Hepatitis C virus mutation affects proteasomal epitope processing

The high incidence of hepatitis C virus (HCV) persistence raises the question of how HCV interferes with host immune responses. Studying a single-source HCV outbreak, we identified an HCV mutation that impaired correct carboxyterminal cleavage of an immunodominant HLA-A2–restricted CD8 cell epitope that is frequently recognized by recovered patients. The mutation, a conservative HCV nonstructural protein 3 (NS3) tyrosine to phenylalanine substitution, was absent in 54 clones of the infectious source, but present in 15/21 (71%) HLA-A2–positive and in 11/24 (46%) HLA-A2–negative patients with chronic hepatitis C. In order to analyze whether the mutation affected the processing of the HLA-A2–restricted CD8 cell epitope, mutant and wild-type NS3 polypeptides were digested in vitro with 20S constitutive proteasomes and with immunoproteasomes. The presence of the mutation resulted in impaired carboxyterminal cleavage of the epitope. In order to analyze whether impaired epitope processing affected T cell priming in vivo, HLA-A2–transgenic mice were infected with vaccinia viruses encoding either wild-type or mutant HCV NS3. The mutant induced fewer epitope-specific, IFN-γ;–producing and fewer tetramer⁺ cells than the wild type. These data demonstrate how a conservative mutation in the flanking region of an HCV epitope impairs the induction of epitope-specific CD8⁺ T cells and reveal a mechanism that may contribute to viral sequence evolution in infected patients.

Hepatitis C virus (HCV) is a 9.6 kb positive-stranded RNA virus of the flavivirus family and the leading cause of chronic hepatitis worldwide. Whereas recovery from acute HCV infection has been associated with multispecific T cell responses that protect upon reexposure to the virus (1, 2), these responses are either not induced or not maintained in the large number of patients who develop chronic infection (3–5).

We have therefore asked whether HCV interferes with the induction of antigen-specific T cells. Induction of CD8⁺ T cells depends on the generation of MHC class I ligands by the proteasome, the major cytosolic proteinase. The proteasome cleaves short peptides from longer polypeptide precursors that are then translocated into the endoplasmic reticulum and bind to newly synthesized MHC class I molecules (6). 26S proteasomes contain a 20S catalytic core, arranged as 2 heptameric outer rings with 7 β-subunits each and 2 heptameric inner rings with 7 α-subunits each. Proteasome activity is closely regulated by cytokines that are produced in viral infections (7–12). In response to IFN-γ;, for example, the constitutive catalytic subunits β1, β2, and β5 are replaced by low molecular weight protein 2 (LMP2) (iβ1), LMP7 (iβ5), and multicatalytic endopeptidase complex-like–1 (MECL-1) (iβ2) to form immunoproteasomes with altered cleavage properties (13).

HCV circulates in an abundant number of quasispecies because of its high replication rate (14) and its lack of polymerase proofreading capacity. Individual HCV sequences have been described as abolishing recognition by T cell receptors (TCRs) and antibodies, thus interfering with the effector arms of the cellular (2, 15, 16) and humoral immune responses (17). In contrast, the possibility that HCV mutations affect the induction of T cell responses has not been investigated. Indirect evidence for this hypothesis stems from descriptive reports that HCV isolates from persistently infected patients often encode less immunogenic sequences than prototype peptides used for in vitro analysis (18, 19). Whether the decreased immunogenicity results from viral mutations or from infection with less immunogenic strains has not been analyzed because the sequence of the infecting virus is not known in most human studies and also because the route of infection and inoculum size differ among the studied individuals.

Having studied a cohort of patients accidentally infected by a single-source HCV with known sequence, we here demonstrate that an HCV mutation located in the flanking region of a frequently recognized HCV epitope (4, 20–25) impairs the induction of HCV-specific CD8⁺ T cells by affecting the sophisticated proteasomal antigen-processing machinery.

The tyrosine/phenylalanine mutation at residue HCV nonstructural protein 3₁₀₈₂ is common in patients who develop persistent infection after a single-source outbreak of HCV. In order to study viral mutations during the natural course of HCV infection in humans, we analyzed a cohort of patients that had accidentally been infected with HCV in 1978/1979 during a single-source outbreak due to a contaminated anti–D immunoglobulin (26). Because the precise time of infection as well as the genotype and the sequence of the original infectious virus were known and identical for all patients, this cohort was suitable for studying candidate mechanisms of viral persistence. As previously described for this and other cohorts, the strength of the cellular immune response correlated with the outcome of infection, and HCV nonstructural protein 3 (NS3) peptides were among the most frequently recognized (3, 5, 20, 27, 28).

