CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope

Abstract

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.

Figure 1. Q9VF/5D-specific CTLs exert a selection pressure on HIV Q9VF gag-overlapping ARF.

(A) Analysis of Q9VF proviral sequences in HIV-infected donors. Using PBMCs, proviral DNA of 20 HIV+ individuals were extracted and the region corresponding to gag-ARF PCR-amplified and cloned. Twenty clones per donor were sequenced. Results are presented as percentage of provirus encoding for Q9VF/5D and 5D variants exhibiting within the epitope an additional AA difference from the consensus sequence, Q9VF/5N and 5N variants, and sequence harboring a stop codon prior the epitope (no epitope). Pies on the right represent percentage of provirus combined for all isolates. Top and bottom panels, results for HLA-B*07+ and HLA-B*07- donors, respectively. (B) Percentage of provirus encoding Q9VF/5D or 5D variants within HLA-B*07+ and HLA-B*07- patients. Each dot represents percentage within the PBMCs of one donor. In HLA-B*07+ patients, variants with 5D are under-represented (P<0.04). (C) Immunogenicity of Q9FV peptide variants. PBMCs of HIV-infected HLA-B*07+ donors were loaded with peptides and T cell activation monitored by IFNγ-ELISot. PBMCs were incubated with HLA-B*07-restricted epitopes: Q9VF/5D, Q9VF/5N, a pool of 3 immunodominant HIV-1 Gag epitopes (SPRTLNAWV, TPQDLNTML, YPLASLRSLF), a CMV-derived epitope (pp65 TPRVTGGGAM) or an HCV-derived epitope as negative control (GPRLGVRAT). Out of 31 HLA-B*07+ patients 5 reacted to Q9VF/5D and Q9VF/5N. Results for the 5 Q9VF reacting patients (Q9VF CTL +, full symbols) and 5 representative Q9VF non-reacting patients (Q9VF CTL-, open symbols) are shown. Data are means of triplicates. Dotted line indicates threshold of significant positive responses.

Figure 2. Q9VF/5D to Q9VF/5N substitution abrogates CTL recognition of HIV-infected cells.

(A) T1-B7 cells were infected with HIV_LAI and HIV_NL-AD8 expressing Q9VF/5D and Q9VF/5N, respectively. Two days p.i., the percentage of HIV-infected cells was monitored by intracellular p24 staining and flow cytometry: 50 and 47% of the cells were infected with HIV_LAI and HIV_NL-AD8, respectively. In an IFNγ-ELISpot assay, infected cells were then used to activate CTL lines specific for Q9VF/5D, Q9VF/5N or an HLA-B*07-restricted HIV-1 Nef epitope (FPVTPQVPLR, F10LR) used as control. For each peptide, specific CTL lines were generated in three different HLA-B*0702 transgenic mice and used in two independent experiments. One representative experiment with one CTL line is shown (mean values of triplicates ±SD). T1-B7 cells loaded with the cognate peptide were used as positive controls. (B) 5N substitution does not affect HIV replication. T1-B7 cells (left panel) and CD4+ activated T cells (right panel) were infected (at 100 and 1 ng/ml respectively) with HIV_LAI and HIV_LAI-5D>5N. HIV_LAI-5D>5N expressing Q9VF/5N was engineered by PCR mutagenesis of the HIV_LAI strain. Whatever the viral input (1, 10 or 100 ng/ml), 5N substitution did not alter the replication capacity of HIV_LAI-5D>5N. T1-B7 cell infection (left panel) was monitored using GFP expression (upon trans-activation of LTR-GFP). Data are representative of at least five independent experiments using various viral inputs. CD4+ T cells infection was monitored using p24-Elisa (right panel) and correspond to the mean values (±SD) of two infections using activated CD4+ T cells from two donors and are representative of two independent experiments (using various viral input). NI: not infected. (C) 5N substitution is sufficient to abrogate CTL recognition of HIV-infected cells. As in (A) using T1-B7 cells infected with HIV_LAI, HIV_NL-AD8 and HIV_LAI-5D>5N. Infection rates were around 30% of p24+ cells.

Figure 3. Q9VF/5N binds TAP pumps and HLA-B*0702 molecules.

