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Journal of Virology, February 2007, p. 2031-2038, Vol. 81, No. 4
0022-538X/07/$08.00+0     doi:10.1128/JVI.00968-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Precise Identification of a Human Immunodeficiency Virus Type 1 Antigen Processing Mutant{triangledown}

Peter Zimbwa,1,{dagger} Anita Milicic,1,{dagger}* John Frater,1 Thomas J. Scriba,1 Antony Willis,2 Philip J. R. Goulder,3 Tilly Pillay,1 Huldrych Gunthard,4 Jonathan N. Weber,5 Hua-Tang Zhang,1 and Rodney E. Phillips1

The James Martin 21st Century School at The Peter Medawar Building for Pathogen Research, Nuffield Department of Clinical Medicine, University of Oxford, South Parks Road, Oxford OX1 3SY, United Kingdom,1 Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom,2 Department of Pediatrics, University of Oxford, Oxford OX3 9DU, United Kingdom,3 University Hospital Zurich, Department of Medicine, Division of Infectious Diseases and Hospital Epidemiology, Ramistrasse 100, CH-8091 Zurich, Switzerland,4 Jefferiss Research Laboratories, Wright-Fleming Institute, Imperial College, St Mary's Hospital, Norfolk Place, London W2 1PG, United Kingdom5

Received 11 May 2006/ Accepted 9 November 2006


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ABSTRACT
 
Human immunodeficiency virus type 1 (HIV-1) evokes a strong immune response, but the virus persists. Polymorphisms within known antigenic sites result in loss of immune recognition and can be positively selected. Amino acid variation outside known HLA class I restricted epitopes can also enable immune escape by interfering with the processing of the optimal peptide antigen. However, the lack of precise rules dictating epitope generation and the enormous genetic diversity of HIV make prediction of processing mutants very difficult. Polymorphism E169D in HIV-1 reverse transcriptase (RT) is significantly associated with HLA-B*0702 in HIV-1-infected individuals. This polymorphism does not map within a known HLA-B*0702 epitope; instead, it is located five residues downstream of a HLA-B*0702-restricted epitope SPAIFQSSM (SM9). Here we investigate the association between E169D and HLA-B*0702 for immune escape via the SM9 epitope. We show that this single amino acid variation prevents the immune recognition of the flanked SM9 epitope by cytotoxic T cells through lack of generation of the epitope, which is a result of aberrant proteasomal cleavage. The E169D polymorphism also maps within and abrogates the recognition of an HLA-A*03-restricted RT epitope MR9. This study highlights the potential for using known statistical associations as indicators for viral escape but also the complexity involved in interpreting the immunological consequences of amino acid changes in HIV sequences.


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INTRODUCTION
 
Cytotoxic T lymphocytes (CTL, CD8+ T cells) have T-cell receptors which specifically recognize antigens presented by HLA class I molecules. CTL have a key role in the immune defense against viral infection and are crucial for the containment of human immunodeficiency virus type 1 (HIV-1) replication (4, 13, 26). In a single individual, HIV variation is enormous. This results from a high proliferative capacity and error-prone reverse transcriptase of HIV (33, 47) and so enables the virus to evade HIV-1-specific CTLs (13, 16, 26). Amino acid variation within HLA class I-restricted epitopes, positively selected by host immune pressure, can lead to escape from CTL recognition (13, 16, 26, 38). Evasion of the CTL recognition and persistence in the face of a vigorous CTL response typifies HIV-1 infection (26, 38, 40). HIV can escape CTL responses when amino acid variation interferes with peptide binding to the HLA class I (2, 10, 12, 15, 19, 27, 39) or alters epitope recognition by the T-cell receptor (24, 41). Emerging evidence shows that a mutation within a CTL epitope can also affect its processing (49).

Antigen processing is often subverted by polymorphisms outside epitopes (48) so that optimal epitope generation is blocked (3, 9) or diminished (30, 42). Optimal epitope generation can be inhibited when proteolytic activity is redirected to novel sites within the variant protein (34). Biochemical descriptions of this phenomenon are emerging, but the significance of this form of immune escape on the pathogenesis of viral infection is very difficult to estimate.