When we compared the HCV NS3_987–1133 sequence of the infectious source with sequences that we isolated from the sera of persistently infected, HLA-A2–positive patients 18 years after the single-source outbreak, we found the highest ratio of nonsynonymous to synonymous HCV mutations at amino acid position NS3₁₀₈₂ (Figure 1). Position NS3₁₀₈₂ was located directly adjacent to the carboxyterminus of an HLA-A2–restricted CD8⁺ T cell epitope recognized by recovered patients of this (20) and other cohorts (4, 20–25). Whereas 54 molecular clones from 3 independent PCRs of the infectious source encoded a tyrosine (Y) in position NS3₁₀₈₂, serum isolates from 15 of 21 (71%) HLA-A2–positive, persistently infected patients encoded a phenylalanine (F) at position NS3₁₀₈₂ (Table 1). Because the Y/F mutant was also observed in a substantial number of HLA-A2–negative, persistently infected patients (11 of 24 [46%]; P = 0.07; data not shown), the epidemiological data alone did not indicate whether HLA-A2–restricted T cell selection pressure could have contributed to the viral sequence evolution in this patient cohort. We therefore decided to analyze the molecular and immunological effects of the Y/F mutation in vitro by studying proteasomal processing of antigen and in vivo by studying its effect on T cell priming in an HLA-transgenic mouse model.

Figure 1

Prevalence of synonymous (open circles) and nonsynonymous (filled circles) HCV mutations within the HCV NS3_987–1133 sequence isolated from HLA-A2 positive, persistently infected HCV patients 18 years after a single-source outbreak of hepatitis C. The arrow indicates the amino acid position with the highest rate of nonsynonymous to synonymous mutations.

Table 1

HCV NS3_1062–1095 sequence of the infectious source and HLA-A2⁺ HCV-infected patients

The Y/F mutation at residue N3₁₀₈₂ impairs carboxyterminal processing of the NS3_1073–1081 epitope. Most MHC class I–restricted peptides are liberated from antigenic precursor sequences by the 20S core particle of the proteasome. To analyze whether the NS3₁₀₈₂ Y/F substitution affected proteasomal processing of the NS3_1073–1081 epitope, we built on our previous demonstration that proteasome-dependent in vitro processing of epitope-harboring polypeptides reflects the in vivo situation with high fidelity (10, 12, 29, 30) and compared proteasome-dependent in vitro processing of the NS3 wild-type polypeptide, designated NS3(Wt)_1062–1095, with processing of the NS3 mutant polypeptide, designated NS3(Mut)_1062–1095. For this purpose, immunoproteasomes were purified from IFN-γ;–stimulated HepG2 human hepatoma cells and from murine mouse embryonal cells–217 (MEC-217) transfected with the immunoproteasome subunits LMP2, LMP7 and MECL-1 (10). Constitutive proteasomes were purified from unstimulated HepG2 cells and from murine MEC-18 cells.

Synthetic NS3(Wt)_1062–1095 and NS3(Mut)_1062–1095 polypeptides (Figure 2a) were then incubated with 20S immunoproteasomes. The digestion products were separated and analyzed by reversed phase–HPLC and mass spectrometry. NS3(Wt)_1062–1095 and NS3(Mut)_1062–1095 polypeptide substrates were digested with the same kinetics with nearly 40–50% turn-over of each substrate within 4–8 hours (Figure 2B). Because longer digestion times resulted in secondary cleavage of processing intermediates, 4-hour and 8-hour digestion times were considered optimal for further biochemical analyses. Processing with constitutive proteasomes yielded the same qualitative results but lower amounts of digest product than processing with immunoproteasomes (not shown).

Figure 2

Digestion of NS3(Wt)_1062–1095 polypeptide and NS3(Mut)_1062–1095 polypeptide with purified 20S immunoproteasomes. (A) The amino acid sequences of the 34-mer NS3(Wt)_1062–1095 and NS3(Mut)_1062–1095 polypeptides are shown in single-letter code. The HLA-A2 restricted CD8⁺ T cell epitope NS3_1073–1081 is framed, and amino acid position NS3₁₀₈₂ containing the wild-type tyrosine or the mutant phenylalanine is circled. The dashed lines indicate the location of proteasomal cuts as deduced from the data shown in panels C–H. (B) NS3(Wt)1062–1095 polypeptide (black bars) and NS3(Mut)_1062–1095 polypeptide (white bars) were digested for 1, 2, 4, and 8 hours with 20S immunoproteasomes. Kinetic analysis of polypeptide substrate turnover is indicated. (C–H) Kinetic analysis of cleavage product generation. Relative abundances of cleavage products derived from immunoproteasomal digestion of NS3(Wt)_1062–1095 polypeptide (filled circles) and NS3(Mut)_1062–1095 polypeptide (open circles) are plotted. The relative abundance of the NS3_1073–1081 epitope could not be assessed due to formation of disulfide bonds that interfered with mass spectrometry analysis.

