Escape mutations alter proteasome processing of major histocompatibility complex class I-restricted epitopes in persistent hepatitis C virus infection

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.

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.

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.

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.

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.

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-γ was measured by flow cytometry.