Within an infected host, immune pressure exerted by the HLA class I alleles leads to selection and the accumulation of virions harboring escape variants (reviewed in reference 25). Statistical approaches, where the frequencies of HLA class I alleles in an HIV-infected population are analyzed for association with viral amino acid polymorphisms, offer a means of surveying a viral genome for escape mutants. Such studies have revealed strong associations between amino acid polymorphisms within HIV-1 epitopes and their restricting HLA class I alleles (21, 31, 50).

Here we show how the same statistical approach can expose antigen-processing HIV mutants. We investigate the statistical association between a polymorphism outside an HLA-B*0702-restricted HIV reverse transcriptase (RT) epitope SPAIFQSSM (SM9) and HLA-B*0702 (31). We demonstrate that this single amino acid polymorphism E169D, five residues downstream of SM9, escapes the CTL immune response through an antigen-processing mechanism. The E169D polymorphism also leads to immune escape through another epitope which is HLA-A*03 restricted (MTKILEPFR, RT 164 to 172); however, no statistical association has been found between E169D and HLA-A*03 (31).


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MATERIALS AND METHODS
 
Patient samples. With the approval of the institutional review body, peripheral blood mononuclear cells (PBMCs) and/or plasma were obtained form 130 HIV-1-infected homosexual men recruited from St. Mary's Hospital, London, England and the SSITT cohort (35). Fresh PBMCs were separated from whole blood by Ficoll-Hypaque (Axis Shield Diagnostics) density gradient centrifugation. All patients were infected with clade B HIV-1 and were on structured treatment interruption therapy. RT was amplified from plasma as part of routine clinical care. RT was also amplified from proviral DNA in four patients, and clones were sequenced. Two molecular viral clones from one patient were identified with 169E and 169D; these were used for HIV constructs. They were isogenic in the sequenced region of residues 2 to 262 other than at following loci: K30N, K49R, R83K K122E, I132V, I135T, E169D, and V245A. All patients were typed as HLA class I.

HLA class I typing. HLA class I typing was done by sequence-specific primer PCR on genomic DNA that was extracted from 3 ml whole blood using a Puregene DNA isolation kit (Gentra) per the manufacturer's instructions (7).

Generation of B-lymphoblastoid cell lines. PBMCs were transformed with Epstein-Barr virus 95.8 stock for 2 h at 37°C in R-10 medium (RPMI 1640, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 10% fetal calf serum) (28). Cyclosporine was then added at 0.5 µg/ml in a 24-well plate and cultured at 37°C for several weeks, with regular changes of media.

Generation of CTL lines. CTL lines were generated from PBMCs of HLA-typed HIV-1-infected patients who responded to peptides RK9, SM9, and ER10. The lines were set up with corresponding synthetic peptides as described previously (23). Briefly, around 3 million PBMCs were resuspended in 2 ml of R-10 medium (RPMI, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 10% fetal calf serum) containing 10 µM peptide and 25 ng/ml interleukin-7 (IL-7; Peprotech) and cultured in a 24-well plate. On day 3, the medium was replaced with R-10 medium containing 200 U/ml IL-2 (proleukin) and 5% T-Stim with phytohemagglutinin (PHA; BD Biosciences). The lines were subsequently grown in the same medium (and restimulated using irradiated mixed heterologous PBMCs and 4 µg/ml PHA) or maintained in R-10 containing 25 ng/ml IL-15 (Peprotech) (5, 32). CTL lines were enriched for specificity by antigen stimulation and positive selection of gamma interferon (IFN-{gamma})-producing cells anti-IFN-{gamma} microbeads per the manufacturer's instructions (Miltenyi Biotec).

We generated CTL lines specific for HLA-A*0301 p17 Gag RLRPGGKKK, HLA-B*0702 RT SPAIFQSSM, and a newly defined HLA-A*3301-restricted Integrase epitope ELKKIIGQVR (P. Zimbwa, A. Milicic, and R. Phillips, unpublished data).