In contrast to the substrates, the relative abundance of the NS3_1073–1081 epitope could not be precisely determined because the epitope’s 2 cysteine residues formed aggregates via disulfide bonds. These aggregates interfered with reliable identification of the NS3_1073–1081 epitope and its immediate precursors by mass spectrometry. As an indirect measure of the generation of the NS3_1073–1081 epitope, we therefore determined the relative abundance of those cleavage products that flanked its amino- and carboxyterminus. Cleavage product NS3_1062–1070, for example, flanked the amino-terminus of the NS3_1073–1081 epitope. Cleavage product NS3_1062–1070 was generated from both the NS3(Wt)_1062–1095 and NS3(Mut)_1062–1095 polypeptide substrates with comparable efficiency (Figure 2C). Generation of cleavage product NS3_1062–1070 was associated with the generation of the complementary cleavage product NS3_1071–1095 (Figure 2D), consistent with a proteasomal cut between amino acid positions 1070 and 1071 in both the NS3(Wt)_1062–1095 and NS3(Mut)_1062–1095 polypeptide substrates (see dotted line in Figure 2A). This proteasomal cut resulted in an amino-terminal elongation of the NS3_1073–1081 epitope by 2 amino acids.

A second cleavage product that flanked the amino-terminus of the NS3_1073–1081 epitope was peptide NS3_1062–1072. As indicated in Figure 2E, cleavage product NS3_1062–1072 was also liberated from both the NS3(Wt)_1062–1095 and NS3(Mut)_1062–1095 polypeptide substrates in comparable amounts (Figure 2E), indicating that the proteasome cut both the NS3(Wt)_1062–1095 and NS3(Mut)_1062–1095 polypeptide between amino acid positions 1072 and 1073 as indicated by the dotted line in Figure 2A. This proteasomal cut resulted in the generation of the correct amino-terminus of the NS3_1073–1081 epitope, irrespective of the presence or absence of the NS3₁₀₈₂ mutation. The demonstration that the NS3_1073–1081 epitope and its amino-terminally elongated forms were generated at the same time is consistent with previous findings for other epitopes (31, 32). In cases of amino-terminally elongated peptides, the precise amino-terminus has been shown to be further defined by postproteasomal trimming by aminoexopeptidases (31, 32).

In contrast to the amino-terminus, the correct carboxyterminus of the NS3_1073–1081 epitope was generated only from the wild-type polypeptide. Figure 2 (F, G, and H) shows the relative abundance of those cleavage products whose generation defined the epitope’s carboxyterminus at amino acid position NS3₁₀₈₂. The generation of cleavage product NS3_1082–1095 indicated the correct processing of the epitope’s carboxyterminus with a proteasomal cut between amino acid positions 1081 and 1082 (Figure 2F). Importantly, cleavage product NS3_1082–1095 was only liberated from the NS3(Wt)_1062–1095and not from the NS3(Mut)_1062–1095 polypeptide substrate (Figure 2F). In contrast, cleavage product NS31083–1095 was liberated from the NS3(Mut)_1062–1095 more efficiently than from the NS3(Wt)_1062–1095 polypeptide substrate (Figure 2G). This result indicated that, in the presence of the NS3₁₀₈₂ mutation, the proteasome cut between amino acid positions 1082 and 1083 rather than between amino acid positions 1081 and 1082 and thus generated a carboxyterminally elongated NS3 epitope. Finally, cleavage product NS3_1084–1095 was liberated from both the NS3(Wt)_1062–1095 and NS3(Mut)_1062–1095 polypeptide substrates in comparable amounts (Figure 2H), indicating that the proteasomal cut between amino acid 1083 and 1084 was not affected by the NS3₁₀₈₂ mutation.

In summary, an incorrect carboxyterminus of the NS3_1073–1081 epitope appeared to be generated from the NS3(Mut)_1062–1095 but not from the NS3(Wt)_1062–1095 polypeptide substrate.

NS31073–1081-specific CD8+ T cells recognize aminoterminally, but not carboxyterminally elongated forms of the minimal optimal epitope. To study recognition of the processing products by CD8⁺ T cells, we generated NS3_1073–1081-specific CD8⁺ T cell lines from the blood of HCV-recovered patients. These T cell lines recognized target cells pulsed with the minimal optimal NS3_1073–1081-epitope in a standard cytotoxicity assay (Figure 3). Epitope-specific T cell lines also recognized the corresponding aminoterminally elongated peptides, but not the carboxyterminally elongated mutant and wild-type peptides (Figure 3), thereby confirming that correct carboxyterminal cleavage by the proteasome was indispensable (33, 34).

Figure 3

NS3_1073–1081 epitope–specific CD8⁺ T cells recognize aminoterminally extended, but not carboxyterminally extended, peptides of the minimal optimal NS3_1073–1081 epitope. NS3_1073–1081–specific CD8⁺ T cells were expanded from PBMCs of an HCV-recovered patient by several weeks of peptide stimulation and tested against amino- and carboxyterminally extended peptides in a standard ⁵¹Cr-release assay. The mutant sequence at AA position 1082 is underlined.

Direct biochemical and immunological detection of the NS31073–1081 epitope in proteasomal digests. To directly assess and quantitate the in vitro generation of the NS3_1073–1081 epitope, it was necessary to prevent the formation of disulfide bonds between the cysteine residues. We therefore synthesized a serine variant of the epitope with a cysteine to serine exchange at position NS31073. This NS3_1073–1081 serine variant was equally well recognized by cytotoxic T cells as the original NS3_1073–1081 epitope (Figure 4A).

