Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function

Replication and Nef Function

Aniqa Shahid,aAlex Olvera,bGursev Anmole,cXiaomei T. Kuang,cLaura A. Cotton,aMontserrat Plana,dChristian Brander,b,e,f Mark A. Brockman,a,c,gZabrina L. Brummea,g

Faculty of Health Sciences, Simon Fraser University, Burnaby, Canadaa

; IrsiCaixa AIDS Research Institute, HIVACAT, UAB, Barcelona, Spainb

; Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, Canadac

; AIDS Research Group, IDIBAPS, HIVACAT, Hospital Clinic, UAB, Barcelona, Spaind

; Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spaine

; University of Vic and Central University of Catalonia, Vic, Barcelona, Spainf

; British Columbia Centre for Excellence in HIV/ AIDS, Vancouver, Canadag

ABSTRACT

HLA-B*13 is associated with superior

in vivo

HIV-1 viremia control. Protection is thought to be mediated by sustained targeting

of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may

con-tribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant

combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression.

Muta-tions were engineered into HIV-1

, and replication capacity was measured using a green fluorescent protein (GFP) reporter T

cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected

target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only

Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The

Gag-I437L-medi-ated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8

resi-dues and terminating at Gag

, was identified in p24

(GQMVHQAI

). No other single or combination Gag, Pol, or

Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef

dou-ble mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover,

target cells infected with HIV-1-Nef

were recognized better by HIV-specific T cells than those infected with HIV-1

or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these

ef-fects may contribute to long-term HIV-1 control by HLA-B*13.

IMPORTANCE

Protective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in

conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of

B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We

also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal

anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven

substitutions E24Q and Q107R in Nef, when present together, substantially impair this protein’s ability to downregulate HLA

class I. This, in turn, increases the visibility of infected cells to HIV-specific T cells. Our results suggest that B*13-associated

es-cape mutations impair HIV-1 replication by two distinct mechanisms, that is, by reducing Gag fitness and dampening Nef

im-mune evasion function.

T

he natural history of HIV-1 is influenced by human leukocyte

antigen (HLA) class I polymorphisms (

1–4

). Notably,

HLA-B*57 and B*27 are associated with viral load control and slower

disease progression (

5

,

6

), but other “protective” HLA class I

al-leles have also been identified (

4

,

7

,

8

). HLA-B*13 is one such

allele, but it remains understudied in part because it is relatively

rare in many populations (expressed in less than 4% of Caucasians

[

9

] and Africans [

10

,

11

] although its frequency exceeds 10% in

some Asian populations [

12–14

]). HLA-B*13, of which the

most common subtype is B*13:02 (

15

), is associated with lower

viral loads in chronic infection (

10

,

11

,

14

,

16

,

17

) and lower

hazard ratios of progression to AIDS in historic natural history

studies (

18

). HLA-B*13 is also enriched among HIV

control-lers (

6

,

19

,

20

).

B*13-mediated protective effects are attributed, at least

par-tially, to sustained targeting of conserved B*13-restricted

cyto-toxic T lymphocyte (CTL) epitopes, particularly in HIV-1 Gag

(

11

) and Nef (

21

). Optimally described B*13 epitopes have also

been identified in Pol (

22

) although these responses appear to

contribute less to control (

11

). Similar to the effects of B*57 (

23–

Received3 August 2015 Accepted31 August 2015

Accepted manuscript posted online9 September 2015

CitationShahid A, Olvera A, Anmole G, Kuang XT, Cotton LA, Plana M, Brander C, Brockman MA, Brumme ZL. 2015. Consequences of HLA-B*13-associated escape mutations on HIV-1 replication and Nef function. J Virol 89:11557–11571. doi:10.1128/JVI.01955-15.

Editor:F. Kirchhoff

Address correspondence to Zabrina L. Brumme, [email protected].

G.A. and X.T.K. contributed equally to this article.

Supplemental material for this article may be found athttp://dx.doi.org/10.1128 /JVI.01955-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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25

) and B*27 (

26

), the protective effects of B*13 may also be

me-diated to some extent by costs to viral replicative fitness incurred

as a result of immune escape within certain CTL epitopes, notably

in Gag (

27

). These replicative costs may limit the net viral

“bene-fit” gained via immune evasion, leading to relative viral control or

reduced pathogenesis. In particular, selection of a Gag-I437L or

-I437M escape mutation at the C-terminal anchor residue of the

B*13-restricted p1

RI9 epitope (the 9-residue Gag sequence at

position 429 to 437, RQANFLGKI

) substantially impaired

in vitro

HIV-1 replicative capacity (

27

,

28

). Likewise, the

B*13-driven Gag-I147L polymorphism, located four codons

down-stream of the p24

VV9 epitope (VQNLQGQMV

135–143

) confers

minor replication defects (

28

).

Though fitness-reducing CTL escape mutations in HIV-1

commonly occur in Gag (

28

,

29

), escape in Pol can also incur

replicative costs (

30

,

31

). In addition, naturally occurring

se-quence variation in Nef (

32–34

), including HLA-driven escape

mutations (

35

,

36

), can modulate this protein’s major functions,

including cell surface CD4 (

37

) and HLA class I (

38

)

downregu-lation. However, the functional costs of B*13-driven immune

es-cape mutations outside Gag and their possible contributions to

B*13-mediated protective effects have not been investigated.

Moreover, the extent to which secondary (compensatory)

poly-morphisms can restore fitness costs of primary B*13-driven

es-cape mutations also remains incompletely known. Such effects,

however, are supported by observations of reduced replication

capacity of patient-derived B*13 Gag sequences in early, but not

chronic, infection (

25

).

To further elucidate the replicative and functional

conse-quences of B*13-driven HIV-1 evolution, we investigated the 10

polymorphisms in Gag, Pol, and Nef that are most commonly

selected in B*13-expressing individuals (

39

). Specifically, we

tested these polymorphisms alone and in biologically observed

combinations for effects on

in vitro

HIV-1 replication capacity

and Nef-mediated CD4 and HLA class I downregulation

activ-ity. Our results extend current knowledge of B*13-driven Gag

replicative costs (

27

,

28

) by identifying Gag-K436R as a

com-pensatory mutation for Gag-I437L. We also provide evidence

for a novel B*13-restricted CTL epitope terminating at Gag

codon 147, B*13’s most frequent escape site in HIV-1 (

39

).

