E2/136L) is a product of mutagenesis at nucleotide 750 of the prototype HCV-1 core coding region (A750->T) that converts 137I (ATA) into 137L (TTA). Plasmid pTM1(NS2-5/1723L) involved mutagenesis of the NS4 coding region (A5508->C) to convert 1723M (ATG) to 1723L (CTG). A change at the same nucleotide position converted 1723M (ATG) to 1723V (GTG) to generate plasmid pTMI(NS2-5/1723V). Expression of wild-type and mutated genes was accomplished by infection of 3 x 105 primary chimpanzee fibroblasts with recombinant vaccinia virus expressing bacterial T7 RNA polymerase (VVT7) at a multiplicity of infection (MOI) of 10 for 1 h. After washing, the cells were transfected with 1.5 µg of the plasmids described above for 3 h, washed, and cultured with Dulbecco's modified Eagle's medium (with 20% fetal bovine serum) for 16 h before 51Cr labeling.


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RESULTS AND DISCUSSION
 
Mutations in class I MHC-restricted HCV epitopes. HCV-specific CTL lines for targeting were derived from the liver of two chimpanzees after 5 (CBO609) or 7 (CH-503) years of persistent infection. The CTL line from the liver of CH-503 efficiently killed target cells expressing the HCV core protein (data not shown) and was found to target a Patr-B1601-restricted epitope spanning aa 131 to 139 (ADLMGYIPL; designated C131) (Fig. 1A). CTL lines from the liver of CBO609 targeted an epitope in NS4B that was mapped to aa 1723 to 1731 (MLAEQFKQK; designated NS4B1723) and presented by the Patr-A*0101 class I molecule. PCR-mediated amplification and sequencing of the HCV quasispecies from the plasma of persistently infected animals revealed a Patr allele-specific pattern of nonsynonymous mutation in these epitopes. For instance, an isoleucine (I137)-to-leucine (L) substitution (I137L) at P7 of the C131 epitope was embedded in the quasispecies master copy of CH-503 but not the Patr-B1601-negative animal, CBO609 (Fig. 2, top panel). Similarly, sequences of multiple molecular clones generated from plasma of two Patr-A0101-positive chimpanzees after 5 years of chronic infection revealed a mutation at P1 of the NS4B1723 epitope in CBO609 (M1723L) and CBO603 (M1723V) (Fig. 2, lower panel). In contrast, the NS4B epitope was intact after several years of persistent virus replication in CH-503, an animal that lacks expression of Patr-A0101.



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  		FIG. 1. Class I MHC restriction of CD8+ CTL lines from chronically infected animals. CD8+ CTL lines specific for the HCV core (A) or NS4B (B) proteins were derived from the livers of chimpanzees CH-503 and CBO609, respectively. A panel of 721.221 target cells transfected with individual class I Patr molecules from (A) CH-503 or (B) CBO609 were infected overnight with recombinant vaccinia viruses expressing the core (A) or NS4B (B) HCV proteins at an MOI of 10. Cytolysis was measured at the indicated E:T cell ratios in a 4-h assay.



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  		FIG. 2. Mutations in the core and NS4B proteins of HCV quasispecies from chronically infected animals. HCV RNA was isolated from plasma collected 7 (CH-503) or 5 (CBO609 and CBO603) years after infection with HCV-1/910. cDNA was prepared, and nested PCR primers were used to amplify viral sequences from the core and NS4B genes. PCR products were ligated into plasmids, and multiple molecular clones were sequenced. The C131 and NS4B1723 epitopes are boxed. +ive, positive;-ive, negative.