Figure 4

Biochemical and immunological analysis of proteasomal digests. (A) NS31073–1081–specific CD8⁺ T cells lyse T2 cells loaded with either the NS31073–1081 serine variant SVNGVCWTV or the NS3_1073–1081 epitope CVNGVCWTV. (B–C) Generation of NS31073–1081 serine variant SVNGVCWTV from NS3(Wt)1062–1095 (filled circles) and NS3(Mut)_1062–1095 (open circles) serine variant polypeptides NS3_1062–1095 by digestion with constitutive 20S proteasome (B) and by digestion with 20S immunoproteasome (C). (D–E) 8-hour–proteasome (D) and immunoproteasome (E) digests of NS3(Wt)1062–1095 and NS3(Mut)1062–1095 serine variant polypeptides were loaded onto TAP-deficient T2-target cells and incubated with NS31073–1081 epitope–specific, cytotoxic T cells at an effector to target ratio of 13 to 1. For comparison, 10-⁵ M NS3_1073–1081 serine variant was loaded onto T2 target cells. Only wild-type and not the mutant polypeptide digests were recognized by NS3_1073–1081 epitope–specific cytotoxic T cells.

When the corresponding NS3(Wt)_1062–1095 and NS3(Mut)_1062–1095 polypeptides with a serine in position NS31073 were subjected to digestion by constitutive proteasomes, the production of the NS3_1073–1081 serine variant could be directly assessed by mass spectrometry. As demonstrated in Figure 4B, a significantly larger amount of the NS3_1073–1081 SVNGVCWTV epitope was generated from the NS3(Wt)_1062–1095 polypeptide than from the NS3(Mut)_1062–1095 polypeptide. For immunological analysis, the complete 8-hour proteasomal digests were then loaded onto transporter associated with antigen processing–deficient (TAP-deficient), T2-target cells and tested for recognition by NS3_1073–1081-specific, cytotoxic T cell lines. Only the wild-type and not the mutant polypeptide digests were recognized by NS3_1073–1081-specific cytotoxic T cells (Figure 4D), thus confirming the biochemical data in Figure 2.

The same qualitative results were obtained when NS3(Wt)_1062–1095 and NS3(Mut)_1062–1095 polypeptides were digested with immunoproteasome instead of constitutive proteasome. Again, a significantly larger amount of the NS3_1073–1081 SVNGVCWTV epitope was generated from NS3(Wt)_1062–1095 than from NS3(Mut)_1062–1095 polypeptide (Figure 4, C and E). Overall, the immunoproteasome appeared to digest the wild-type polypeptide more rapidly than the constitutive proteasome did, as indicated by a plateau-phase of epitope liberation from the wild-type polypeptide in the biochemical analysis (Figure 4C) and by the higher cytotoxicity in the immunological analysis (Figure 4E).

The HCV NS31082 Y/F mutant reduces the induction of HCV NS31073–1081-specific CD8+ T cells in HLA-A2 transgenic mice. To analyze whether the altered proteasomal cleavage of the NS3₁₀₈₂ Y/F mutant affected the generation of the NS3_1073–1081 epitope in vivo, we employed a humanized mouse model. Specifically, we used transgenic mice that expressed the α1 and α2 chains of the human HLA-A2 molecule and the α3 chain of the murine K^d molecule (35). Upon immunization, these mice generate T cells against the same HLA-A2–restricted epitopes as HLA-A2–positive humans (36). In contrast to what occurs in human studies, however, this mouse model allows the induction of T cells in the context of a single, defined HLA molecule and a single, defined viral sequence. Therefore, the HLA-A2–transgenic mouse model is not subject to the influences of additional factors such as selection pressure in the context of other HLA molecules and epitopes and/or preservation of viral replication fitness as may be operating in vivo in infected humans.

To take advantage of this model, we immunized HLA-A2–transgenic mice with vaccinia viruses that encoded full-length HCV NS3 sequences with either the Y wild type or the F mutant at amino acid position NS3₁₀₈₂. Two weeks after immunization, spleen cells were isolated, and the frequency of NS3_1073–1081-specific T cells was assessed by ex vivo intracellular IFN-γ;–staining and by tetramer analysis. As shown in Figure 5A for individual mice and in Figure 5, B and C, for all mice, the frequency of NS3_1073–1081-specific, IFN-γ;–producing and tetramer⁺ cells was significantly higher in mice infected with wild-type than in those infected with mutant NS3 encoding virus. In contrast, the frequency of CD8⁺ cells that recognized either a vaccinia virus epitope or an unrelated HCV NS3 epitope did not differ between both groups of mice, thus confirming the specificity of the observation.