Notably, although B*13-associated substitutions in Pol and

Nef did not impair viral replication, the B*13-associated Nef

poly-morphisms E24Q-Q107R, when expressed together, reduced

Nef’s ability to downregulate HLA class I from the infected cell

surface. This, in turn, enhanced the recognition of infected cells by

HIV-1-specific T cells

in vitro

. Though this mutation combination

is observed in less than 5% of B*13-expressing persons, it

never-theless represents a novel consequence of CTL escape in HIV-1.

Specifically, our results suggest that B*13-mediated immune

con-trol could also be attributable, at least in part, to CTL

escape-driven attenuation of Nef’s immune evasion function.

MATERIALS AND METHODS

Definition of HLA-B*13-associated polymorphisms. Ten published HLA-B*13 and/or B*13:02-associated polymorphisms in HIV-1 Gag (n⫽

4), Pol (n⫽4), and Nef (n⫽2), originally identified via statistical asso-ciation with phylogenetic correction in the International HIV Adaptation Collaborative (IHAC) cohort comprising ⬎1,800 antiretroviral-naive chronically HIV-1 subtype B-infected individuals (39), were selected for analysis (Table 1andFig. 1A; see also Fig. S1 in the supplemental

mate-rial). These polymorphisms represent all B*13/B*13:02-associated sites in Gag, Pol, and Nef for which a specific “adapted” (CTL escape form) amino acid was identified at aPvalue of⬍0.0001 andqvalue of⬍0.05 in the original study (39). At two of these codons (protease 63 and reverse trans-criptase [RT] 369), the original study identified two possible adapted forms; the one with the lowerPvalue was selected for the present analysis. The extent to which these polymorphisms are enriched in B*13:02-posi-tive (B*13:02⫹) versus B*13:02-negative (B*13:02⫺) individuals in the original study (39), along with their locations respective to published or bioinformatically predicted B*13-restricted CTL epitopes, is shown in Fig. S1 in the supplemental material.

Characterization of escape rates and frequencies in B*13ⴙ individ-uals.The frequencies and kinetics of selection of B*13-associated poly-morphisms in acute/early HIV-1 subtype B infection were defined using longitudinal plasma HIV-1 RNA sequences from nine B*13:02⫹ serocon-verters from cohorts in Berlin (Jessen-Praxis Medical Clinic, Berlin, Ger-many), New York (Aaron Diamond AIDS Research Center, New York, NY, USA), Boston (Massachusetts General Hospital, Boston, MA, USA), and Vancouver (British Columbia Centre for Excellence in HIV/AIDS; Vanguard and Vancouver Injection Drug Users Study [VIDUS], Canada) (40,41). All patients provided written informed consent, and the cohort studies were approved by their respective institutional review boards. For four patients, HIV-1 sequences were already published (41). For others, HIV-1 RNA was extracted from plasma, and Gag, Pol, and Nef were am-plified in independent nested reverse transcription-PCRs (RT-PCRs) us-ing HIV-1-specific primers. Amplicons were bidirectionally sequenced on a 3130xl automated DNA sequencer (Applied Biosystems, Inc.), and chro-matograms were analyzed using Sequencher, version 5.0 (Genecodes).

TABLE 1Single and combination mutations in HIV-1 Gag, Pol, and Nef evaluated in this study

Protein and

mutation type Mutation(s)

Mutant namea

Gag

Single A146S

S---I147L

-L--K436R

--R-I437L ---L

Combination I147L-I437L -L-L

A146S-I147L SL--I147L-K436R -LR-I147L-K436R-I437L -LRL A146S-I147L-K436R SLR-K436R-I437L --RL A146S-I147L-K436R-I437L SLRL A146S-I147L-I437L SL-L Pol

Single L63S (protease)

S---Q334N (RT)

-N--T369A (RT)

--A-K374R (RT) ---R

Combination L63S-T369A-K374R S-AR

T369A-K374R --AR

L63S-T369A

S-A-L63S-K374R S--R

Nef

Single E24Q

Q-Q107R -R

Combination E24Q-Q107R QR

a

Mutants are named according to the scheme shown at the bottom ofFig. 1BtoD. Combination mutations are listed in descending order based on their prevalencesin vivo.

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FIG 1HLA-B*13-associated polymorphisms in HIV-1 Gag, Pol, and Nef. (A) Locations of 10 published HLA-B*13-associated polymorphisms in HIV-1 subtype B Gag, Pol, and Nef (HXB2 codon numbering) (39). (B to D) Top panels show the rates of escape of these polymorphisms, calculated from longitudinal plasma HIV-1 RNA sequences from nine B*13:02 seroconverters using Kaplan-Meier methods. Note that curves for Gag-A146S-K436R and Pol-L63S-T369A are superimposed. Prevalences of these polymorphisms in B*13:02⫹individuals in early (1 year postinfection;n⫽9) and chronic (n⫽69) infection are shown in the middle panels. Frequencies of these polymorphisms, alone and in combination, in 69 B*13:02⫹individuals with chronic infection are shown in the bottom panels. Polymorphisms are listed in decreasing frequency, with hyphens denoting the wild-type (nonescaped) form. Single and combination mutants indicated with a dot were engineered into NL4.3 and tested forin vitroviral RC. PR, protease.

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Sequences were aligned to the HIV-1 subtype B reference strain HXB2 (GenBank accession numberK03455) using an algorithm based on the HyPhy platform (42). Time from estimated infection date to the first detection of the specific B*13-associated polymorphism was determined using Kaplan-Meier methods; these data were also used to calculate escape prevalence after 1 year of infection. The prevalence of these mutations in chronic infection was calculated using published plasma HIV-1 RNA se-quences from 69 B*13:02⫹individuals in the IHAC cohort (39). Note that cryopreserved peripheral blood mononuclear cells (PBMCs) were not available from these patients. Patient HIV and HLA data are available upon request in accordance with the Providence Health Care/University of British Columbia Institutional Review Board protocols.

