HIV-1 envelope protein binds to and signals throughintegrin a4b7, the gut mucosal homing receptor forperipheral T cells

James Arthos1,6, Claudia Cicala1,6, Elena Martinelli1,2,6, Katilyn Macleod1, Donald Van Ryk1, Danlan Wei1,Zhen Xiao3, Timothy D Veenstra3, Thomas P Conrad3, Richard A Lempicki4, Sherry McLaughlin5,Massimiliano Pascuccio1, Ravindra Gopaul1, Jonathan McNally1, Catherine C Cruz1, Nina Censoplano1,Eva Chung1, Kristin N Reitano1, Shyam Kottilil1, Diana J Goode1 & Anthony S Fauci1

Infection with human immunodeficiency virus 1 (HIV-1) results in the dissemination of virus to gut-associated lymphoid tissue.

Subsequently, HIV-1 mediates massive depletion of gut CD4+ T cells, which contributes to HIV-1-induced immune dysfunction.

The migration of lymphocytes to gut-associated lymphoid tissue is mediated by integrin a4b7. We demonstrate here that the

HIV-1 envelope protein gp120 bound to an activated form of a4b7. This interaction was mediated by a tripeptide in the V2 loop

of gp120, a peptide motif that mimics structures presented by the natural ligands of a4b7. On CD4+ T cells, engagement of

a4b7 by gp120 resulted in rapid activation of LFA-1, the central integrin involved in the establishment of virological synapses,

which facilitate efficient cell-to-cell spreading of HIV-1.

Gut-associated lymphoid tissue (GALT) is the principal site wherehuman immunodeficiency virus 1 (HIV-1) replicates1–6. Regardless ofthe route of transmission, HIV-1 rapidly establishes infection inGALT1. It has been postulated that the distinct tropism of HIV-1for GALT results from the high frequency of activated CD4+CCR5+

T cells, which, it is argued, provide a target-rich environment6. YetCCR5 expression, as measured by flow cytometry, does not correlatewith the frequency of infected memory CD4+ T cells in the gut5.Moreover, infection of resting memory CD4+ T cells in the gut isthought to be vital to the establishment of infection7. Soon afterHIV-1 seeds the gut, it undergoes substantial depletion of CD4+

T cells. It has been proposed that this depletion represents anirreversible insult to the immune system, which ultimately results inAIDS8. The mechanisms underlying CD4+ T cell depletion in the gutremain controversial. It results from direct infection6 or, alternatively,through bystander effects mediated by the HIV-1 envelope4. It isapparent that much is not yet understood about HIV-1 replication inthe gut and the concomitant depletion of CD4+ T cells from the gut.Yet understanding of these events is fundamental to an accuratedescription of HIV-1 pathogenesis and possibly to the developmentof an effective HIV-1 vaccine.

Here we show that the HIV-1 envelope protein gp120 bound to andsignaled by means of an activated form of integrin a4b7 on CD4+

T lymphocytes. We further show that gp120 rapidly activated LFA-1,an integrin that facilitates HIV-1 infection, on CD4+ T cells in ana4b7-dependent way. Functioning principally as a homing receptor,a4b7 mediates the migration of leukocytes to and retention ofleukocytes in the lamina propria of the gut9,10. Thus, in the tissuewhere HIV-1 ‘preferentially’ replicates, its envelope interacts directlywith an adhesion receptor that is specifically linked to the function ofCD4+ T cells in that tissue.

RESULTS

Integrin a4b7 mediates gp120 binding to natural killer cells

It has been reported that HIV-1 gp120 disrupts the cytolytic activity ofnatural killer (NK) cells11, but the mechanisms underlying thisphenomenon are unknown, as freshly isolated NK cells do not havedetectable expression CD4. In pursuing this further, we determinedthat a panel of recombinant HIV-1 gp120 envelope proteins obtainedfrom genetically diverse HIV-1 viruses bound NK cells, but only inthe presence of Ca2+ (Fig. 1a), which suggested that gp120 wasbinding to a C-type lectin receptor expressed on the NK cells.However, none of the known gp120-specific C-type lectins areexpressed on these cells.

To identify the receptor(s) that mediated the binding of gp120 toNK cells, we passed lysate derived from 1 � 109 uncultured NK cells

Received 17 October 2007; accepted 14 January 2008; published online 10 February 2008; doi:10.1038/ni1566

1Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. 2Centre ofExcellence for Biomedical Research, University of Genoa, Genoa, Italy. 3Laboratory of Proteomics and Analytical Technologies and 4Laboratory of Immunopathogenesis andBioinformatics, Science Applications International Corporation, Frederick, Maryland 21702, USA. 5Department of Microbiology, University of Washington School ofMedicine, Seattle, Washington 98195, USA. 6These authors contributed equally to this work. Correspondence should be addressed to J.A. (jarthos@niaid.nih.gov).