CD4+ T-cell lines. The SupT1 cell line used in making the HIV recombinants is a human lymphoma T-cell lymphoblast line obtained from J. A. Hoxie through the Centralized Facility for AIDS Reagents supported by EU Programme EVA/MRC and the Medical Research Council, Hertfordshire, United Kingdom. Jurkat E6-1 is a human T-cell lymphoblast clone (HLA-A*0301/-B*0702/*3501 Cw*0401/*0701) that was obtained from R. A. Weiss through the same facility. The MT-2 cell line used for titration of HIV is an human T-cell leukemia virus type 1-infected human leukemic T-cell lymphoblast line obtained from G. Farrar through the European Collection of Cell Cultures, United Kingdom. These cell lines were maintained in R-10 medium.

ELISpot assay. Antigen-specific responses from PBMCs, CTL lines, or clones were measured using synthetic peptides corresponding to the relevant epitopes and a standard enzyme-linked immunospot (ELISpot) assay for IFN-{gamma}, as described previously (22). Fresh or cryopreserved PBMCs (25,000 to 50,000 per well) or CTLs (500 to 2,500 per well) were used in overnight or 4-h assays. Jurkat E6-1 cells, either infected with HIV-1 or pulsed with exogenous peptide (Bio-Synthesis, TX) at 2.5 µM were added as antigen-presenting targets. All assays were performed in duplicate or triplicate, and positive (PHA) and appropriate negative controls were included in every assay. Spot quantification was automated and standardized using an ELISpot plate reader (software version 3.2.3; Autoimmun Diagnostika, Germany).

Cell surface and intracellular staining for IFN-{gamma} and p24. PBMCs (≥500,000) or CTLs (≥100,000) were incubated with either 5 µM peptide, peptide-pulsed B-lymphoblastoid cell line (BCL), or HIV-1-infected Jurkat E6-1 cells for 90 min at 37°C in 200 µl of R-10 medium (RPMI 1640 containing 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine), with 0.2 µg/ml of each anti-CD28 and anti-CD49d antibody (Becton Dickinson). Brefeldin A (Sigma-Aldrich) was added at 10 µg/ml to prevent egress of IFN-{gamma} from the Golgi apparatus, and cells were incubated for a further 5 h at 37°C. Cells were then washed and stained for surface CD4, CD8, and HLA-A,B,C at 4°C for 20 min. After washing, cells were fixed and permeabilized with Cytofix/Cytoperm (Becton Dickinson). Cells were washed again before staining for IFN-{gamma} (30 min at 4°C) or p24 (20 min at room temperature). After further washing, cells were resuspended in phosphate-buffered saline and analyzed on a FACScalibur flow cytometer (Becton Dickinson).

Peptide binding assay. The competitive fluorescent binding assay was performed as described previously (20, 29). Briefly, BCL expressing HLA-A*0301 were stripped of their naturally bound self-peptides by acid elution for 90 s (pH = 2.9). The B cells were then incubated for 24 h at 2 to 8°C with an HLA-specific reference peptide conjugated to a fluorescein label and a test peptide titrated between 200 µM and 20 nM. After the incubation, each sample was stained with 7-amino-actinomycin D (Viaprobe; BD Biosciences) to exclude dead cells from subsequent analysis, fixed, and analyzed by flow cytometry. All assays were done three times. The inhibition of fluorescein-labeled reference peptide binding, through competition with the test peptide, is a measure of the binding affinity of the test peptides. Inhibition was determined as described previously (20).

Generation of HIV-1 RT fragments by PCR. RT fragments were amplified from proviral DNA by nested PCR with outer primers RT18 (5'-GGA AAC CAA AAA TGA TAG GGG GAA TTG GAG G-3', nucleotides 2376 to 2406) and RT21 (5'-CTG TAT TTC TGC TAT TAA GTC TTT TGA TGG G-3', nucleotides 3538 to 3508) and inner primers RT19 (5'-GGA CAT AAA GCT ATA GGT ACA G-3', nucleotides 2453 to 2474) and RT21. Initial denaturing at 95°C for 2 min was followed by 35 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 1 min. A final extension at 72°C was run for 5 min. PCR products were checked for size on a 1% agarose gel, and 2 µl of the PCR product, purified using a DNA purification kit (QIAGEN), was ligated into the TOPO TA plasmid using a TOPO TA cloning kit per the manufacturer's instructions (Invitrogen). The RT insert and TOPO TA vector were extracted from the Escherichia coli cells using a DNA extraction kit (QIAGEN) per the manufacturer's instructions. After EcoRI digestion, the presence of the insert was confirmed using a 1% agarose gel.