Generation of mutant HIV-1.B*13-associated polymorphisms were engineered into the HIV-1 subtype B reference strain NL4.3 (NIH AIDS Research and Reference Reagent Program, catalog 114; contributed by Malcolm Martin [43]). First, pNL4.3gag,pol(covering protease and the first 400 codons of reverse transcriptase), andnefwere subcloned into pCR2.1-TOPO (Life Technologies). Individual mutations were intro-duced into these plasmids using a QuikChange II XL site-directed mu-tagenesis kit (Stratagene). Combination mutants were generated by suc-cessive rounds of mutagenesis. All mutants were confirmed by DNA sequencing. Mutant subcloned genes were then used to generate recom-binant NL4.3 viruses by homologous recombination (25). Briefly, mutant

gag,pol, andnefwere reamplified from subclones using 100-mer primers complementary to NL4.3. After verification on an agarose gel, each am-plicon was cotransfected, along with the relevant linearized pNL4.3 vector (⌬gag,⌬pol, or⌬nefvector), into a Tat-inducible CEM-GFP-reporter (where GFP is green fluorescent protein) T cell line (GXR25) (44) via electroporation. Cells were incubated for 10 days at 37°C in R20⫹ me-dium (RPMI meme-dium with phenol red, supplemented with 20% [vol/vol] fetal bovine serum [FBS] and 1% [vol/vol] penicillin-streptomycin) to allow the generation and propagation of infectious virions. GFP ex-pression (indicating HIV-1 infection) of these cultures was monitored daily by flow cytometry (Guava easyCyte 8HT; Millipore); once this reached ⬎15%, supernatants containing infectious mutant virions were harvested, aliquoted, and frozen at⫺80°C. The median time to harvest was 13 days (range, 11 to 14 days). HIV-1 RNA from each virus stock was reextracted (Invitrogen PureLink Genomic DNA/RNA kit; Life Technologies) and reamplified, and the relevant gene was rese-quenced to confirm the presence of the desired substitution(s) and the absence of any others.

In vitroHIV-1 replication capacity assays.Viral titers were assessed as described previously by infecting GXR25 reporter cells in order to de-termine the volume necessary to achieve a multiplicity of infection (MOI) of 0.003 (0.3% HIV-infected cells) at 2 days after infection (25,31,45,46). For subsequent viral replication assays, GXR25 cells were infected with control (wild-type NL4.3 [wtNL4.3]) and mutant viruses at an MOI of 0.003, after which the percentage of GFP-positive (GFP⫹; HIV-infected) cells was monitored daily by flow cytometry until day 6. For each virus, the natural log of the slope of virus spread (measured as percent infected cells) was calculated during the exponential phase. This value was then normal-ized by the mean rate of spread of wtNL4.3 such that replication capacity (RC) values of⬍1.0 or⬎1.0 indicate a rate of viral spread lower than or higher than that of NL4.3, respectively. Each virus was assayed in a mini-mum of two independent experiments with each containing three repli-cates.

Characterization of a novel putative HLA-B*13:02-restricted epitope in p24 Gag.The presence of a novel B*13:02-restricted epitope overlapping Gag₁₄₆and Gag₁₄₇was investigated bioinformatically and experimentally. First, the HIV-1 consensus B amino acid sequence at Gag135–155was scanned for the presence of B*13:02-restricted 8- to

11-mer epitopes using NetMHCpan, version 2.8 (47,48) (http://www.cbs .dtu.dk/services/NetMHCpan/). This algorithm identifies candidate epitopes based on their predicted half-maximal inhibitory concentrations (IC50), where values of⬍500 nM, 500 to 5,000 nM, and⬎5,000 nM are

predictive of strong binders, weak binders, and nonbinders, respectively (47,48). After candidate epitopes were experimentally verified, binding predictions were repeated for variants containing B*13-associated poly-morphisms at Gag146and/or Gag147. PBMCs from seven B*13:02⫹

chron-ically HIV-1 subtype B-infected individuals recruited at the Hospital Clinic Barcelona (n⫽4) and at IrsiCaixa AIDS Research Institute, Bad-alona (n⫽3), Spain, collected with written informed consent, were screened for responses against C- and N-terminally extended versions of the predicted optimal epitope using a gamma interferon (IFN-␥) enzyme-linked immunospot (ELISpot) assay as described previously (31). The threshold for positive responses was defined as the highest of the following three criteria: a minimum of five spots per well, the mean number of spot-forming cells (SFC) of the negative-control wells plus three times its standard deviation, or three times the mean of negative-control well val-ues (49). Plasma samples were also available from three of the patients; these were subjected to HIV-1 RNA Gag sequencing.

Assessment of Nef-mediated CD4 and HLA class I downregulation in infected cells.Nef-mediated CD4 and HLA class I downregulation was assessed in a GXR25 cell line stably transduced to express HLA-A*02:01 (GXR-A*02). Briefly, one million GXR-A*02 cells were infected with Nef control and mutant viruses at an MOI of 0.01. Control viruses included wtNL4.3, NL4.3 with a deletion of Nef (⌬Nef), and NL4.3 with an M20A mutation in Nef (NefM20A) (the latter two kindly provided by Takamasa

Ueno, Kumamoto University), as well as an HIV-uninfected culture. The NefM20Avirus was used as a control since it is impaired for HLA class I

downregulation (50). The NL4.3-⌬Nef virus was pseudotyped with vesic-ular stomatitis virus G protein (VSV-G) to enhance infection in GXR-A*02 cells (51). Once HIV-1 infection reached⬇10 to 15%, 500,000 cells were stained with allophycocyanin (APC)-labeled anti-CD4 and phyco-erythrin (PE)-labeled anti-HLA-A*02 antibodies (BD Biosciences), and cell surface expression of these molecules was measured by flow cy-tometry. The level of Nef-mediated CD4 downregulation of each virus was expressed as the ratio of the median fluorescence intensity (MFI) of CD4 expression in GFP⫹(HIV-infected) cells to the MFI of CD4 in the uninfected cultures (the latter was used because CD4 downregula-tion precedes GFP expression in GXR25 cells, leading to lower MFIs in the GFP⫺gate in infected cultures). These values were then normal-ized to the value of the wtNL4.3 control. The calculation was as fol-lows: [(1⫺(MFI of GFP⫹_{nef mutant}/MFI of GFP⫺_uninfected))/(1⫺(MFI of GFP⫹wtNL4.3/MFI of GFP⫺uninfected))]⫻100. As such, normalized

values of⬍100% and⬎100% indicate CD4 downregulation functions lower than or higher than values for wtNL4.3, respectively. The same calculation was used to quantify Nef-mediated HLA-A*02 downregula-tion. Viruses were assessed in a minimum of three independent experi-ments.