(A) Q9VF/5N and Q9VF/5D peptides exhibit similar affinities for HLA-B*0702. (Left panel) Q9VF/5D, Q9VF/5N and their natural EGF Nt-extended precursors were loaded O/N at RT on T2-B7 cells. An HLA-B*07-restricted CMV-derived reference epitope (pp65 RPHERNGFTV, R10TV) and an HLA-A*02-restricted HIV-1-derived epitope (p17 SLYNTVATL, SL9) were also used as positive and negative control, respectively. HLA-B*0702 binding was monitored using ME-1 antibody and flow cytometry. Based on the reference peptide R10TV, a relative affinity (RA) was calculated. Data are representative of three different experiments (mean values of triplicates ±SD). (Right panel) T2-B7 were cultured O/N at 26°C to increase peptide-receptive cell surface molecules, pulsed with the indicated peptides for 2 h in presence of β2-microglobulin and BFA to stop delivery of newly synthesized MHC-I molecules. Cells were then shifted to 37°C for 1 h, washed to remove unbound peptides and incubated at 37°C in presence of BFA (0.5 µg/ml) which is considered as time “zero”. At the indicated time points, samples were removed to 0°C, stained on ice using ME.1 Ab and analyzed by FACS. Data are mean values of two independent experiments. The capacity of each peptide to stabilize HLA-B*0702 (t _1/2) was compared using exponential regression. T_1/2 of HLA-B*0702 pulsed with the irrelevant peptide (S9L) was 22 min while binding of Q9VF/5D and Q9VF/5N peptides prolonged the t_1/2 to 211 and 641 min respectively. T_1/2 of CMV (pp65 TPRVTGGGAM, T10AM) and Gag (p24 TPQDLNTML, T9ML) peptides used as positive were 552 and 124 min respectively. (B) Human TAP transporter binding assay. Microsomes from insect cells expressing human TAPs were incubated with the labeled reference reporter peptide (RRYNASTEL, R9L) then loaded with serial dilutions of unlabeled reference peptide or tested peptides with or without EGF Nt-extension. TAP affinities were determined as the concentrations required to inhibit 50% of reporter peptide binding (IC₅₀) and data are presented as 1/IC₅₀ ratios: the highest the ratio, the stronger the affinity. Results are mean values (±SD) from three independent experiments. (C) Q9VF/5D epitope generation is dependent on proteasomal processing. T1-B7 cells were infected with HIV_LAI (as in Figure 1), monitored for HIV infection by flow cytometry, treated or not (unTx) with epoxomicin (6 h at 37°C). To remove residual MHC-peptide complexes, cells were then treated with a citrate-phosphate buffer, washed and used as targets to activate Q9VF/5D-specific CTLs in IFNγ-ELISpot assay (8h). Note that epoxomycin inhibition affected neither MHC-density (as monitored by FACS, not shown) nor the capacity of treated cells to present exogenous peptide (0.1 µg/ml) (right panel). Results are mean values (±SD) of triplicates and representative of three different Q9VF/5D CTL clones. Mock, non infected cells (left panel) or loaded with the irrelevant HCV peptide (right panel).

Figure 4. 5N introduces an aberrant proteasomal cleavage site within the epitope.

(A) 5N introduces a strong cleavage site within Q9VF epitope. 27mer synthetic peptides encompassing Q9VF/5D or Q9VF/5N were submitted to in vitro immunoproteasome (IP) digestion. Resulting peptide fragments were analyzed by mass-spectrometry. Proteasome cleavage patterns are presented as C-terminal cleavages to a specific AA (horizontal axis) of Q9VF/5D (upper panel) and Q9VF/5N (lower panel) substrates. The percentage of C-terminal cuts at each AA is indicated. The most frequent fragments at 1 h IP digestion are depicted. Data represent one of two independent experiments. (B) The overall production of Q9VF epitope is drastically reduced by the 5N substitution. Q9VF/5D (upper panel) and Q9VF/5N (lower panel) encompassing peptides were digested by IP from 0 h to 18 h. Resulting peptide fragments were analyzed by MS/MS, as in (A). Proteasome cleavage patterns are presented as the estimated percentage of peptide fragments corresponding to either the substrate (M1-P27), the epitope Q9VF (Q11-F19), precursors with a C-terminal cut at F19, peptide fragments with a cleavage within the epitope most likely abolishing epitope production (referred to as “Antitopes”), or other fragments, with the sum of all fragments intensities set as 100%.