Sequencing of proviral DNA. The cloned HIV-1 sequences were determined from both directions using primers M13F and M13R located in the TOPO TA plasmid on either side of the insert (Invitrogen). Cycling conditions were 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. Sequences were analyzed on an ABI 3700 automated analyzer.

Generation of recombinant HIV by electroporation. The recombinant HIVs were made as previously described (6). In short, RT sequences from proviral DNA were purified by gel extraction (QIAGEN) per the manufacturer's instructions and used to generate recombinant viruses with the RT-deleted HXB2-based proviral molecular clone pHXB2{Delta} 2-261RT. The amount of product was determined by spectrophotometry. One microgram of the amplified RT was mixed with 1 µg of SmaI-linearized plasmid pHXB2{Delta} 2-261RT in 5 x 106 SupT1 cells in 250 µl of cold R-10. Cotransfection was achieved by applying a 40-ms electric pulse with a Gene Pulser II set (Bio-Rad) at 250 V and 950 µF. After electroporation, 500,000 more SupT1 cells were added and cells cultured in 25-cm3 and 75-cm3 flasks in R-10. The cultures were examined on days 5, 7, 10, 12, 14, 17, 19, and 21 for syncytium formation (45, 46). If most or all cells were forming syncytia, the supernatant was harvested by centrifugation and stored at –80°C for subsequent titration. Recombinant viral titers (50% tissue culture infectious dose) were determined in MT-2 cells using the Spearman-Karber formula (41a).

Infection of Jurkat E6-1 cells. Jurkat E6-1 (2 x 106) cells were pelleted in 15-ml conical tubes at 1,000 x g for 5 min and then resuspended in 10 ml R-10 containing 2 µg/ml hexadimethrine bromide (Polybrene; Sigma-Aldich). Cells were then centrifuged at 1,000 x g for 5 min before resuspension in 1 ml R-10 with 2 µg/ml Polybrene-containing recombinant virus stock. Aliquots of culture supernatant (250 µl) and resuspended cells (2 ml) were stored at –80°C for subsequent viral sequencing and cellular assays every other day until day 13 when most cells had formed syncytia.

Proteasome digestion. Wild-type (QGWKGSPAIFQSSMTKILEPFRKQNPD) or mutant (QGWKGSPAIFQSSMTKILDPFRKQNPD) RT peptide (5 µg) was added to 300 µl buffer (20 mM HEPES-KOH, pH 7.8, 2 mM MgAc2, 2 mM dithiothreitol [Sigma]). (The SM9 epitope is underlined.) The immuno or constitutive 20S proteasome (Immatics) was then added at 2 µg per reaction mixture and incubated at 37°C. Aliquots of the reaction mix were taken at times 0, 4, 6, 8, 12, 18, 24, and 48 h and added to acetic acid (10% final concentration) to terminate the reaction (43). Digestion experiments were set up in triplicate, and the assay was performed twice for each oligomer and proteasome. Digests were then analyzed by mass spectrometry (Ettan matrix-assisted laser desorption ionization-time of flight; Amersham Biosciences) and sequences inferred using PAWS (Protein Analysis Work Sheet).


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RESULTS
 
Identification of a polymorphism associated with HLA-B*0702 but outside a known epitope. A study by Moore et al. (31) reported a strong association between the E169D polymorphism within the RT protein of HIV-1 and HLA-B*0702 (odds ratio = 12.57) in 473 patients infected with HIV-1. We tested this finding in 130 HIV patients from St. Mary's Hospital, London, and the Swiss-Spanish intermittent treatment trial (SSITT) cohort (36). We sequenced the viral RT in these patients and analyzed the statistical association between HLA-B*0702 and E169D: 3 of 24 (12.5%) of HLA-B*0702+ patients had E169D compared to 4 of 106 (3.8%) HLA-B*0702– patients (odds ratio = 3.3, P = 0.016, Fisher's exact test), thus confirming the findings of Moore et al.