Impact of Nef-mediated HLA class I downregulation on infected-cell recognition by HIV-1-specific effector T infected-cells.To determine the im-pact of Nef-mediated HLA-A*02 downregulation on the recognition of infected target cells by HIV-1-specific effector T cells, we employed a reporter T cell coculture assay. Briefly, 1 million GXR-A*02 target cells were infected with control (wtNL4.3-Nef,⌬Nef, or NefM20A) or Nef

mu-tant virus at an MOI of 0.01 in R20⫹medium without phenol red. Cul-tures were monitored daily by flow cytometry and used as “targets” when the proportion of GFP⫹(HIV-infected) cells reached⬇10 to 15%. HLA-A*02-restricted reporter “effector” T cells were generated using a modifi-cation of previously described methods (52,53). Briefly, Jurkat T cells were cotransfected with four expression plasmids: 3 ␮g each of pSELECTGFPZeo(Invivogen) containing T cell receptor␣(TCR␣) and

TCR␤genes isolated from a CTL clone specific for the A*02-restricted p24Gag_FK10Gag_{epitope (FLGKIWPSYK, Gag residues 433 to 442) (kindly}

provided by Mario Ostrowski and Brad Jones, University of Toronto); 5 ␮g of a CD8␣expression plasmid (Invivogen); and 10␮g of a luciferase reporter plasmid driven by nuclear factor of activated T cells (NFAT) (Panomics/Affymetrix). Reporter effector T cells were electroporated us-ing a Gene Pulser Xcell Electroporation System (Bio-Rad Laboratories)

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(square-wave protocol; 500 V, 2,000␮F, 3 ms, and 1 pulse), recovered at room temperature for 10 min, and incubated for 18 h in R10⫹ me-dium (RPMI 1640 meme-dium, 10% FBS, and 1% [vol/vol] penicillin-streptomycin) to allow the expression of TCR. A total of 50,000 HIV-1-infected target cells were cocultured with 50,000 effector cells (1:1 effector/target [E/T] ratio) for 6 h, and TCR-mediated signaling was detected as NFAT-driven luciferase expression using a Steady-Glo lu-ciferase system (Promega) and quantified using a Tecan Infinite M200 plate reader. Prior studies have indicated that this signal is dependent on endogenously processed HIV-1 Gag FK10Gag_{peptide antigen}

pre-sented on HLA-A*02.

Western blotting.Western blotting was performed as described pre-viously (36). GXR-A*02 cells were infected with control or mutant vi-ruses. When cultures reached⬇10 to 15% infection, 800,000 cells were pelleted and lysed. Due to the similar molecular masses (⬇27 kDa) of Nef and GFP (HIV-1 infection control), lysates were split in half, and two blots were processed in parallel (with Nef and the␤-actin housekeeping gene control on one and GFP on the other). Cell lysates were subjected to SDS-PAGE using Mini-Protean TGX 4% to 20% gels (Bio-Rad Laborato-ries), and proteins were electroblotted onto a polyvinylidene difluoride (PVDF) membrane (GE Healthcare). Nef was detected using a polyclonal rabbit antibody (NIH AIDS Research and Reference Reagent Program, catalog 2949; contributed by Ronald Swanstrom) (1:4,000) (54), followed by staining with secondary donkey anti-rabbit antibody (GE Healthcare) (1:30,000). Expression levels of␤-actin and GFP were assessed using pri-mary mouse anti-actin (Sigma) (1:20,000) and pripri-mary mouse anti-GFP (1:10,000), respectively, followed by secondary goat anti-mouse (Jackson ImmunoResearch) (1:20,000). Western blots were developed using Clar-ity Western ECL Substrate (Bio-Rad Laboratories), based on the chemi-luminescence detection method. Blots were developed with ImageQuant LAS 400 (GE Healthcare).

Statistical analyses.The one-samplettest was used to assess whether replication capacities and CD4/HLA-A*02 downregulation functions of mutant NL4.3 viruses differed significantly from those of the wtNL4.3 reference strain (whose function was set to 1.0 for RC or 100% for CD4/ HLA downregulation). A Bonferroni correction was used to adjust for multiple tests. Spearman’s correlation was used to assess the relationship between Nef-mediated HLA-A*02 downregulation in HIV-infected target cells and NFAT-mediated TCR signaling in HIV-specific effector T cells. Statistical analyses were performed in Prism, version 5.0 (GraphPad Soft-ware). All tests of significance were two tailed.

RESULTS

Rates and frequencies of B*13-mediated escape in HIV-1 Gag,

Pol, and Nef.

Ten published HLA-B*13 and/or

B*13:02-associ-ated polymorphisms in HIV-1 were investigB*13:02-associ-ated: A146S, I147L,

K436R, and I437L in Gag; L63S in protease; Q334N, T369A, and

K374R in reverse transcriptase; and E24Q and Q107R in Nef (

Ta-ble 1

and

Fig. 1A

) (

39

). These polymorphisms are significantly

enriched in B*13:02

individuals with chronic HIV-1 subtype B

infection, and nine of them lie within or near published or

pre-dicted B*13-restricted CTL epitopes (see Fig. S1 in the

supple-mental material). In addition, Gag-I437L and, to a lesser extent,

Gag-K436R are experimentally verified B*13-driven escape

muta-tions within the RI9

epitope (RQANFLGKI

429 – 437

) (

11

) while

Nef-Q107R confers escape from CTL responses against the RI9

epitope (RQDILDLWI

) (

21

). Together, these observations

support these 10 polymorphisms as commonly selected B*13

es-cape mutations

in vivo

.

To infer the extent and timing of HLA-B*13-driven escape

during HIV-1 infection, we analyzed longitudinal plasma HIV-1

RNA sequences from nine B*13:02

seroconverters (

40

,

41

) and

cross-sectional sequences from 69 B*13:02

individuals with

chronic infection (

39

). In HIV-1 Gag, 36% and 13% of B*13:02

individuals harbored Gag-I147L and -I437L, respectively, at 1 year

postinfection; by the chronic stage, 69% and 26% of B*13:02

individuals harbored these substitutions (

Fig. 1B

, top and

mid-dle). In contrast, Gag-A146S and Gag-K436R emerged later in

infection (reaching 17% and 26%, respectively, by the chronic

stage). For HIV-1 Pol, the protease-L63S, T369A, and

RT-K374R substitutions were observed in

⬇

12% of B*13:02

individ-uals in early infection and in 25 to 37% of B*13:02

individuals in

chronic infection (

Fig. 1C

, top and middle). RT-Q334N frequency

was higher in early (25%) than in chronic (9%) infection

se-quences, suggesting it as a possible transient escape form in some

individuals (

40

); however, our ability to conclude this is limited

since the data are derived from independent cross-sectional early

and chronic cohorts rather than from longitudinal samples from

the same individuals. For HIV-1 Nef, E24Q was observed in 0%

and 12% of early and chronic infection sequences whereas Q107R

was observed in 25% and 33% of sequences at these stages,

respec-tively (

Fig. 1D

, top and middle).