Figure 5

Immunization of HLA-A2 transgenic mice with the wild-type HCV NS3 sequence induces more IFN-γ;–secreting and cytotoxic HCV NS3_1073–1081–specific CD8 T cells than immunization with the mutant HCV NS3 sequence. (A and B) Ex vivo analysis of NS31073–1081–specific, IFN-γ; producing CD8 T cells demonstrated a significantly greater response in mice immunized with wild-type than of mice immunized with mutant NS3 encoding vaccinia virus. In contrast, the response against the vaccinia virusH3L (VVH3L) epitope and the HCV NS3_1406–1414 epitope did not differ between both groups of mice. (A) Dot plots from individual mice tested in the same experiment. (B) Mean and standard deviation of the results of all 8 mice per group. (C) Frequency of NS31073–1081-tetramer–specific T cells is higher in mice immunized with wild-type than in mice immunized with mutant HCV NS3 sequences. Mean and standard deviation of the results of 8 mice per group are shown. (D) NS3_1073–1081-specific T cell lines from mice immunized with wild-type HCV NS3 sequences (WT) display greater cytotoxicity than those derived from mice immunized with mutant HCV NS3 (Mut). Mean and standard deviation of the results of 11 mice per group are shown.

In separate experiments, additional immunizations with recombinant DNA-expression vectors were performed to increase the number of NS3_1073–1081-specific T cells and to establish T cell lines suitable for cytotoxicity analyses. At all effector/target ratios, cytotoxic T cell responses of mice that had been immunized with wild-type NS3 sequences were significantly stronger than cytotoxic T cell responses of mice immunized with mutant NS3 sequences (Figure 5D). Collectively, these results demonstrate that the NS3₁₀₈₂ Y/F substitution reduced the generation of the NS3_1073–1081 epitope in vitro when polypeptides were digested with purified 20S proteasomes. In addition, the NS3₁₀₈₂ substitution impaired the induction of epitope-specific T cells in vivo when full-length NS3 protein was endogenously expressed, ubiquitinylated, and processed by the 26S proteasome in the presence of additional cytosolic proteases.

Most MHC class I ligands are liberated from strings of polypeptides and ubiquitinylated proteins by the proteasome, the main cytosolic protease (13). Whereas the amino-terminus of each epitope can be further defined by postproteasomal aminoexopeptidases (31, 32, 37), the carboxyterminus needs to be defined precisely with the first cut (33, 34). Studying a single source outbreak of HCV, we identified an HCV mutation that interfered with the correct carboxyterminal cleavage of an immunodominant, HLA-A2 restricted HCV epitope from its mutated polypeptide precursor.

The emergence of viral mutations in immunogenic sequences has long been discussed as a potential immune escape mechanism. As regards HCV, sequences that do not bind to the MHC and/or the T cell receptor and thus are not recognized by HCV-specific CD8⁺ T cells have been observed in chimpanzees and in humans (15, 16, 18). In addition, mutations that generate partial agonists or antagonists to the T cell receptor and downregulate wild-type–specific T cell responses have been described in HCV (18) as well as in hepatitis B virus (HBV) (38) and HIV infections (39). Perhaps even more efficient mechanisms of viral escape are mutations in epitope-flanking residues that interfere with antigen processing and presentation of MHC class I–restricted epitopes because, in these cases, the induction phase rather than the effector phase of HCV-specific T cell responses can be impaired. Indeed, the literature provides several examples showing that amino acid residues in the flanking regions of T cell epitopes impair proteasomal processing of those epitopes (40–43). On the other hand, there are also examples showing that extensive sequence changes in the flanking regions of other immunodominant CD8 T cell epitopes do not influence antigen processing (44). In our study, we observed impairment of antigen processing by an exchange of two very similar amino acids. Although the Y/F substitution is a conservative one, it impaired correct carboxyterminal cleavage of the NS3_1073–1081 epitope not only by constitutive proteasomes, but also by immunoproteasomes. Importantly, the same effects were observed in vitro when polypeptides were digested with purified 20S proteasomes and in vivo when full length HCV NS3 protein was endogenously expressed in an animal model, ubiquitinylated, and processed by the 26S proteasome in the presence of additional cytosolic proteases. Thus, the data demonstrate a mechanism by which a conservative HCV mutation can interfere with the induction of epitope-specific CD8⁺ T cells.

The observation that this specific HCV mutation was also found in a substantial number of HLA-A2–negative patients is consistent with a recent report on an HIV mutation in an HLA-B51–restricted epitope, which was also less frequent in HLA-B*51-negative than in HLA-B*51-positive persons, but not completely absent in HLA-B*51-negative persons. Overall, 29% of HLA-B*51-negative persons carried the HIV mutation as compared to 98% of HLA-B*51-positive persons (43). Notably, with more than 400 patients enrolled, the HIV study was much larger than our study, and differences in the prevalence of the HIV mutation between patient subgroups were remarkable and statistically significant (43). In the HIV study as well as in our study, however, the presence of the mutation in a subpopulation of patients without the relevant HLA haplotype is consistent with the influence of multiple selection forces that drive the evolution of viral sequences in humans. These selection forces include pressure on additional, overlapping, or adjacent T cell epitopes that are presented in the context of other MHC class I and II alleles as well as selection pressure to preserve viral replication fitness (45). Because the HCV NS31073–₁₀₈₂ sequence is located directly downstream of the HCV protease domain, it is, for example, possible that the Y/F mutation affects HCV replication and was therefore also found in HLA-A2–negative patients. For these reasons, the HLA-A2–transgenic mouse model provides a valuable tool for analyzing the effect of a single mutation on the induction of epitope-specific CD8⁺ cells in the context of a single, defined HLA molecule.