Position E169D lies within an HLA-A*0301 epitope MTKILEPFR (amino acids [aa] 164 to 172) but not within any known HLA-B*07 epitopes (http://www.hiv.lanl.gov/content/immunology/). However, an HLA-B*0702-restricted epitope, SPAIFQSSM (aa 156 to 164), maps five residues upstream of the E169D polymorphism. Another HLA-A*03 epitope, AIFQSSMTK (aa 158 to 166), also maps upstream of the E169D polymorphism (Fig. 1).


Figure 1
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  		FIG. 1. Position of three epitopes with different HLA restrictions within the HIV RT protein: HLA-B*0702-restricted SPAIFQSSM (SM9, RT aa 156 to 164), HLA-A*0301-restricted AIFQSSMTK (AK9, RT aa 158 to 166), and HLA-A*0301-restricted MTKILEPFR (MR9, RT aa 164 to 172).

Alleles HLA-A*03 and HLA-B*0702 are in strong linkage disequilibrium (8, 17). The original study by Moore et al. (31) did not detect an association between HLA-A*0301 and E169D independent of HLA-B*0702. In our patient group, all of the HLA-B*0702+ patients were also HLA-A*03+, so we could not dissect this finding with respect to HLA-A*03. Notwithstanding the lack of a statistical association between E169D and HLA-A*03, we investigated the effect of this variant on the CTL recognition of the HLA-A*03-restricted epitope MTKILEPFR (MR9).

E169D mutation abolishes recognition of the HLA-A*0301-restricted RT epitope MR9 (aa 164 to 172). We compared recognition of peptides MTKILEPFR and MTKILDPFR in IFN-{gamma} ELISpot assays directly ex vivo using PBMCs from an HLA-A*0301-positive patient. In contrast to the wild-type MTKILEPFR peptide, the MTKILDPFR variant did not activate CTL (Fig. 2), indicating that the variant MTKILDPFR is unlikely to be recognized in HLA-A*0301 positive individuals infected with HIV-1. We also tested the relative binding affinities of these two peptides to HLA-A*0301, using a competitive binding assay (20), and found a 44% reduction in the binding of the variant peptide MTKILDPFR in comparison with wild-type peptide MTKILEPFR (the E169D polymorphism is underlined).


Figure 2
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  		FIG. 2. MTKILDPFR variant evades recognition by CTL specific for the MTKILEPFR epitope. The antigenicity of the peptide MTKILEPFR and its variant MTKILDPFR were tested at different peptide concentrations using PBMCs from an HLA-A*0301-positive patient (50,000 PBMCs per well) directly ex vivo in a 16-h IFN-{gamma} ELISpot assay. The assay was performed in duplicate; data show means ± standard errors of the means.

Construction of recombinant pathogenic HIV-1 clones bearing RT 169E and 169D. The E169D change maps close to the HLA-B*0702-restricted epitope SPAIFQSSM, and we wanted to test the possibility that E169D might interfere with the processing of this epitope. We have shown previously that a single amino acid change can interfere with CTL recognition of epitopes restricted by two distinct HLA alleles (29). To investigate whether E169D could also interfere with the generation of the HLA-B*0702 peptide, we first screened 130 B-clade RT sequences from HLA-typed patients. Of these, 123 had E (wild type) and 7 had D (mutant) at position 169. We then generated RT fragments (aa 2 to 261) which were inserted into an HIV-1 backbone to yield recombinant HIV-1 molecular clones HIV-1 (169E) (wild type) and HIV-1 (169D) (mutant), as described in Materials and Methods.

Both viruses were pathogenic, as demonstrated by the formation of syncytia in infected SupT1 cell line by day 7 after infection (Fig. 3a to c). When Jurkat E6-1 cells were infected with an identical titer (multiplicity of infection [MOI] = 10–5) of wild-type or mutant viruses, both recombinant forms expressed comparable levels of p24 Gag and down-regulated the surface expression of CD4 on Jurkat cells (Fig. 3d to f).


Figure 3
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  		FIG. 3. Both wild-type and mutant recombinant HIV form syncytia in SupT1 cells and down-regulate CD4 expression on infected Jurkat cells. SupT1 cells (a to c) or Jurkat E6-1 cells (d to f) were infected with either wild-type (E6-1.6.0) or mutant (E6-1.6.1) recombinant HIV. On day 5 of culture, the cells were investigated under the microscope for syncytium formation and stained for surface CD4 and intracellular HIV-1 p24. (a and d) Uninfected cells; (b and e) cells infected with wild-type virus; (c and f) cells infected with mutant recombinant virus. Down-regulation of the CD4 molecule as a result of HIV infection can be seen. The percentages represent the proportions of infected (p24 positive) cells.