T-cell recognition of wild-type and mutated peptides. The presence of mutations localized to restricted but not unrestricted HCV epitopes was consistent with our earlier findings in these animals and suggested that they were selected to circumvent CTL recognition (10). To assess whether the mutations impaired epitope recognition by CD8+ T cells, we compared killing of target cells loaded with synthetic nonameric peptides representing the wild-type and mutant sequences. Target cells sensitized with various concentrations of the wild-type C131 peptide were killed in a dose-dependent manner by the core-specific CTL line from CH-503 (Fig. 3A). Unexpectedly, however, target cells sensitized with variant with an I137L substitution were recognized with equal efficiency (Fig. 3A). Similar observations were made with the NS4B1723 P1 substitution peptides. The M1723L and M1723V mutations observed in viruses from chimpanzees CBO609 and CBO603 also did not reduce cytolysis when compared with the wild-type NS4B1723 epitope (Fig. 3B). These results indicated that the mutations in core and NS4B epitopes had no effect on T-cell receptor recognition of the altered epitopes.



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  		FIG. 3. Recognition of target cells sensitized with wild-type or mutant HCV peptides. Patr-B1601 (A)- or A0101 (B)-transfected 721.221 target cells were sensitized for 1 h with various concentrations of nonameric C131 or NS4B1723 wild-type and mutant peptides. They were then cocultured with the (A) C131 or (B) NS4B1723-specific CTL lines at an E:T ratio of 20:1 in a 4-h 51Cr release assay. The target cells were also incubated with the (C) C131 or (D) NS4B1723 peptides (10 µM) for 1 h, washed three times, and then cultured for an additional 1, 16, 48, or 96 h before labeling with 51Cr. Peptide-sensitized targets were then cocultured with the core or NS4-specific CTL lines for 4 h at a 20:1 E:T ratio.

We could not exclude the possibility that these amino acid substitutions resulted in reduced stability of peptide-Patr class I complexes that might not be revealed in a short-term cytotoxicity assay. Patr-B1601-transfected target cells were therefore sensitized with the HCV core C131 and I137L substitution peptides for 1 h and then washed three times to remove unbound peptide. After 1, 16, 48, and 96 h of culture, the target cells were labeled with 51Cr tested for lysis by the core-specific CTL line. As shown in Fig. 3C, the peptide-Patr class I complex was remarkably stable because killing of target cells sensitized with the wild-type and variant C131 peptides was not reduced even after 4 days of culture. Identical results were obtained for NS4B1723 epitopes with the M1723L and M1723V substitutions (Fig. 3D). Taken together, these results demonstrated that mutations localized to the C131 and NS4B1723 epitopes affected neither TCR recognition nor MHC class I binding.

T-cell recognition of targets expressing mutated HCV proteins. Mutations within epitopes or flanking sequences can also alter class I MHC processing of epitopes. These changes would not necessarily be detected with synthetic peptides, so we assessed CTL killing of target cells that expressed mutated HCV core or NS4B proteins. Fibroblast cell lines from chimpanzees CH-503 and CBO609 were infected with VVT7 and then transfected with plasmids expressing structural or nonstructural HCV proteins regulated by the T7 promoter. Plasmid pTMI(C-E2wt) contained the wild-type HCV-1 core, E1, and E2 genes. Site-directed mutagenesis was used to introduce the I137L substitution into the core gene of this plasmid [designated pTMI(C-E2/L137)]. Importantly, both plasmids also expressed the same control Patr-A0401-restricted epitope (designated E2588; KHPDATYSR) (17) located in E2. An E2588-specific CTL line derived from chimpanzee CH-503 killed fibroblasts transiently transfected with the TMI(C-E2wt) or pTMI(C-E2/137L) plasmids with equivalent efficiency (Fig. 4A). This indicated that expression of the structural proteins was not altered by introduction of the core I137L mutation. As expected, the C131-specific CTL line also efficiently lysed the pTMI(C-E2wt)-transfected fibroblasts expressing the wild-type epitope (Fig. 4B). In contrast, lysis of targets expressing the core protein with the I137L substitution was reduced by about 75% (Fig. 4B), indicating that the antigen processing apparatus of the cell impaired presentation of the mutated C131 epitope.