Although this study demonstrated a mechanism of altered antigen processing and impaired induction of HCV-specific T cells, significantly larger, population-based studies will be required to analyze the contribution of this mechanism to the overall selection pressure that drives HCV sequence evolution in infected patients. As a starting point for these studies, mutations in the flanking region of this particular HCV NS3_1073–1081 epitope are intriguing for several reasons. In addition to being the single most vigorously recognized CD8⁺ T cell epitope in all published studies (4, 20–25), the NS3_1073–1081 epitope is one of only a few described epitopes that are recognized by circulating T cells as well as by nonspecifically expanded intrahepatic T cells (46). Moreover, it is also one of only two epitopes for which a TCR antagonist based on an intraepitope mutation has been described in persistently infected patients (18). The Y/F mutation in the flanking region of the epitope described here should, however, not be regarded as a main cause of HCV persistence. Rather, it should be regarded as an example of a viral escape mechanism that might also occur in the flanking regions of other CD4⁺ and CD8⁺ T cell epitopes. If many of these mutations occur throughout the HCV polyprotein, they may collectively contribute to the evolution of HCV quasispecies in persistently infected patients and to the characteristic weakness of the HCV-specific immune response.

The authors thank L. Müller and S. Bigl (Landesuntersuchungsanstalt, Chemnitz, Germany) for patient samples; M. Roggendorf (Universitätsklinikum Essen, Germany) for PCR products of the infectious source; and A. Elstner for expert technical assistance. The HLA-A2 tetramer was produced at the National Institute of Allergy and Infectious Diseases Tetramer Facility of the NIH AIDS Research and Reference Reagent Program. This study was supported by grant Se885/1-1 of the Deutsche Forschungsgemeinschaft, Bonn, Germany, to U. Seifert; grant 1297 (01KI 9659/5 Pathogenesis of HCV infection) of the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF) to B. Rehermann; Sonderforschungsbereich 421 to P. Kloetzel and H. Hengel; and by the NIDDK, NIH, Department of Health and Human Services (DHHS) intramural research program.

Heiner Wedemeyer’s present address is: Department of Gastroenterology, Hepatology and Endocrinology, Medizinische Hochschule Hannover (MHH), Hannover, Germany.

Thomas Ruppert’s present address is: Zentrum für Molekulare Biologie Heidelberg, University of Heidelberg, Heidelberg, Germany.

Alice Sijts’s present address is: David H. Smith Center for Vaccine Biology and Immunology, Aab Institute for Biomedical Sciences, University of Rochester Medical Center, Rochester, New York, USA.

Nonstandard abbreviations used: hepatitis C virus (HCV); nonstructural protein 3 (NS3); phenylalanine (F); tyrosine (Y).

Conflict of interest: The authors have declared that no conflict of interest exists.