To optimize the time course and viral titer for infection, the recombinant wild-type HIV-1 (169E) virus was used to infect Jurkat E6-1 cells (which are HLA-A*0301 and HLA-B*0702 positive) at an MOI of 1.6 x 10–3. The infected and uninfected cells were sampled on alternate days and stained for intracellular p24 Gag. The proportion of p24 Gag-positive infected cells increased from 4.6% on day 1 to 50.2% on day 5; the proportion of p24 Gag-positive cells on day 5 also increased with the titer of infecting virus from 2.3% at an MOI of 10–5 to 58.5% at an MOI of 3.2 x 10–3 (data not shown).

The successful presentation of HLA class I-restricted HIV-1 epitopes on the surface of the Jurkat cells infected with the wild-type HIV-1 construct was confirmed by IFN-{gamma} ELISpot. We used CTL specific for three epitopes: HLA-A*0301-restricted p17 Gag RLRPGGKKK (RK9), HLA-B*0702-restricted RT SPAIFQSSM (SM9), and HLA-A*3301-restricted ELKKIIGQVR (ER9), as a negative control. Jurkat cell clone E6-1 does not express HLA-A*3301, and the CTL response to the ER9 epitope was absent, as expected (Fig. 4). CTL recognition of the RK9 and SM9 epitopes was readily detected 5 days after infection of the target cells and remained high (Fig. 4).


Figure 4
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  		FIG. 4. Infected Jurkat E6-1 cells are recognized by CTL after day 5 of infection. Jurkat E6-1 cells infected with wild-type virus were used as targets in 16-h IFN-{gamma} ELISpot assays after the indicated number of days postinfection; each assay contained 250,000 infected cells and 2,500 CTL. The HLA-A*3301 ER10-specific CTL line represents the negative control, as Jurkat E6-1 cells do not express HLA-A*3301. RK9, p17 Gag RLRPGGKKK (HLA-A*0301 restricted); SM9, RT SPAIFQSSM (HLA-B*0702 restricted); ER10, integrase ELKKIIGQVR (HLA-A*3301 restricted).

EI69D mutation abolishes the CTL recognition of the neighboring HLA-B*0702-restricted RT epitope SM9 (aa 156 to 164). To investigate whether E169D influenced the CTL recognition of SM9, we infected Jurkat E6-1 cells with either wild-type (169E) or mutant (169D) virus and used them as targets for the three CTL lines in ELISpot assays on day 5 of infection. Both mutant and wild-type targets were recognized equally well by the p17 Gag RLRPGGKKK-specific CTL line (P > 0.5, Fisher's exact test), and neither was recognized by the HLA-A*3301-restricted ELKKIIGQVR-specific CTL line (negative control). There was strong CTL recognition of the HLA-B*0702-restricted SPAIFQSSM epitope in the wild-type virus-infected cells, which was absent when tested against mutant HIV-1-infected target cells (P = 9.3 x 10–7, Fisher's exact test) (Fig. 5).


Figure 5
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  		FIG. 5. Flanking mutation E169D specifically abolishes recognition of the RT epitope SM9 (RT aa 156 to 164). Jurkat E6-1 cells, uninfected or infected with wild-type or mutant recombinant viruses (day 5), were used in IFN-{gamma} ELISpot assays with CTL lines specific for three epitopes. RK9, HLA-A*0301-restricted p17 Gag RLRPGGKKK (positive control); ER10, HLA-A*3301-restricted integrase ELKKIIGQVR (negative control); SM9, HLA-B*0702-restricted RT SPAIFQSSM. Each assay was carried out using 100,000 targets and 1,000 CTL per well.