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  		FIG. 4. Expression of wild-type and mutated HCV proteins in target cells. Fibroblast cell lines from chimpanzee CH-503 were infected with VVT7 and then transiently transfected with plasmids pTMI(C-E2wt) or pTMI(C-E2/137L) that expressed the wild-type proteins or core proteins with I137L substitution, respectively. They were then incubated with CTL lines specific for the E2588 epitope that was intact in both plasmids (A) or the C131 epitope (B). Fibroblast targets from CBO609 were similarly transfected with plasmids pTMI(NS2-5wt) or pTMI(NS2-5/L1723 or pTMINS2-5/V1723). They were then incubated with a CTL line against the control (i.e., intact) epitope NS4B1939 (C) or the CTL line targeting NS4B1723 (D). Effector and 51Cr-labeled target cells were cocultured at a ratio of 20:1 for 4 h. Results are representative of three replicate experiments.

Similar results were obtained for the NS4B proteins with M1723L and M1723V substitutions. A plasmid expressing the wild-type NS4B protein [pTMI(NS2-5wt)] was mutated to incorporate the M1723L or M1723V substitutions fixed at P1 of the NS4B1723 epitope [plasmids designated pTMI(NS2-5/L1723) and pTMI(NS2-5/V1723, respectively]. A CTL line specific for a control Patr-B1701-restricted NS4 epitope (NS41939;DAAARVTAIL) (10) that was intact in both plasmids was used to confirm equivalent presentation of the wild-type and mutated NS4B genes (Fig. 4C). The NS4B1723-specific CTL line also efficiently killed target cells expressing the wild-type NS4B (1723M) protein (Fig. 4b). However, killing was reproducibly reduced by 40 to 50% by introduction of the M1723L and M1723V mutations into NS4B (Fig. 4D). Thus, processing of the core and NS4B proteins through the class I MHC pathway revealed an inhibitory effect of mutations on CTL lysis that was not evident when cells were sensitized with synthetic peptides.

Proteasome processing of wild-type and mutated HCV epitopes. Mutation of HCV epitopes could cause a block at multiple points within the antigen processing pathway. These include efficient digestion of proteins into epitopic peptides by the proteasome or other proteases associated with the antigen processing pathway and transport of the peptides between the cytosolic and endoplasmic reticulum (ER) compartments. We favored a role for destruction of the mutated epitopes by proteasomes because a predictive algorithm (PAProC) (21) indicated the potential for new cleavage sites within the mutated C131 and NS4B1723 epitopes. The I137L substitution in the C131 epitope was predicted to result in a unique cut between amino acids P6 (Y136) and P7 (L137) by the constitutive proteasome (12, 20). Similarly, the M1723L and M1723V changes introduced two potential cleavage sites after amino acids P1 and P2 of the mutated but not wild-type NS4B1723 epitopes.

To test this prediction, we used MALDI-TOF mass spectrometry to identify NH2-terminal peptide fragments generated by proteasome digestion of synthetic polypeptides containing the NS4B1723 wild-type or M1723L substitution epitopes. Proteasome processing of polypeptides that are wild-type or with the M1723L substitution generated a partially overlapping set of peptide fragments that had NH2-terminal serine (S1712) residues but variable COOH termini (Fig. 5A). An 11-aa fragment spanning residues S1712 to M1722 was found after digestion of both the wild-type and mutant polypeptides. Proteasome cleavage therefore occurred immediately before the P1 amino acid of the wild-type (M1723) and mutant (L1723) epitopes to create a correct NH2 terminus. However, this cut immediately before the P1 amino acid was clearly favored for the wild-type polypeptide, as the S1712-M1722 fragment represented approximately 67% of all NH2-terminal peptides generated by proteasome digestion (as measured by relative absorbance intensity). In contrast, S1712-M1722 represented only 12% of all peptides generated by digestion of the mutant polypeptide. Instead the mutant polypeptide was fragmented predominantly by cuts after amino acids Q1720, G1721, M1722, and L1723. The latter two represent cleavage within the epitope after the P1 and P2 residues and were not found after proteasome treatment of the wild-type polypeptide. Relative absorbance intensity as determined by MALDI-TOF mass spectrometry provides an imperfect measure of true peptide abundance, and thus it is difficult to accurately rank each fragment or cut site generated by the proteasome. Nevertheless, the sharp decline in the relative abundance of fragment S1712-M1722 after digestion of the mutant versus wild-type polypeptide and the appearance of fragments corresponding to cuts after the P1 and P2 residues support the notion that mutation of this epitope altered proteasome digestion patterns within HCV-infected cells.