Chronic HIV-1 sequences were additionally analyzed for the

presence of naturally occurring mutant combinations (

Fig. 1B

,

C

,

and

D

, bottom). We began by engineering all combinations

ob-served at

⬎

5% frequency (plus the Nef double E24Q-Q107R

mu-tation, observed at 4% frequency) into an HIV-1

backbone.

Later, we engineered two additional Gag combinations for our

analyses of compensatory mutation pathways. Together a total of

23 mutant viruses (10 single and 13 combination mutations) were

constructed (

Table 1

).

Impact of HLA-B*13-associated polymorphisms in Gag on

viral RC.

Mutant HIV-1

viruses were evaluated using a

mul-ticycle

in vitro

replication capacity (RC) assay using the GXR25

GFP reporter cell line (

25

,

44

). Representative data are shown in

Fig. 2

, including flow cytometry results, viral growth curves, and

wtNL4.3-normalized replication capacity measurements for

un-infected cultures and those un-infected with wtNL4.3 and Gag-I437L

virus (which is known to impair

in vitro

RC) (

27

,

28

).

When engineered alone into HIV-1

, two of the four

B*13-associated Gag polymorphisms, Gag-I147L and Gag-I437L,

re-duced RC by 5% and 17%, respectively (

P

⬍

0.001), while

repli-cation capacities of Gag-A146S and Gag-K436R viruses were

comparable to the wtNL4.3 level (

Fig. 3A

and

B

). The RC

mea-surements for Gag-I147L, Gag-K436R, and Gag-I437L viruses are

consistent with those of previous studies conducted in Jurkat T

cells and CD8

-depleted peripheral blood mononuclear cells

(PBMCs) (

27

,

28

). To our knowledge, no studies have previously

investigated A146S for RC costs (though A146P, which is selected

by HLA-B*57 and other alleles [

39

,

55

], does not carry a replicative

cost in PBMCs [

28

,

55

,

56

]).

B*13-associated polymorphisms have not been evaluated

pre-viously in combination for their effects on RC; in particular, it is

unclear whether they all represent primary escape mutations or

may also include secondary (compensatory) changes. Eight

B*13-associated polymorphism combinations in Gag were therefore

as-sessed (

Fig. 3C

and

D

). The two combinations that contained

Gag-I437L in the absence of the adjacent K436R (i.e., I147L-I437L

and A146S-I147L-I437L) exhibited 9% and 17% reductions in

RCs, respectively. These values were significantly lower than the

wtNL4.3 value (

P

⬍

0.001) but not significantly different than the

value for Gag-I437L alone, indicating that neither A146S nor

I147L compensates for Gag-I437L-mediated replicative costs. In

contrast, mutant combinations that included Gag-I437L in the

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presence of K436R (K436R-I437L, I147L-K436R-I437L, and

A146S-I147L-K436R-I437L) exhibited RCs comparable to the

wtNL4.3 value, indicating that the replicative cost of Gag-I437L

was compensated by the adjacent K436R. Similarly, the three

combination mutants with Gag-I147L in the presence of the

adja-cent A146S and/or the downstream K436R also exhibited RCs that

were not significantly different from the wtNL4.3 value. This

sug-gests that the modest replicative cost of Gag-I147L in the absence

of I437L might be compensated by A146S or K436R although

further study will be required to elucidate these mechanisms,

par-ticularly in the case of K436R, which lies in a different Gag protein

subunit (p1).

A novel putative B*13-restricted epitope in p24 Gag.

The

re-gion spanning Gag

is dense in CTL epitopes (

22

). It has

previously been hypothesized that Gag-I147L confers escape from

CTL targeting the upstream B*13:02-restricted VV9 epitope (VQ

NLQGQMV

) (

Fig. 4A

) via an antigen-processing

mecha-nism (

11

). Another possibility is that Gag-A146S and/or -I147L

confers escape from an undiscovered B*13:02-restricted epitope

at this site, a hypothesis that is consistent with the higher

preva-FIG 2HIV-1 replication capacity: control data. (A) Representative flow plots of virus spread in culture (measured as percent GFP⫹cells) for uninfected controls and cells infected with wtNL4.3 and mutant Gag-I437L viruses. (B) Growth curves for the data shown in panel A, expressed as the fold increase in infected cells from the day 2 value. (C) Mean RC value for Gag-I437L normalized to that of the wtNL4.3 control, from one representative experiment containing three replicates. Histograms and error bars denote the means and standard deviations, respectively.

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lence of escape within (versus outside) CTL epitopes (

39

). To

investigate this, we utilized NetMHCpan, version 2.8, to predict

B*13:02-restricted epitopes in the HIV-1 subtype B consensus

se-quence at Gag

. We identified two overlapping candidate

epitopes, LI10 (Gag

) and GI8 (Gag

) (

Fig. 4A

), where

the latter is fully embedded in the former. Predicted half-maximal

(IC

) HLA binding affinities were 3,324 nM (LI10) and 3,181 nM

(GI8), classifying them as weak binders of potentially low

immu-nogenicity (

57

) (

Fig. 4A

). However, for context, the published

adjacent VV9 epitope has a predicted IC

of 9,174 nM, suggesting

that responses to both LI10 and GI8 are feasible. Of note,

Gag-A146S and -I147L lie at the penultimate and C-terminal residues

of both LI10 and GI8.

IFN-

␥

ELISpot assays were performed using PBMCs from two

chronically infected B*13:02

individuals with responses to this

region. In both cases, GI8 elicited the strongest response to a series

of N-terminally extended peptide sequences that included LI10

(

Fig. 4B

). Removal of the C-terminal isoleucine abrogated

re-sponses, suggesting that this position served as the F-pocket

an-chor residue for HLA-B*13 (data not shown). Limited cell

num-bers precluded HLA restriction confirmation and experimental

validation of A146S and/or I147L as bona fide escape mutations.

Still, the predicted IC

values of GI8 variants harboring

Gag-I147L were nearly 3-fold higher than the wild-type value,

support-ing I147L as a potential escape mutation (

Fig. 4C

). This

conclu-sion is reinforced by the observation that responding patient 1

FIG 3Replication capacities of NL4.3 viruses encoding B*13-associated polymorphisms in Gag. (A) Representative growth curves of single Gag mutants, expressed as the fold increase in infected cells from the day 2 value. (B) NL4.3-normalized RC values of each single Gag mutant, derived from two independent experiments, each containing triplicate measurements. Histograms and error bars denote the means and standard deviations, respectively. ***,P⬍0.001. (C and D) Experiments are the same as those described for panels A and B, respectively, but with data for combination Gag mutants.