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over a gp120 affinity column in the presence of Ca2+. We then elutedbound proteins with EDTA and identified them by tandem massspectrometry. Unexpectedly, no C-type lectin receptors were eluted.However one integrin, b7, was among the proteins identified. On NKcells, b7 pairs with a4 (CD49d); the activity of this heterodimer isregulated by divalent cations, such that binding to its natural ligands isenhanced when Ca2+ is replaced with Mn2+. With that in mind, weincubated NK cells with a panel of biotin-labeled gp120 proteins in thepresence of Mn2+ and noted enhanced binding for each (Fig. 1b).Soluble MadCAM, an addressin specific solely for a4b7, efficientlycompetitively inhibited gp120 binding, whereas a control immuno-globulin fusion protein, NKG2D-Ig, did not (Fig. 1c), which suggestedthat gp120 was binding to a4b7 on the NK cells. To demonstrate suchan interaction directly, we cotransfected 293T human fibroblasts witha4 and b7 expression vectors; both receptors were highly expressed, asshown by the reactivity of transfected cells with monoclonal anti-bodies (mAbs) specific for a4 and b7 (Fig. 1d). Biotin-labeled gp120bound much more to a4b7-transfected 293T fibroblasts than to mock-transfected cells, and this interaction was blocked by unlabeledMadCAM-Ig (Fig. 1e). We did additional characterization of thecapacity of gp120 to mediate biological responses through a4b7

in NK cells and determined that gp120-a4b7 interactions led tophosphorylation of the p38 mitogen-activated protein kinase (Sup-plementary Fig. 1 online). These data collectively indicate that HIV-1envelope protein gp120 binds to an activated form of integrin a4b7.

Activated a4b7 on T cells recognizes HIV-1 gp120

We next sought to determine whether gp120 also bound a4b7 onT cells. Although circulating T cells express both a4 and b7, mostheterodimers are present in an inactive conformation. Specializeddendritic cells in mesenteric lymph nodes and Peyer’s patches convertvitamin A to retinoic acid, which acts directly on T cells to ‘imprint’ agut-homing phenotype that includes a higher activation state of a4b7

(ref. 12). A GALT-like phenotype, in which both CD4+ and CD8+

T cells have high expression of active a4b7, can be induced by cultureof peripheral blood mononuclear cells (PBMCs) in the presence ofretinoic acid12. We cultured PBMCs in the presence of retinoic acidand noted higher expression of a4 and b7 (Supplementary Fig. 2online). To measure CD4-independent binding on CD4+ T cells, wedepleted PBMC samples of CD8+ cells and incubated the resultantcells with biotin-labeled gp120 in the presence of a CD4-specific mAb(Leu3A) that inhibits the binding of gp120 to CD4. We noted

substantial CD4-independent binding, as indicated by residualbinding in the presence of Leu3A, and this binding was abrogatedwhen divalent cations were excluded (Fig. 2a). We noted little CD4-independent binding for cells cultured in the absence of retinoic acid(Fig. 2a). CD4-independent gp120 binding was also inhibited by bothMadCAM-Ig and a fusion protein of the adhesion molecule VCAMand immunoglobulin, but not by the closely related E-cadherin–Igfusion protein, which is specific for integrin aEb7 (Fig. 2b).

The principal contact sites for the natural ligands of a4b7 reside onthe a-chain13. Three a4-specific mAbs, HP2/1, L25 and 2B4-3 alsoinhibited binding of gp120 to a4b7 (Fig. 2c). These mAbs map to sitesbetween residue 152 and residue 203 of a4 and recognize epitopes nearthe MadCAM binding site14. The two b7-specific mAbs we testedpartially inhibited gp120 binding (Fig. 2c), whereas three b1-specificmAbs had a minimal effect. Of note, Act-1, a mAb specific for a4b7

heterodimers15, also inhibited g120 binding. These results demon-strate that gp120 binds to a4b7 at a sites or sites near the epitopesrecognized by its natural ligands. We also assayed binding to CD8+

T cells and found that gp120 bound these cells in a way that was cationdependent and was inhibited by mAb HP2/1 (Fig. 2d). As a furtherspecificity control, we determined that immune sera from HIV-1-infected patients could, to variable degrees, inhibit gp120-a4b7 inter-actions on CD8+ T cells (Supplementary Fig. 3 online). HIV-1 gp120appears on virions as trimeric ‘spikes’; thus, we determined that bothHIV-1 gp120 trimers and intact HIV-1 virions bound a4b7 (Supple-mentary Fig. 4 online). We note that avidity effects resident in theenvelope trimer substantially enhance apparent affinity for a4b7.

Although gp120 envelope proteins from HIV-1 subtypes A, B, C andD all bound a4b7, we repeatedly noted distinct differences in theirreactivity (Figs. 1a,b and 2d). With further investigation, we deter-mined that substantial differences in a4b7 binding activity were evident,even when we compared closely related gp120 proteins derived from asingle patient (Supplementary Fig. 5 online). The main MadCAM andVCAM contact sites on a4b7 reside in a4. We determined that for thosemonomeric envelope proteins with the strongest a4b7 binding activity,we were able to detect small amounts of binding to a4b1 (Supplemen-tary Fig. 6 online). The reactivity of gp120 proteins, and in particulartrimeric gp120 proteins, with a4b1 remains to be determined.

