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

0022-538X/07/$08.00

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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

䌤

Peter Zimbwa,

† Anita Milicic,

†* John Frater,

Thomas J. Scriba,

Antony Willis,

Philip J. R. Goulder,

Tilly Pillay,

Huldrych Gunthard,

Jonathan N. Weber,

Hua-Tang Zhang,

and Rodney E. Phillips

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

; Department of

Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom

; Department of Pediatrics,

University of Oxford, Oxford OX3 9DU, United Kingdom

; University Hospital Zurich, Department of Medicine,

Division of Infectious Diseases and Hospital Epidemiology, Ramistrasse 100, CH-8091 Zurich,

Switzerland

; and Jefferiss Research Laboratories, Wright-Fleming Institute, Imperial College,

St Mary’s Hospital, Norfolk Place, London W2 1PG, United Kingdom

Received 11 May 2006/Accepted 9 November 2006

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-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.

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

contain-ment of human immunodeficiency virus type 1 (HIV-1)

repli-cation (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

en-ables 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

pep-tide 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

gener-ation can be inhibited when proteolytic activity is redirected to

novel sites within the variant protein (34). Biochemical

de-scriptions of this phenomenon are emerging, but the

signifi-cance 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

associ-ation 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

polymor-phisms 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 SPAIF

QSSM (SM9) and HLA-B*0702 (31). We demonstrate that

* Corresponding author. Mailing address: The James Martin 21st

Century School at The Peter Medawar Building for Pathogen

Re-search, Nuffield Department of Clinical Medicine, University of

Ox-ford, South Parks Road, Oxford OX1 3SY, United Kingdom. Phone:

44 794 1969 255. Fax: 44 1865 617028. E-mail: anita.milicic@clinpharm

.ox.ac.uk.

† P.Z. and A.M. contributed equally to this study.

Published ahead of print on 15 November 2006.

2031

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to 172); however, no statistical association has been found

between E169D and HLA-A*03 (31).

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 centrifuga-tion. 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 iden-tified 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␮g/ml streptomycin, 2 mML-glutamine, 10% fetal calf

serum) (28). Cyclosporine was then added at 0.5␮g/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 previ-ously (23). Briefly, around 3 million PBMCs were resuspended in 2 ml of R-10

medium (RPMI, 100 U/ml penicillin, 100␮g/ml streptomycin, 2 mML-glutamine,

10% fetal calf serum) containing 10␮M 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 subse-quently grown in the same medium (and restimulated using irradiated mixed

heterologous PBMCs and 4␮g/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-␥

)-produc-ing cells anti-IFN-␥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, un-published data).

CD4ⴙT-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-␥, as

de-scribed 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␮M 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 auto-mated and standardized using an ELISpot plate reader (software version 3.2.3; Autoimmun Diagnostika, Germany).

(Sigma-Aldrich) was added at 10␮g/ml to prevent egress of IFN-␥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-␥(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 per-formed 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␮M 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

␮l 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 man-ufacturer’s instructions (Invitrogen). The RT insert and TOPO TA vector were

extracted from theEscherichia colicells 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 instruc-tions and used to generate recombinant viruses with the RT-deleted

HXB2-based proviral molecular clone pHXB2⌬2-261RT. The amount of product was

determined by spectrophotometry. One microgram of the amplified RT was

mixed with 1␮g of SmaI-linearized plasmid pHXB2⌬2-261RT in 5⫻106

SupT1

cells in 250␮l 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␮F. 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⫻106_{) cells were pelleted in}

15-ml conical tubes at 1,000⫻gfor 5 min and then resuspended in 10 ml R-10

containing 2␮g/ml hexadimethrine bromide (Polybrene; Sigma-Aldich). Cells

were then centrifuged at 1,000⫻gfor 5 min before resuspension in 1 ml R-10

with 2␮g/ml Polybrene-containing recombinant virus stock. Aliquots of culture

supernatant (250␮l) 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 (QGWKGSPAIFQSSMTKILEPFRKQ

NPD) or mutant (QGWKGSPAIFQSSMTKILDPFRKQNPD) RT peptide (5

␮g) was added to 300␮l buffer (20 mM HEPES-KOH, pH 7.8, 2 mM MgAc2,2

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mM dithiothreitol [Sigma]). (The SM9 epitope is underlined.) The immuno or

constitutive 20S proteasome (Immatics) was then added at 2␮g 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).

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

poly-morphism 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

Hospi-tal, 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-HLA-B*0702

patients

had E169D compared to 4 of 106 (3.8%) HLA-B*0702

pa-tients (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 MT

KILEPFR (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, SP

AIFQSSM (aa 156 to 164), maps five residues upstream of the

E169D polymorphism. Another HLA-A*03 epitope, AIFQS

SMTK (aa 158 to 166), also maps upstream of the E169D

poly-morphism (Fig. 1).