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  		FIG. 5. Proteasome processing of synthetic NS4B peptides. (A) Two synthetic peptides spanning amino acids S1712 to R1741 of NS4B containing either the HCV-1 wild-type (M1723) or mutant (L1723) sequences were synthesized and purified by RP-HPLC. NS4B1723 epitopes are boxed. Both peptides were processed with the constitutive proteasomes (CP) or immunoproteasomes (IP) for 16 h and MALDI-TOF mass spectrometry was used to identify fragments. Fragments generated by cleavage of peptide bonds immediately before or after the NH2 terminus of the wild-type (WT) and mutant (MT) epitopes are shown. Asterisks indicate unique cleavage sites detected only in the peptide with M1723L substitution by MALDI-TOF mass spectrometry. (B) Wild-type peptide or peptides with M1723L substitution were mock digested in buffer alone (0 h) or buffer containing constitutive proteosomes or immunoproteasomes for 4, 8, or 16 h. Aliquots of the processed peptides (equivalent to approximately 60 µM of the wild-type or mutant substrate) were used to sensitize target cells for lysis by the NS4B1723-specific CTL line. CTL activity was measured at a 20:1 E:T ratio in a 4-h assay. The percentage of specific lysis of target cells sensitized with the mock-digested wild-type or mutant peptides was normalized to 0, and the fold increase or decrease in cytolytic activity with the immunoproteasome-digested peptides was calculated. The percentages of lysis of targets sensitized with mock-digested wild-type and mutant polypeptides were 20 and 35%, respectively. Four percent lysis was detected against unsensitized targets. (C) Wild-type peptides or peptides with M1723L substitution were processed with the immunoproteasome for 16 h as described above, and serial dilutions of the product, equivalent to 20, 10, and 5 µM concentrations of the starting substrate, were used to sensitize target cells. The fold change in cytolytic activity after immunoproteasome treatment was calculated as described above. The percentages of lysis of targets treated with mock-digested wild-type and mutant polypeptide were 22 and 50%, respectively. (D) Wild-type or mutant NS4b1723 peptides were mock digested or treated with the immunoproteasome for 16 h. BLCL cells sensitized with the processed products were cocultured with the NS4B1723-specific CTL line. The frequency of T cells producing IFN-{gamma} was measured by flow cytometry.

It is important to emphasize the fibroblast target cells expressing the mutated form of the epitope were partially lysed by cytotoxic T cells (Fig. 4D). Indeed, detection of the minor peptide corresponding to amino acids S1712 to M1722 does suggest that an epitope with a functional (L1723) NH2 terminus was generated, albeit inefficiently, despite the mutation. Two minor peptide fragments generated by cuts after Q1720 or G1721 might also lead to generation of a functional epitope after trimming of these NH2-terminal amino acids by the ER peptidase (25, 36). Identical results were obtained with immunoproteasomes that are formed in response to IFN-{gamma} and display a discrete but partially overlapping amino acid cleavage pattern when compared with constitutive proteasomes (3, 30).

To evaluate the functional significance of these changed digestion patterns, wild-type and mutated polypeptides were treated for 0, 4, 8, or 16 h with constitutive or immunoproteasomes. Processed peptides were then used to sensitize 51Cr-labeled target cells, and lysis by the NS4B1723-specific T-cell line was compared. As shown in Fig. 5B, treatment of the wild-type peptide with either proteasome resulted in a time-dependent increase in target cell lysis when compared with mock-digested peptide. In contrast the M1723L variant peptide showed a modest though significant decrease in activity that was apparent after 4 h of treatment with either proteasome (Fig. 5B). To confirm these trends, the substrate polypeptides were incubated with the immunoproteasome for 16 h and various concentrations of the product were titrated on target cells. Target cell lysis was enhanced three- to fourfold at all three concentrations (20, 10, and 5 µM) of wild-type peptide (Fig. 5C). In contrast, we observed a dose-dependent reduction in killing of targets with the processed M1723L variant. Indeed, at the 5 µM dose, killing was reduced about 3.4-fold compared to that with mock-digested peptide (Fig. 5C). These results were recapitulated by intracellular cytokine (IFN-{gamma}) production by the CD8+ T-cell line as an endpoint (Fig. 5D). Enhanced IFN-{gamma} production was observed when NS4B1723-specific CD8+ T cells were stimulated with immunoproteasome-processed wild-type peptide, and the frequency of responders was reduced with the processed NS4B1723 M1723L substitution peptide (Fig. 5D).