1. Shoukry, N, et al. Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection. J. Exp. Med. 2003. 197:1645-1655.
   View this article via: PubMed CrossRef Google Scholar
2. Grakoui, A, et al. HCV persistence and immune evasion in the absence of memory T cell help. Science. 2003. 302:659-662.
   View this article via: PubMed CrossRef Google Scholar
3. Diepolder, HM, et al. Possible mechanism involving T lymphocyte response to non-structural protein 3 in viral clearance in acute hepatitis C virus infection. Lancet. 1995. 346:1006-1007.
   View this article via: PubMed CrossRef Google Scholar
4. Lechner, F, et al. CD8+ T lymphocyte responses are induced during acute hepatitis C virus infection but are not sustained. Eur. J. Immunol. 2000. 30:2479-2487.
   View this article via: PubMed CrossRef Google Scholar
5. Thimme, R, et al. Determinants of viral clearance and persistence during acute hepatitis C virus infection. J. Exp. Med. 2001. 194:1395-1406.
   View this article via: PubMed CrossRef Google Scholar
6. Pamer, E, Cresswell, P. Mechanisms of MHC class I-restricted antigen processing. Annu. Rev. Immunol. 1998. 16:323-358.
   View this article via: PubMed CrossRef Google Scholar
7. Khan, S, et al. Immunoproteasomes largely replace constitutive proteasomes during an antiviral and antibacterial immune response in the liver. J. Immunol. 2001. 167:6859-6868.
   View this article via: PubMed Google Scholar
8. Schwarz, K, et al. Overexpression of the proteasome subunits LMP2, LMP7, and MECL-1, but not PA28 alpha/beta, enhances the presentation of an immunodominant lymphocytic choriomeningitis virus T cell epitope. J. Immunol. 2000. 165:768-778.
   View this article via: PubMed Google Scholar
9. Groettrup, M, Khan, S, Schwarz, K, Schmidtke, G. Interferon-gamma inducible exchanges of 20S proteasome active site subunits: why? Biochimie. 2001. 83:367-372.
   View this article via: PubMed CrossRef Google Scholar
10. Sijts, AJ, et al. Efficient generation of a hepatitis B virus cytotoxic T lymphocyte epitope requires the structural features of immunoproteasomes. J. Exp. Med. 2000. 191:503-514.
    View this article via: PubMed CrossRef Google Scholar
11. Groettrup, M, et al. The interferon-gamma-inducible 11 S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20 S proteasome in vitro. J. Biol. Chem. 1995. 270:23808-23815.
    View this article via: PubMed CrossRef Google Scholar
12. Sijts, AJ, et al. MHC class I antigen processing of an adenovirus CTL epitope is linked to the levels of immunoproteasomes in infected cells. J. Immunol. 2000. 164:4500-4506.
    View this article via: PubMed Google Scholar
13. Kloetzel, PM. Antigen processing by the proteasome. Nat. Rev. Mol. Cell Biol. 2001. 2:179-187.
    View this article via: PubMed CrossRef Google Scholar
14. Neumann, AU, et al. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-alpha therapy. Science. 1998. 282:103-107.
    View this article via: PubMed CrossRef Google Scholar
15. Weiner, A, et al. Persistent hepatitis C virus infection in a chimpanzee is associated with emergence of a cytotoxic T lymphocyte escape variant. Proc. Natl. Acad. Sci. U. S. A. 1995. 92:2755-2759.
    View this article via: PubMed CrossRef Google Scholar
16. Erickson, AL, et al. The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes. Immunity. 2001. 15:883-895.
    View this article via: PubMed CrossRef Google Scholar
17. Farci, P, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science. 2000. 288:339-344.
    View this article via: PubMed CrossRef Google Scholar
18. Chang, KM, et al. Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus. J. Clin. Invest. 1997. 100:2376-2385.
    View this article via: JCI PubMed CrossRef Google Scholar
19. Tsai, SL, et al. Hepatitis C virus variants circumventing cytotoxic T lymphocyte activity as a mechanism of chronicity. Gastroenterology. 1998. 115:954-965.
    View this article via: PubMed CrossRef Google Scholar
20. Takaki, A, et al. Cellular immune responses persist, humoral responses decrease two decades after recovery from a single source outbreak of hepatitis C. Nat. Med. 2000. 6:578-582.
    View this article via: PubMed CrossRef Google Scholar
21. Lechner, F, et al. Analysis of successful immune responses in persons infected with hepatitis C virus. J. Exp. Med. 2000. 191:1499-1512.
    View this article via: PubMed CrossRef Google Scholar
22. Gruener, NH, et al. Association of hepatitis C virus-specific CD8+ T cells with viral clearance in acute hepatitis C. J. Infect. Dis. 2000. 181:1528-1536.
    View this article via: PubMed CrossRef Google Scholar
23. Cerny, A, et al. Cytotoxic T lymphocyte response to hepatitis C virus - derived peptides containing the HLA A2.1 binding motif. J. Clin. Invest. 1995. 95:521-530.
    View this article via: JCI PubMed CrossRef Google Scholar
24. Cucchiarini, M, et al. Vigorous peripheral blood cytotoxic T cell response during the acute phase of hepatitis C virus infection. Cell Immunol. 2000. 203:111-123.
    View this article via: PubMed CrossRef Google Scholar
25. Prezzi, C, et al. Virus-specific CD8(+) T cells with type 1 or type 2 cytokine profile are related to different disease activity in chronic hepatitis C virus infection. Eur. J. Immunol. 2001. 31:894-906.
    View this article via: PubMed CrossRef Google Scholar
26. Wiese, M, Berr, F, Lafrenz, M, Porst, H, Oesen, U. Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in Germany: a 20-year multicenter study. Hepatology. 2000. 32:91-96.
    View this article via: PubMed CrossRef Google Scholar