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harbored wild-type I147 whereas patient 2 harbored I147L in

plasma (data not shown): the latter’s lower GI8 response is thus

consistent with waning of CTL responses to the wild-type epitope

following

in vivo

escape.

HLA-B*13-associated polymorphisms in Pol and Nef do not

impair viral RC.

We next examined the effects of B*13-associated

polymorphisms in Pol (L63S in protease and Q334N, T369A, and

K374R in RT) and in Nef (E24Q and Q107R) on viral replication.

None of the Pol substitutions significantly altered

in vitro

RCs

when engineered alone into NL4.3 (

Fig. 5A

and

B

), and the four

most common Pol mutant combinations observed in natural

se-quences also did not significantly affect RC (

Fig. 5C

and

D

).

Sim-ilarly, the two B*13-associated Nef polymorphisms did not impair

the RC, either alone or in combination (

Fig. 6A

and

B

). Note that

the Nef observations are not simply attributable to the lack of

requirement of this protein for viral replication

in vitro

as

⌬

Nef

virus replicated poorly in GXR25 cells (in experiments initiated at

an MOI of 0.01, HIV-1

and VSV-G-pseudotyped HIV-1

exhibited 45% and 32% reductions in RCs, respectively,

com-pared to the HIV-1

level).

Impact of HLA-B*13-associated Nef polymorphisms on CD4

and HLA class I downregulation.

Naturally occurring sequence

variation in Nef can modulate its CD4 and HLA class I

downregu-lation activities (

33–36

,

58

). We therefore investigated the impact

of B*13-associated polymorphisms on Nef function. To this end,

the GXR25 cell line was stably transduced to express HLA-A*02:01

(GXR-A*02) as a representative HLA class I molecule. GXR-A*02

cells were infected with control viruses (wtNL4.3,

⌬

Nef, and

Nef

; the last is impaired for HLA class I downregulation [

50

]

but replicates comparably to wtNL4.3 [data not shown]) or

mu-tant Nef viruses (E24Q, Q107R, and E24Q-Q107R) and stained

for surface CD4 and A*02 expression once infection reached

⬇

10

to 15%. Representative data for uninfected and control-infected

cultures are shown in

Fig. 7A

and

B

. Compared to the activity of

wtNL4.3,

⌬

Nef virus displayed a modest (5%) reduction in CD4

downregulation that was nevertheless statistically significant and a

substantial (66%) defect in HLA class I downregulation activity

(

P

ⱕ

0.01) (

Fig. 7C

and

D

). Efficient downregulation of CD4 by

⌬

Nef virus is likely due to the actions of Vpu and Env in our

culture system (

59

). The ability of Nef

to downregulate CD4

was comparable to that of wtNL4.3, but the HLA downregulation

function of Nef

was impaired by 60% (

P

⬍

0.01). Neither

Nef-E24Q nor -Q107R significantly affected CD4 or HLA class I

downregulation alone; moreover, the CD4 downregulation

func-tion of the E24Q-Q107R double mutant was comparable to that of

wtNL4.3. However, the ability of the double mutant to

downregu-late HLA class I was 42% reduced compared to that of wtNL4.3

(

P

⫽

0.007). Intracellular Nef expression was readily detectable in

cultures infected with control and mutant HIV-1, suggesting that

the attenuated HLA downregulation activity of the Nef double

mutant was not simply due to poor expression or to a stability

defect (

Fig. 7E

).

Impaired HLA class I downregulation of Nef double mutant

renders infected cells more susceptible to recognition by

HIV-1-specific T cells.

An impaired ability of the Nef-E24Q-Q107R

double mutant to downregulate viral peptide/HLA complexes

im-plies that cells infected with this virus should become more visible

to HIV-specific CD8

T cells, regardless of HLA restriction. To

FIG 4Prediction of a novel putative B*13-restricted CTL epitope in p24Gag_{. (A) Locations and sequences of published and bioinformatically predicted}

B*13:02-restricted epitopes are shown above the HIV-1 consensus B amino acid sequence spanning Gag135–155. (B) IFN-␥ELISpot responses, measured as the

number of spot-forming cells (SFC) per million PBMCs, to predicted epitopes and their N-terminal extensions in two B*13:02⫹chronically HIV-1 subtype B-infected patients who responded to this region. (C) NetMHCpan-predicted IC50for GI8 and variants harboring the B*13:02-associated polymorphisms

Gag-A146S and/or -I147L.

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test this hypothesis, we used a novel coculture assay that

fea-tures target cells (GXR-A*02 cells infected with control or

mu-tant HIV-1) and HIV-1-specific effector cells transiently

ex-pressing an A*02-restricted FK10

-specific TCR

␣

/

␤

, CD8

␣

,

and an NFAT-driven luciferase reporter construct (see Fig. S2

in the supplemental material). In this assay, TCR-dependent

signaling is quantified based on luminescence following

cocul-ture with A*02/FK10

-expressing target cells.

Downregula-tion of HLA-A*02 by wild-type Nef from the infected target cell

surface is expected to lead to reduced TCR signaling in effector

cells, while target cells infected with HIV-1 containing

defec-tive Nef sequences that fail to downregulate HLA-A*02 should

lead to increased TCR signaling.

Consistent with this, we observed a significant negative

corre-lation between the extent of Nef-mediated HLA-A*02

downregu-lation on target cells and TCR-driven luciferase signal in

HIV-1-specific effector cells (Spearman’s

R

,

⫺

0.88;

P

⫽

0.03) (

Fig. 8

).

This was confirmed in three independent experiments

(Spear-man’s

R

,

⫺

0.82 to 0.89;

P

⫽

0.03 to 0.06). Importantly, control

experiments using uninfected GXR-A*02 target cells and parental

GXR25 target cells (lacking A*02) did not induce luciferase upon

coculture with effector cells (data not shown), indicating that

tar-FIG 5Replication capacities of NL4.3 viruses encoding B*13-associated polymorphisms in Pol. (A) Representative growth curves of single Pol mutants, expressed as the fold increase in infected cells from the day 2 value. (B) NL4.3-normalized RC values of each Pol single mutant, obtained from two independent experiments, each containing triplicate measurements. Histograms and error bars denote the means and standard deviations, respectively. (C and D) Experi-ments are the same as those described for panels A and B, respectively, but with data for combination Pol mutants.