The potential of virus-specific CD8+ T cells to select for variant epitopes that are poorly processed in infected cells was predicted by several observations. For instance, whether viral and tumor epitopes are efficiently released by proteasome digestion of protein substrate is influenced by flanking amino acids, particularly those near the COOH terminus (2, 22, 27, 34). Proof that this has practical consequences for virus persistence was only recently revealed in studies of human immunodeficiency virus (HIV) infection. Appearance of mutations immediately adjacent to HIV and HCV epitopes was associated with impaired CD8+ T-lymphocyte recognition of target cells 7, 24). Processing mutations are not localized exclusively to flanking regions, as they also appear within viral epitopes, apparently as a result of CD8+ T-cell selection pressure. In a murine influenza virus infection model, mutation of a dominant epitope prevented cytolysis of virus infected cells, even though a synthetic peptide representing the escape variant sensitized target cells for recognition by CD8+ T cells (23). This mechanism of immune evasion is also operational in HIV infections, where amino acid substitutions within epitopes subverted recognition of target cells producing the mutated proteins (35). Our study extends this observation to HCV infection. More importantly, it establishes a unique correlation between recognition of target cells expressing wild-type or mutated HCV proteins and in vitro patterns of proteasome-mediated digestion. It is likely that these patterns of digestion obtained with proteasomes purified from B lymphocytes will be recapitulated with proteasomes from hepatocytes, although this remains to be formally demonstrated. While our results indicate that the proteasome was clearly involved in destruction of the NS4B epitopes with P1 substitution (M1723L or M1723V) in two persistent infections, it is possible that mutations inhibiting additional functions of the antigen processing pathway, including other necessary proteases or those that impair transport of mutated peptides between cellular compartments by molecules like the transporter associated with antigen presentation (TAP), are also operational for other HCV epitopes.

Finally mutations in MHC class I-restricted epitopes of HCV have also been found in persistently infected humans (5, 15, 31). At least some of them did not impair CD8+ T-cell recognition of peptide-loaded target cells, so their potential impact on immune escape and persistence was not established (5). Findings from this study suggest that these virus variants may nonetheless be the product of CD8+ T-cell selection pressure and could influence the outcome of HCV infection in humans.


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ACKNOWLEDGMENTS
 
This work was supported by Public Health Service grants (RO1 A147367 and U19 AI48231) to C. M. Walker.

We thank Dana Hasselschwert and Neal Smith of the New Iberia Research Center, New Iberia, La., for outstanding veterinary and technical support. We also gratefully acknowledge the support of the National Center for Research Resources and Ray O'Neill for these studies. We thank David Bowen for helpful discussions and proofreading of the manuscript and Kari Green-Church of The Ohio State University for assistance with the mass spectrometry.


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FOOTNOTES
 
* Corresponding author. Mailing address: Children's Hospital, WA4011, 700 Children's Dr., Columbus, OH 43205. Phone: (614) 722-2692. Fax: (614) 722-3680. E-mail: WalkerC{at}pediatrics.ohio-state.edu. Back