E169D impairs the generation of the SM9 epitope by the proteasome. These results show that D at position 169, five residues downstream from the SPAIFQSSM epitope (aa 156 to 164), specifically interferes with SM9-specific CTL recognition. In line with our previous study (30), we hoped to determine whether the SM9 epitope is proteasome dependent, but the SM9-specific T-cell clone did not survive long enough to complete these experiments. Instead, we performed in vitro assays of proteasome processing. We investigated the effect of the E169D substitution on digestion of oligopeptides corresponding to a part of the HIV-1 RT protein, similar to our earlier study (30).

Two 27-mer synthetic oligopeptides corresponding to the aa residues 151 to 177 of the wild-type (QGWKGSPAIFQSSMTKILEPFRKQNPD) and mutant (QGWKGSPAIFQSSMTKILDPFRKQNPD) RT protein were digested in vitro using the constitutive and immuno 20S proteasome (see Materials and Methods). (The E169D polymorphism is underlined.) Aliquots were taken at 0, 4, 6, 8, 12, 18, 24, and 48 h of digestion, and products were analyzed using mass spectrometry. The digestion with the constitutive 20S proteasome resulted in very few fragments whereas the 20S immunoproteasome produced a wide array of digestion products. A similar observation has been reported previously for HIV-1 RT (44). Lactacystin, an inhibitor of the 20S proteasome, completely blocked the substrate-specific proteasomal activity.

The products of the in vitro cleavage of the wild-type and mutant oligopeptides and the position of the three epitopes in this region of RT are illustrated in Fig. 6. Digestion of the wild-type (169E) oligopeptide within 6 h released an intermediate peptide QGWKGSPAIFQSSM, which has the appropriate carboxyl terminus (M) for the HLA-B*0702-restricted epitope SM9. Importantly, this fragment was absent following the digestion of the mutant oligopeptide (169D) even after 48 h, by which time nonspecific proteasomal degradation of the oligomers starts to occur. The correct carboxyl termini for the HLA-A*0301-restricted AIFQSSMTK and MTKILEPFR epitopes were detected after 6 h of digestion of both the wild-type and mutant synthetic oligopeptides (Fig. 6).


Figure 6
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  		FIG. 6. In vitro proteasome digestion of RT peptides. (a) Products detected after 6 h of in vitro 20S immunoproteasome digestion of 27-mer oligopeptides corresponding to wild-type (169E) and mutant (169D) RT sequence. The correct precursor for the SPAIFQSSM (SM9) epitope is shown in boldface type; this fragment was absent when the mutant 169D sequence was digested. Sequences in blue represent digestion products detected only with the mutant oligomer. The fragments shown were identified in triplicate in two independent experiments. (b) A diagram showing the three RT epitopes: red, HLA-B*0702-restricted SPAIFQSSM (SM9, RT aa 156 to 164); green, HLA-A*0301-restricted AIFQSSMTK (AK9, RT aa 158 to 166); yellow, HLA-A*0301-restricted MTKILEPFR (ER9, RT aa 164 to 172). Arrows indicate digestion products detected after 24 h of incubation with the 20S immunoproteasome. Colored arrows correspond to the correct carboxyl termini for the appropriate epitopes. The correct restriction site for epitope SM9 is marked with a red arrow. The variant position E169D is shown within the blue box.

These results provide an explanation for the lack of recognition of the mutant recombinant HIV-1 virus by SPAIFQSSM-specific CTL (Fig. 5). They strongly suggest that the E169D mutation abolishes CTL recognition of SM9 by blocking the correct proteasomal cleavage.


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DISCUSSION
 
CTL escape due to intraepitope amino acid alteration can occur through various mechanisms. It can affect peptide binding to the HLA class I or recognition of the peptide/HLA class I complex by the T-cell receptor. Variation within a CTL epitope can also affect antigen processing (12, 34, 49). Studies which evaluated the effect of artificially altered epitope-flanking residues on antigen processing predicted that naturally occurring flanking variants could also interfere with epitope presentation. Recent reports have confirmed this prediction (3, 9, 30, 42).

Processing mutants can be detected by searching for evidence that amino acid polymorphisms in the vicinity of a known epitope are positively selected during an infection. Our group has detected this phenomenon in one case (30). In this study, we show that escape mutants can also be predicted from statistical surveys which associate HLA class I molecules and amino acid polymorphisms adjacent to CTL epitopes (21, 31, 50).