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.

Not-withstanding the lack of a statistical association between

E169D and HLA-A*03, we investigated the effect of this

vari-ant 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 MTKIL

E

PFR and MTKIL

D

PFR in

IFN-

␥

ELISpot assays directly ex vivo using PBMCs from an

HLA-A*0301-positive patient. In contrast to the wild-type MT

KIL

E

PFR peptide, the MTKIL

D

PFR variant did not activate

CTL (Fig. 2), indicating that the variant MTKIL

D

PFR is

un-likely to be recognized in HLA-A*0301 positive individuals

infected with HIV-1. We also tested the relative binding

affin-ities of these two peptides to HLA-A*0301, using a

competi-tive binding assay (20), and found a 44% reduction in the

binding of the variant peptide MTKIL

D

PFR in comparison

with wild-type peptide MTKIL

E

PFR (the E169D

polymor-phism is underlined).

Construction of recombinant pathogenic HIV-1 clones

bear-ing 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

inves-tigate 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

for-mation 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

)

of wild-type or mutant viruses, both recombinant forms

ex-pressed comparable levels of p24 Gag and down-regulated the

surface expression of CD4 on Jurkat cells (Fig. 3d to f).

To optimize the time course and viral titer for infection, the

recombinant wild-type HIV-1 (169E) virus was used to infect

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).

FIG. 2. MTKILDPFR variant evades recognition by CTL specific

for the MTKILEPFR epitope. The antigenicity of the peptide MTKI

LEPFR and its variant MTKIL

D

PFR 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-

␥

ELISpot

assay. The assay was performed in duplicate; data show means

⫾

standard errors of the means.

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Jurkat E6-1 cells (which are HLA-A*0301 and HLA-B*0702

positive) at an MOI of 1.6

⫻

10

. The infected and uninfected

cells were sampled on alternate days and stained for

intracel-lular 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

to 58.5% at an MOI of 3.2

⫻

10

(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-

␥

ELISpot.

We used CTL specific for three epitopes:

HLA-A*0301-re-stricted p17 Gag RLRPGGKKK (RK9),

HLA-B*0702-re-stricted RT SPAIFQSSM (SM9), and HLA-A*3301-reHLA-B*0702-re-stricted

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).

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.

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EI69D mutation abolishes the CTL recognition of the

neigh-boring HLA-B*0702-restricted RT epitope SM9 (aa 156 to

164).

To investigate whether E169D influenced the CTL

rec-ognition 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

⫻

10

,

Fisher’s exact test) (Fig. 5).

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

com-plete these experiments. Instead, we performed in vitro assays

of proteasome processing. We investigated the effect of the

E169D substitution on digestion of oligopeptides

correspond-ing 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 (QGWKGSPAIFQSSMT

KILEPFRKQNPD) and mutant (QGWKGSPAIFQSSMTKIL

DPFRKQNPD) 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

diges-tion 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

sub-strate-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

interme-diate peptide QGWKGSPAIFQSSM, which has the

appropri-ate 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).

These results provide an explanation for the lack of

recog-nition of the mutant recombinant HIV-1 virus by SPAIFQSS

M-specific CTL (Fig. 5). They strongly suggest that the E169D

mutation abolishes CTL recognition of SM9 by blocking the

correct proteasomal cleavage.

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-

␥

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 ELKKII

GQVR (HLA-A*3301 restricted).

FIG. 5. Flanking mutation E169D specifically abolishes recognition

of the RT epitope SM9 (RT aa 156 to 164). Jurkat E6-1 cells,

unin-fected or inunin-fected with wild-type or mutant recombinant viruses (day

5), were used in IFN-

␥

ELISpot assays with CTL lines specific for three

epitopes. RK9, HLA-A*0301-restricted p17 Gag RLRPGGKKK

(pos-itive control); ER10, HLA-A*3301-restricted integrase ELKKII

GQVR (negative control); SM9, HLA-B*0702-restricted RT SPAIFQ

SSM. Each assay was carried out using 100,000 targets and 1,000 CTL

per well.

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DISCUSSION

CTL escape due to intraepitope amino acid alteration can

occur through various mechanisms. It can affect peptide

bind-ing 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-flank-ing residues on antigen processepitope-flank-ing 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

evi-dence 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

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.

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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

asso-ciated 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

down-stream 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-

␥

assay, we showed that E169D confers

escape from pressure mediated through

HLA-A*0301-re-stricted 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

pro-cessing.

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

scru-tiny 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

pro-cessed 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

express-ing the HIV-1 (169D) mutant virus (

P

⫽

6.7

⫻

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

immunoprotea-some. 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.

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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