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REFERENCES
 
    1
  1. Appay, V., P. R. Dunbar, M. Callan, P. Klenerman, G. M. Gillespie, L. Papagno, G. S. Ogg, A. King, F. Lechner, C. A. Spina, S. Little, D. V. Havlir, D. D. Richman, N. Gruener, G. Pape, A. Waters, P. Easterbrook, M. Salio, V. Cerundolo, A. J. McMichael, and S. L. Rowland-Jones. 2002. Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat. Med. 8:379-385.[CrossRef][Medline]
    2. 2
  2. Beekman, N. J., P. A. van Veelen, T. van Hall, A. Neisig, A. Sijts, M. Camps, P. M. Kloetzel, J. J. Neefjes, C. J. Melief, and F. Ossendorp. 2000. Abrogation of CTL epitope processing by single amino acid substitution flanking the C-terminal proteasome cleavage site. J. Immunol. 164:1898-1905.[Abstract/Free Full Text]
    3. 3
  3. Cardozo, C., and R. A. Kohanski. 1998. Altered properties of the branched chain amino acid-preferring activity contribute to increased cleavages after branched chain residues by the "immunoproteasome." J. Biol. Chem. 273:16764-16770.[Abstract/Free Full Text]
    4. 4
  4. Cerny, A., J. G. McHutchison, C. Pasquinelli, M. E. Brown, M. A. Brothers, B. Grabscheid, P. Fowler, M. Houghton, and F. V. Chisari. 1995. Cytotoxic T lymphocyte response to hepatitis C virus-derived peptides containing the HLA A2.1 binding motif. J. Clin. Investig. 95:521-530.
    5. 5
  5. Chang, K. M., B. Rehermann, J. G. McHutchison, C. Pasquinelli, S. Southwood, A. Sette, and F. V. Chisari. 1997. Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus. J. Clin. Investig. 100:2376-2385.[Medline]
    6. 6
  6. Cooper, S., A. L. Erickson, E. J. Adams, J. Kansopon, A. J. Weiner, D. Y. Chien, M. Houghton, P. Parham, and C. M. Walker. 1999. Analysis of a successful immune response against hepatitis C virus. Immunity 10:439-449.[CrossRef][Medline]
    7. 7
  7. Draenert, R., S. Le Gall, K. J. Pfafferott, A. J. Leslie, P. Chetty, C. Brander, E. C. Holmes, S. C. Chang, M. E. Feeney, M. M. Addo, L. Ruiz, D. Ramduth, P. Jeena, M. Altfeld, S. Thomas, Y. Tang, C. L. Verrill, C. Dixon, J. G. Prado, P. Kiepiela, J. Martinez-Picado, B. D. Walker, and P. J. Goulder. 2004. Immune selection for altered antigen processing leads to cytotoxic T lymphocyte escape in chronic HIV-1 infection. J. Exp. Med. 199:905-915.[Abstract/Free Full Text]
    8. 8
  8. Eckels, D. D., H. Wang, T. H. Bian, N. Tabatabai, and J. C. Gill. 2000. Immunobiology of hepatitis C virus (HCV) infection: the role of CD4 T cells in HCV infection. Immunol. Rev. 174:90-97.[CrossRef][Medline]
    9. 9
  9. Eisenlohr, L. C., J. W. Yewdell, and J. R. Bennink. 1992. A transient transfection system for identifying biosynthesized proteins processed and presented to class I MHC restricted T lymphocytes. J. Immunol. Methods 154:131-138.[CrossRef][Medline]
    10. 10
  10. Erickson, A. L., Y. Kimura, S. Igarashi, J. Eichelberger, M. Houghton, J. Sidney, D. McKinney, A. Sette, A. L. Hughes, and C. M. Walker. 2001. The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes. Immunity 15:883-895.[CrossRef][Medline]
    11. 11
  11. Farci, P., A. Shimoda, A. Coiana, G. Diaz, G. Peddis, J. C. Melpolder, A. Strazzera, D. Y. Chien, S. J. Munoz, A. Balestrieri, R. H. Purcell, and H. J. Alter. 2000. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science 288:339-344.[Abstract/Free Full Text]
    12. 12