In HIV-1 RT, HLA-B*0702 was found to be strongly associated with E169D (31). There have been no reports to indicate that the E169D variant is associated with antiretroviral drug treatment. Although no HLA-B*0702-restricted epitope maps to this polymorphism, position 169 lies 5 amino acids downstream of an HLA-B*0702-restricted epitope, SM9 (aa 156 to 164). Position 169 also maps within an overlapping HLA-A*0301-restricted epitope, MR9 (aa 164 to 172). Despite the strong linkage disequilibrium between HLA-A*0301 and B*0702 and the high frequency of HLA A*03 in the population (8, 17), statistical association between E169D and HLA-A*0301, independent of HLA-B*0702, has not been found.

Using ELISpot IFN-{gamma} assay, we showed that E169D confers escape from pressure mediated through HLA-A*0301-restricted MR9-specific CTL. The statistical association with HLA-B*0702 suggested that the same polymorphism might also confer viral escape in HLA-B*0702+ individuals infected with HIV. In HLA-B*0702+ patients, the E169D mutation could allow viral escape in one of two ways: position 169 might lie within an as yet unidentified HLA-B*0702-restricted epitope or, alternatively, E169D might confer escape from SM9-mediated CTL pressure by interfering with antigen processing.

Previous studies on HIV-1 antigen processing have utilized systems in which HIV-1 antigens were expressed in BCLs by recombinant vaccinia virus (30) or HIV-1 mRNA inserts (1, 9). We have established a novel HIV-1 CD4+ target assay to investigate the effect of E169D on the processing of the SM9 epitope. We reconstituted HIV-1 with an RT sequence derived from a patient. This clone bore the polymorphism under scrutiny in its natural context, which would ensure viral viability (11, 14, 19, 37). We infected CD4+ Jurkat E6-1 cells, a natural HIV-1 host, allowing HIV-1 protein to be expressed and processed in a more physiological manner. Initial in vitro studies showed both recombinant viruses, HIV-1 (169E) and HIV-1 (169D), to be equally viable and infectious.

When the HIV recombinant virus-infected CD4+ cells were presented to SM9-specific CTLs, the targets expressing HIV-1 (169E) were recognized significantly better than those expressing the HIV-1 (169D) mutant virus (P = 6.7 x 10–10). Yet there was no disparity in recognition by a control line specific for the p17 Gag epitope RK9 which is isogenic in both 169E and 169D HIV constructs (P < 0.51). This suggested that epitope SM9 was not being presented at the surface of the Jurkat E6-1 cell. We investigated whether E169D mutation might inhibit proteasome cleavage of the SM9 epitope and subjected 27-mer synthetic polypeptides with either E or D at position 169 to in vitro digestion by the 20S immunoproteasome. Digestion of the 169E polypeptide liberated the correct carboxyl terminals for the HLA-B*0702-restricted epitope SM9, as well as HLA-A*0301-restricted epitopes MR9 and AK9. However, digestion of the 169D polypeptide liberated the appropriate fragments for the HLA-A*0301-restricted epitopes only; the potential precursor of the SM9 epitope was extended at the carboxyl end.

This study shows that statistical approaches can be a way of identifying antigen-processing escape mutants. A single amino acid mutation, E169D, can confer escape from dual attack by CTL, governed by either HLA-A*0301 and B*0702, though by different mechanisms. However, it also highlights how difficult it would be to predict the effect of HIV variation on CTL recognition on purely statistical evidence, without a detailed functional analysis of individual variants.


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ACKNOWLEDGMENTS
 
This work was funded by the Wellcome Trust, United Kingdom.

We thank Andy Sewell for help and support during the preparation of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: The James Martin 21st Century School at The Peter Medawar Building for Pathogen Research, Nuffield Department of Clinical Medicine, University of Oxford, South Parks Road, Oxford OX1 3SY, United Kingdom. Phone: 44 794 1969 255. Fax: 44 1865 617028. E-mail: anita.milicic{at}clinpharm.ox.ac.uk. Back

{triangledown} Published ahead of print on 15 November 2006. Back

{dagger} P.Z. and A.M. contributed equally to this study. Back


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Journal of Virology, February 2007, p. 2031-2038, Vol. 81, No. 4
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