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get cell recognition was HIV-1 epitope and HLA specific. Taken

together, our results suggest that the impaired HLA class I

down-regulation activity of the B*13:02-associated Nef-E24Q-Q107R

double mutant could render HIV-1-infected cells more

vulnera-ble to CD8

T cell responses

in vivo

.

DISCUSSION

We engineered a total of 23 mutant HIV-1

recombinant

vi-ruses harboring B*13-associated polymorphisms either alone

(

n

⫽

10) or in naturally occurring combinations (

n

⫽

13) and

investigated their effects on HIV-1 replication and Nef protein

function. Our results extend current knowledge in multiple ways.

First, we confirmed that escape via Gag-I437L incurs a substantial

(17%) fitness cost (

27

,

28

), and we extend this observation by

demonstrating that the adjacent K436R mutation rescues

in vitro

RC to wild-type levels. This compensatory relationship is

corrob-orated by the time course of selection of these polymorphisms

in

vivo

(I437L is ultimately selected in 26% of B*13:02

individuals;

of these, 33% also developed K436R), the highly significant

nega-tive association reported between I437L and wild-type K436 in

HIV-1 subtype B sequences (

39

), and evidence that despite its

strong association with B*13:02, K436R alone does not confer

escape in the majority of HIV-infected individuals (

27

,

60

).

Sim-ilarly, we confirmed that Gag-I147L, the most rapidly and

fre-quently selected B*13:02 escape mutation in HIV-1 (

39

), incurs a

minor (5%) replicative cost, which appears to be rescued by the

adjacent A146S and/or distal K436R mutation. Again, these

puta-tive compensatory relationships are corroborated by their time

course of selection (I147L is ultimately selected in 70% of B*13:02

individuals; of these, 13% also develop the A146S or K436R

mu-tation). Taken together, we hypothesize that replicative costs of

Gag-I147L and -I437L escape mutations, which usually occur

within the first year of infection and may be compensated only

later in fewer than half of cases, contribute to the long-term

en-hanced viremia control observed in B*13:02-expressing persons.

While B*13-mediated escape in Pol and Nef does not

substan-tially impair viral RC, the B*13-driven Nef-E24Q-Q107R double

mutant conferred a

⬎

40% reduction in Nef’s ability to

downregu-late cell surface HLA class I. This defect was nearly as profound as

that conferred by

⌬

Nef or the Nef-M20A mutation (which does

not occur naturally) in our assay system. Moreover, the inability of

Nef-E24Q-Q107R to downregulate HLA class I enhanced the

rec-ognition of infected cells by HIV-1-specific T cells

in vitro

. In other

words, this naturally occurring mutant combination, selected

in

vivo

under B*13-mediated CTL pressure, conferred a functional

cost to Nef’s immune evasion activity. Although attenuation of

Nef’s HLA class I downregulation function as a consequence of

CTL escape has been described previously (

35

,

36

), the mutations

required to confer these effects are rare

in vivo

. The

HLA-B*35-driven Nef-R75T and -Y85F escape mutations, when present

to-gether, reduced Nef’s HLA class I downregulation activity nearly

2-fold, but this combination occurs in

⬍

0.2% of natural HIV-1

sequences (

35

). Similarly, a Nef clone exhibiting

⬇

15% lower

HLA downregulation activity was isolated from an acutely

HIV-infected individual who subsequently controlled HIV-1 viremia

spontaneously to

⬍

2,000 copies/ml; however, this defect required

the selection of four Nef mutations in the context of two

indepen-dent HLA alleles (

36

). Although the E24Q-Q107R double mutant

is observed in only 4% of B*13-expressing individuals and

al-though this combination tends to arise late in infection, it

never-theless represents the most frequent

in vivo

CTL escape pathway

identified to date that significantly compromises this Nef

func-tion. Importantly, once this double mutant arises

in vivo

, its HLA

downregulation defect would serve to boost the levels of viral

epitopes complexed with all HLA-A and HLA-B alleles expressed

by the individual (not just those restricted by HLA-B*13), thus

rendering infected cells more susceptible to recognition by

nu-merous HIV-1-specific T cells (or at least those specific for viral

epitopes that had not yet developed CTL escape mutations). In

fact, we hypothesize that the reason this double mutant is

ob-served only rarely

in vivo

is because of its substantial functional

cost.

Several limitations of the study merit comment. First, we

fo-cused on escape in Gag, Pol, and Nef because all known

B*13-FIG 6Replication capacities of NL4.3 viruses encoding B*13-associated polymorphisms in Nef. (A) Representative growth curves of single and double Nef mutants, expressed as the fold increase in infected cells from the day 2 value. (B) NL4.3-normalized RC values of each Nef mutant, obtained from two independent experiments, each containing triplicate measurements. Histograms and error bars denote the means and standard deviations, respectively.

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restricted epitopes lie in these proteins. However, undiscovered

B*13 epitopes are likely to exist in other regions, as indicated by

the existence of B*13-associated polymorphisms in gp41, Tat, Vif,

and Vpr (

39

), and their impact on HIV-1 replication and protein

function remains unknown. Second, due to the relatively large

number of mutants assessed here, all replication capacity assays

were performed in an immortalized GFP reporter T cell line.

However, the magnitude of replication defects observed for

Gag-I437L and Gag-I147L corroborated previous results in PBMCs

(

27

,

28

), indicating that RC measurements in our assay system are

generally representative of those in other cell types. Third, due to

insufficient PBMCs from B*13

individuals, we were unable to

fully characterize the novel GI8 epitope in p24

although its

bioinformatically guided discovery (combined with reduced

pre-dicted binding affinities of its escape variants) supports it as such.

Fourth, we assessed the impact of Nef-E24Q and/or Nef-Q107R

on only three of Nef’s

in vitro

functions (viral replication and

CD4/HLA class I downregulation), but the impact of Nef-E24Q

and Nef-Q107R on other Nef functions, including upregulation of

HLA class II invariant chain (CD74) (

61

), enhancement of virion

infectivity (

62

), and alteration of T cell receptor signaling (

63

),

remains to be determined. Moreover, to evaluate the effect of Nef

FIG 7Impact of B*13-associated polymorphisms on Nef-mediated CD4 and HLA class I downregulation. (A and B) Representative flow plots of CD4 (A) and HLA class I (B) downregulation for uninfected cells and cells infected with wtNL4.3,⌬Nef, and NefM20Acontrol viruses. Median fluorescence intensities (MFIs)

of CD4 and HLA class I expression (yaxis) are shown for GFP⫹(HIV-infected; red gates) versus GFP⫺(HIV-1 uninfected; black gates) cells. (C and D) Impact of control viruses and those harboring B*13-associated Nef polymorphisms on CD4 and HLA-A*02 downregulation, normalized to wtNL4.3 levels. Receptor downregulation values were obtained from a minimum of three independent experiments, each containing one measurement. Histograms and error bars denote the means and standard deviations, respectively. *,Pⱕ0.01. (E) Detection of Nef, GFP, and␤-actin protein expression by Western blotting in GXR-A*02 reporter T cells infected with NL4.3 viruses encoding control or mutant Nef sequences.