  12. Goldberg, A. L., P. Cascio, T. Saric, and K. L. Rock. 2002. The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides. Mol. Immunol. 39:147-164.[CrossRef][Medline]
    13. 13
  13. Grakoui, A., N. H. Shoukry, D. J. Woollard, J. H. Han, H. L. Hanson, J. Ghrayeb, K. K. Murthy, C. M. Rice, and C. M. Walker. 2003. HCV persistence and immune evasion in the absence of memory T cell help. Science 302:659-662.[Abstract/Free Full Text]
    14. 14
  14. Gruener, N. H., F. Lechner, M.-C. Jung, H. Diepolder, T. Gerlach, G. Lauer, B. Walker, J. Sullivan, R. Phillips, G. R. Pape, and P. Klenerman. 2001. Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus. J. Virol. 75:5550-5558.[Abstract/Free Full Text]
    15. 15
  15. Kaneko, T., T. Moriyama, K. Udaka, K. Hiroishi, H. Kita, H. Okamoto, H. Yagita, K. Okumura, and M. Imawari. 1997. Impaired induction of cytotoxic T lymphocytes by antagonism of a weak agonist borne by a variant hepatitis C virus epitope. Eur. J. Immunol. 27:1782-1787.[Medline]
    16. 16
  16. Kato, N. 2000. Genome of human hepatitis C virus (HCV): gene organization, sequence diversity, and variation. Microb. Comp. Genomics 5:129-151.[Medline]
    17. 17
  17. Kowalski, H., A. L. Erickson, S. Cooper, J. D. Domena, P. Parham, and C. M. Walker. 1996. Patr-A and B, the orthologues of HLA-A and B, present hepatitis C virus epitopes to CD8+ cytotoxic T cells from two chronically infected chimpanzees. J. Exp. Med. 183:1761-1775.[Abstract/Free Full Text]
    18. 18
  18. Lechner, F., D. K. Wong, P. R. Dunbar, R. Chapman, R. T. Chung, P. Dohrenwend, G. Robbins, R. Phillips, P. Klenerman, and B. D. Walker. 2000. Analysis of successful immune responses in persons infected with hepatitis C virus. J. Exp. Med. 191:1499-1512.[Abstract/Free Full Text]
    19. 19
  19. Neumann, A. U., N. P. Lam, H. Dahari, D. R. Gretch, T. E. Wiley, T. J. Layden, and A. S. Perelson. 1998. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science 282:103-107.[Abstract/Free Full Text]
    20. 20
  20. Niedermann, G. 2002. Immunological functions of the proteasome. Curr. Top. Microbiol. Immunol. 268:91-136.[Medline]
    21. 21
  21. Nussbaum, A. K., C. Kuttler, K. P. Hadeler, H. G. Rammensee, and H. Schild. 2001. PAProC: a prediction algorithm for proteasomal cleavages available on the WWW. Immunogenetics 53:87-94.[CrossRef][Medline]
    22. 22
  22. Ossendorp, F., M. Eggers, A. Neisig, T. Ruppert, M. Groettrup, A. Sijts, E. Mengede, P. M. Kloetzel, J. Neefjes, U. Koszinowski, and C. Melief. 1996. A single residue exchange within a viral CTL epitope alters proteasome-mediated degradation resulting in lack of antigen presentation. Immunity 5:115-124.[CrossRef][Medline]
    23. 23
  23. Price, G. E., R. Ou, H. Jiang, L. Huang, and D. Moskophidis. 2000. Viral escape by selection of cytotoxic T cell-resistant variants in influenza A virus pneumonia. J. Exp. Med. 191:1853-1867.[Abstract/Free Full Text]
    24. 24
  24. Seifert, U., H. Liermann, V. Racanelli, A. Halenius, M. Wiese, H. Wedemeyer, T. Ruppert, K. Rispeter, P. Henklein, A. Sijts, H. Hengel, P. M. Kloetzel, and B. Rehermann. 2004. Hepatitis C virus mutation affects proteasomal epitope processing. J. Clin. Investig. 114:250-259.[CrossRef][Medline]
    25. 25
  25. Serwold, T., F. Gonzalez, J. Kim, R. Jacob, and N. Shastri. 2002. ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum. Nature 419:480-483.[CrossRef][Medline]
    26. 26