27. Wertheimer, AM, et al. Novel CD4+ and CD8+ T-cell determinants within the NS3 protein in subjects with spontaneously resolved HCV infection. Hepatology. 2003. 37:577-589.
    View this article via: PubMed CrossRef Google Scholar
28. Wedemeyer, H, et al. Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection. J. Immunol. 2002. 169:3447-3458.
    View this article via: PubMed Google Scholar
29. Seifert, U, et al. An essential role for tripeptidyl peptidase in the generation of an MHC class I epitope. Nat. Immunol. 2003. 4:375-379.
    View this article via: PubMed CrossRef Google Scholar
30. Kuckelkorn, U, et al. Link between organ-specific antigen processing by 20S proteasomes and CD8(+) T cell-mediated autoimmunity. J. Exp. Med. 2002. 195:983-990.
    View this article via: PubMed CrossRef Google Scholar
31. Mo, XY, Cascio, P, Lemerise, K, Goldberg, AL, Rock, K. Distinct proteolytic processes generate the C and N termini of MHC class I-binding peptides. J. Immunol. 1999. 163:5851-5859.
    View this article via: PubMed Google Scholar
32. Beninga, J, Rock, KL, Goldberg, AL. Interferon-gamma can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase. J. Biol. Chem. 1998. 273:18734-18742.
    View this article via: PubMed CrossRef Google Scholar
33. Niedermann, G, et al. Contribution of proteasome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility complex class I molecules. Immunity. 1995. 2:289-299.
    View this article via: PubMed CrossRef Google Scholar
34. Craiu, A, Akopian, T, Goldberg, A, Rock, KL. Two distinct proteolytic processes in the generation of a major histocompatibility complex class I-presented peptide. Proc. Natl. Acad. Sci. U. S. A. 1997. 94:10850-10855.
    View this article via: PubMed CrossRef Google Scholar
35. Newberg, MH, et al. Importance of MHC class 1 alpha2 and alpha3 domains in the recognition of self and non-self MHC molecules. J. Immunol. 1996. 156:2473-2480.
    View this article via: PubMed Google Scholar
36. Wedemeyer, H, et al. Oral immunization with HCV-NS3-transformed salmonella: induction of HCV-specific CTL in a transgenic mouse model. Gastroenterology. 2001. 121:1158-1166.
    View this article via: PubMed CrossRef Google Scholar
37. Stoltze, L, et al. Two new proteases in the MHC class I processing pathway. Nat. Immunol. 2000. 1:413-418.
    View this article via: PubMed CrossRef Google Scholar
38. Bertoletti, A, et al. Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells. Nature. 1994. 369:407-410.
    View this article via: PubMed CrossRef Google Scholar
39. Klenerman, P, et al. Cytotoxic T-cell activity antagonized by naturally occurring HIV-1 Gag variants. Nature. 1994. 369:403-407.
    View this article via: PubMed CrossRef Google Scholar
40. Del Val, M, Schlicht, HJ, Ruppert, T, Reddehase, MJ, Koszinowski, UH. Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends on its neighboring residues in the protein. Cell. 1991. 66:1145-1153.
    View this article via: PubMed CrossRef Google Scholar
41. Theobald, M, et al. 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. 1998. 188:1017-1028.
    View this article via: PubMed CrossRef Google Scholar
42. Beekman, NJ, et al. Abrogation of CTL epitope processing by single amino acid substitution flanking the C-terminal proteasome cleavage site. J. Immunol. 2000. 164:1898-1905.
    View this article via: PubMed Google Scholar
43. Moore, CB, et al. Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population level. Science. 2002. 296:1439-1443.
    View this article via: PubMed CrossRef Google Scholar
44. Brander, C, et al. Efficient processing of the immunodominant, HLA-A*0201-restricted human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitope despite multiple variations in the epitope flanking sequences. J. Virol. 1999. 73:10191-10198.
    View this article via: PubMed Google Scholar
45. Friedrich, TC, et al. Reversion of CTL escape-variant immunodeficiency viruses in vivo. Nat. Med. 2004. 10:275-281.
    View this article via: PubMed CrossRef Google Scholar
46. Koziel, MJ, et al. HLA class I-restricted cytotoxic T lymphocytes specific for hepatitis C virus. Identification of multiple epitopes and characterization of patterns of cytokine release. J. Clin. Invest. 1995. 96:2311-2321.
    View this article via: JCI PubMed CrossRef Google Scholar
47. Drexler, I, et al. Identification of vaccinia virus epitope-specific HLA-A*0201-restricted T cells and comparative analysis of smallpox vaccines. Proc. Natl. Acad. Sci. U. S. A. 2003. 100:217-222.
    View this article via: PubMed CrossRef Google Scholar
48. Rehermann, B, et al. Quantitative analysis of the peripheral blood cytotoxic T lymphocyte response, disease activity and viral load in patients with chronic hepatitis C virus infection. J. Clin. Invest. 1996. 98:1432-1440.
    View this article via: JCI PubMed CrossRef Google Scholar
49. Rehermann, B, et al. Differential cytotoxic T lymphocyte responsiveness to the hepatitis B and C viruses in chronically infected patients. J. Virol. 1996. 70:7092-7102.
    View this article via: PubMed Google Scholar
50. Rispeter, K, Lu, M, Lechner, S, Zibert, A, Roggendorf, M. Cloning and characterization of a complete open reading frame of the hepatitis C virus genome in only two cDNA fragments. J. Gen. Virol. 1997. 78:2751-2759.
    View this article via: PubMed Google Scholar
51. Schlicht, HJ, Schaller, H. The secretory core protein of human hepatitis B virus is expressed on the cell surface. J. Virol. 1989. 63:5399-5404.
    View this article via: PubMed Google Scholar
52. Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press. Plainview, New York, USA. 37–84.