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mutations on HLA downregulation and its consequences for T

cell-mediated recognition of infected cells, we used HLA-A*02

and an FK10

-specific TCR as representative indicators of this

process. Since HLA-B molecules may generally be more resistant

to Nef-mediated downregulation than HLA-A molecules (

64

) and

since the impact of Nef-mediated HLA downregulation may be to

some extent HIV gene and/or epitope specific (

65–67

),

recogni-tion of other CTL epitopes might differ from that observed here

for A*02-FK10

. However, using a transient-transfection

sys-tem, we previously demonstrated a strong correlation between the

ability of natural Nef sequences to downregulate HLA-A*02 and

HLA-B*07 (

34

), and Gag-specific CTL in general may be less

sus-ceptible to Nef-mediated HLA downregulation than CTL

target-ing other HIV-1 proteins (

67

), possibly due to presentation of Gag

epitopes before Nef-mediated HLA downregulation is complete

(

68

). Together, these observations suggest that our luciferase

re-porter T cell approach should be broadly representative of CTL

recognition and may be a conservative measure of Nef function.

Finally, while we have demonstrated enhanced recognition of

target cells infected with Nef-E24Q-Q107R mutant virus using a

reporter T cell assay, we have not performed inhibition studies or

assessed other effector functions directly with primary CTL

clones.

The mechanism whereby Nef-E24Q and -Q107R together

con-fer such a profound HLA downregulation defect requires further

study. While these codons have not been explicitly identified as

being critical for Nef function, both lie within or adjacent to

im-portant motifs. Nef-E24 is located in the N-terminal alpha helix

within a region (amino acids 17 to 26) that, when deleted and

engineered along with a V10E substitution, renders Nef defective

for HLA downregulation (

69

); it also lies four codons downstream

of M20, which, when artificially mutated to alanine, produces a

severe HLA downregulation defect (

50

). Nef-Q107 lies adjacent to

arginine residues R105/R106 that are essential for several Nef

functions, including dimerization (

70–72

); it is therefore possible

that the creation of a triple arginine (RRR

) via a Q107R

substitution may alter this protein-protein interface. Mutations at

the adjacent residue 106 (e.g., R106K and R106L) also confer

modest to severe HLA class I downregulation defects (

73

).

More-over, a recent crystal structure of Nef in complex with HLA class I

suggests that Nef codons 24 and 107 lie nearby and within

(respec-tively) neighboring antiparallel alpha helices, suggesting that their

presence in combination could modulate interactions between

these helices and possibly impact HLA downregulation function

(

74

). Further studies will be required to test this hypothesis and

identify compensatory pathways in natural sequences.

Taken together with the previous literature, our results suggest

that B*13-mediated viremia control is achieved via multiple

mechanisms. The major mechanism, demonstrated by other

stud-ies, is likely to be effective sustained targeting of CTL epitopes in

multiple HIV-1 proteins (

11

,

21

). Fitness costs of the resulting

viral escape mutations, which presumably limit the net viral

“ben-efit” gained from evading the CTL response, also likely contribute.

Specifically, B*13 escape mutations may impair HIV-1 by two

distinct mechanisms: by reducing Gag fitness and by dampening

Nef’s immune evasion function by compromising its HLA

down-regulation activity. Functional costs of CTL escape in Nef have

been reported by others (

35

,

36

,

75

); our results extend these

ob-servations by suggesting that such costs may be biologically

rele-vant. More broadly, our study highlights the potential utility of

HIV-1 T cell vaccines designed to target vulnerable epitopes in Nef

where escape mutations impair its viral immune evasion activity.

ACKNOWLEDGMENTS

We thank Denis Chopera for engineering the pNL4.3-⌬nef vector used in the homologous recombination procedure, Takamasa Ueno for provid-ing control mutant Nef plasmids, Bemuluyigza Baraki for generatprovid-ing the GXR-A*02 cell line, and Mario Ostrowski and Brad Jones for providing the 5B2 CTL clone used to generate the FK10Gag_{-specific TCR. We thank}

Chanson Brumme for assistance with data analysis. We also thank Mina John, Simon Mallal, P. Richard Harrigan, Martin Markowitz, Bruce Walker, Heiko Jessen, Evan Wood, Kanna Hayashi, and M.-J. Milloy for specimen and/or data access.

This work was supported by operating grants from the Canadian In-stitutes for Health Research (CIHR) (MOP-93536 and HOP-115700 to Z.L.B. and M.A.B.). The VIDUS and ACCESS studies were supported by the U.S. National Institutes of Health (VIDUS, R01DA011591 and U01DA038886; ACCESS, R01DA021525). This research was undertaken, in part, thanks to funding from the Canada Research Chairs program through a Tier 1 Canada Research Chair in Inner City Medicine which supports Evan Wood. X.T.K. was supported by a Master’s Scholarship from the CIHR Small Health Organizations Partnership Program (SHOPP), in partnership with the Canadian Association for HIV Re-search in partnership with Bristol-Myers Squibb Canada and ViiV Healthcare, and currently holds a CIHR Frederick Banting and Charles Best Canada Graduate Scholarship (doctoral). A.O. was funded by the Instituto de Salud Carlos III (PI12/00529). M.A.B. holds a Canada Re-search Chair (Tier 2) in Viral Pathogenesis and Immunity from the Can-ada Research Chairs Program. Z.L.B. was supported by a CIHR New In-vestigator Award and currently holds a Scholar Award from the Michael Smith Foundation for Health Research.

The funders of this study played no role in determining the content of the manuscript or the authors’ decision to publish.

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FIG 8Impact of the Nef double mutant E24Q-Q107R on recognition of HIV-infected target cells by HIV-specific effector T cells. Representative data de-picting a negative correlation between the Nef-mediated ability to downregu-late HLA-A*02 on GFP reporter target cells infected with control and mutant Nef viruses and TCR-driven luciferase signal in A*02-FK10Gag_{-specific Jurkat}

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