  26. Shoukry, N. H., A. Grakoui, M. Houghton, D. Y. Chien, J. Ghrayeb, K. A. Reimann, and C. M. Walker. 2003. Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection. J. Exp. Med. 197:1645-1655.[Abstract/Free Full Text]
    27. 27
  27. Theobald, M., T. Ruppert, U. Kuckelkorn, J. Hernandez, A. Haussler, E. A. Ferreira, U. Liewer, J. Biggs, A. J. Levine, C. Huber, U. H. Koszinowski, P. M. Kloetzel, and L. A. Sherman. 1998. The sequence alteration associated with a mutational hotspot in p53 protects cells from lysis by cytotoxic T lymphocytes specific for a flanking peptide epitope. J. Exp. Med. 188:1017-1028.[Abstract/Free Full Text]
    28. 28
  28. Thimme, R., J. Bukh, H. C. Spangenberg, S. Wieland, J. Pemberton, C. Steiger, S. Govindarajan, R. H. Purcell, and F. V. Chisari. 2002. Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease. Proc. Natl. Acad. Sci. USA 99:15661-15668.[Abstract/Free Full Text]
    29. 29
  29. Thimme, R., D. Oldach, K. M. Chang, C. Steiger, S. C. Ray, and F. V. Chisari. 2001. Determinants of viral clearance and persistence during acute hepatitis C virus infection. J. Exp. Med. 194:1395-1406.[Abstract/Free Full Text]
    30. 30
  30. Toes, R. E., A. K. Nussbaum, S. Degermann, M. Schirle, N. P. Emmerich, M. Kraft, C. Laplace, A. Zwinderman, T. P. Dick, J. Muller, B. Schonfisch, C. Schmid, H. J. Fehling, S. Stevanovic, H. G. Rammensee, and H. Schild. 2001. Discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products. J. Exp. Med. 194:1-12.[Abstract/Free Full Text]
    31. 31
  31. Tsai, S. L., Y. M. Chen, M. H. Chen, C. Y. Huang, I. S. Sheen, C. T. Yeh, J. H. Huang, G. C. Kuo, and Y. F. Liaw. 1998. Hepatitis C virus variants circumventing cytotoxic T lymphocyte activity as a mechanism of chronicity. Gastroenterology 115:954-965.[CrossRef][Medline]
    32. 32
  32. Urbani, S., C. Boni, G. Missale, G. Elia, C. Cavallo, M. Massari, G. Raimondo, and C. Ferrari. 2002. Virus-specific CD8+ lymphocytes share the same effector-memory phenotype but exhibit functional differences in acute hepatitis B and C. J. Virol. 76:12423-12434.[Abstract/Free Full Text]
    33. 33
  33. Wedemeyer, H., X. S. He, M. Nascimbeni, A. R. Davis, H. B. Greenberg, J. H. Hoofnagle, T. J. Liang, H. Alter, and B. Rehermann. 2002. Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection. J. Immunol. 169:3447-3458.[Abstract/Free Full Text]
    34. 34
  34. Yellen-Shaw, A. J., E. J. Wherry, G. C. Dubois, and L. C. Eisenlohr. 1997. Point mutation flanking a CTL epitope ablates in vitro and in vivo recognition of a full-length viral protein. J. Immunol. 158:3227-3234.[Abstract]
    35. 35
  35. Yokomaku, Y., H. Miura, H. Tomiyama, A. Kawana-Tachikawa, M. Takiguchi, A. Kojima, Y. Nagai, A. Iwamoto, Z. Matsuda, and K. Ariyoshi. 2004. Impaired processing and presentation of cytotoxic-T-lymphocyte (CTL) epitopes are major escape mechanisms from CTL immune pressure in human immunodeficiency virus type 1 infection. J. Virol. 78:1324-1332.[Abstract/Free Full Text]
    36. 36
  36. York, I. A., S. C. Chang, T. Saric, J. A. Keys, J. M. Favreau, A. L. Goldberg, and K. L. Rock. 2002. The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 8-9 residues. Nat. Immunol. 3:1177-1184.[CrossRef][Medline]

Journal of Virology, April 2005, p. 4870-4876, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4870-4876.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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