Integrin a4b7 binds to the gp120 V2 loop

Madcam, VCAM and fibronectin all bind a4b7 through structurallyhomologous binding motifs16. In MadCAM, the minimal essential

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Figure 1 Binding of gp120 to a4b7 on NK cells. (a,b) Flow cytometry of freshly isolated NK cells incubated with biotinylated recombinant gp120 proteins

derived from 92Th14-12 (HIV-1 subtype B), 92Ug021-9 (HIV-1 subtype A), 93MW959 (HIV-1 subtype C) and AN1, binding in the presence of Ca2+

(a) or Mn2+ (b). Strep-PE, streptavidin-phycoerythrin. (c) Binding of biotinylated 93MW959 gp120 to NK cells in the presence of a fivefold molar excess

of MadCAM-Ig or NKG2D-Ig. (d,e) Flow cytometry of the binding of phycoerythrin-conjugated mAb specific for a4 (PE-a4) or b7 (PE-b7; d) or of biotinylatedAN1 gp120 (e) to 293T cells mock transfected or transfected with cDNA expressing a4 and b7 (a4b7). (e) + MadCAM-Ig, fivefold molar excess of

MadCAM-Ig. Data are representative of three or more independent experiments.

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epitope is a short tripeptide loop, Leu-Asp-Thr, whereas in fibronectinthe sequence is Leu-Asp-Val and in VCAM it is Ile-Asp-Ser. Ineach, the core aspartic acid residue is pivotal, and its removalabrogates binding16. We sought to determine whether the cyclichexapeptide Cys-Trp-Leu-Asp-Val-Cys (CWLDVC) containing thecore Leu-Asp-Val loop, which has been shown to inhibit the bind-ing of all three natural ligands to a4b7 (ref. 17), would also inhibitthe binding of gp120 to a4b7. The binding of HIV-1 gp120 to CD8+

T cells cultured in retinoic acid was inhibited by CWLDVC in adose-dependent way (Fig. 3). This suggested to us that HIV-1might recognize a4b7 by mimicking the tripeptide loop present inits natural ligands. Inspection of the HIV-1 envelope sequence showeda single consensus Leu-Asp-Val sequence in the V2 loop, at positions182–184 (numbering for HIV-1 clone HXB2; Fig. 4a). The asparticacid at position 183 is conserved in over 98% of the 976 gp120sequences in the 2006 Los Alamos HIV database of alignedenvelope sequences.

We constructed and expressed a mutant of a synthetic ancestralsubtype B gp120 envelope protein (AN1 gp120) in which the leucineat position 182 and the aspartic acid at position 183 were replacedwith alanine residues. By surface plasmon resonance assay, we deter-mined that this ‘alanine mutant’ recognized soluble CD4 and severalconformationally sensitive gp120-specific mAbs with binding kinetics

similar to that of wild-type AN1 gp120, which indicated that it wasconformationally intact (Supplementary Fig. 7 online). In cell surfacestaining assays, the alanine mutant bound CD4+ T cells to a greaterextent than did its wild-type ‘parent’; however, unlike wild-typegp120, the alanine mutant showed only minor CD4-independentbinding (Fig. 3). Consistent with that observation, wild-type butnot mutant gp120 bound to CD8+ T cells (Fig. 3). We conclude that

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Figure 2 Integrin a4b7 mediates CD4-independent binding of gp120 on retinoic acid–treated T cells. (a–c) Flow cytometry of PBMC samples depleted of

CD8+ cells and activated with OKT3 and IL-2, cultured in the presence of retinoic acid except where noted otherwise (– RA), and stained with biotinylated

AN1 gp120 alone or in the presence of the CD4-blocking mAb Leu3A (+ Leu3A), or with Leu3A in the absence of retinoic acid (– RA) or divalent cations(– Ca2+ & Mn2+; a); stained with biotinylated gp120 in the presence of Leu3A plus E-cadherin–Ig, MadCAM-Ig or VCAM-1–Ig (b); or stained with biotinylated

gp120 in the presence of Leu3A and mAb specific for a4, b7 or b1 (c). Binding is presented as percent inhibition relative to binding of gp120 in the

absence of antibody to integrin (set as 100%); error bars, s.d. of three independent binding inhibitions. (d) Flow cytometry of activated CD8+ T cells cultured

in the presence of retinoic acid, stained with AN1 gp120, 93MW959 gp120 or 92Ug021-9 gp120 with or without divalent cations; some samples were

then blocked with the a4-specific mAb HP2/1 (+ HP2/1). Far left, neutravidin binding in the absence of biotinylated gp120. Data are representative of three

or more independent experiments.

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Figure 3 Binding to a4b7 uses a Leu-Asp-Val sequence in the gp120 V2

loop. (a) Flow cytometry of CD8+ T cells cultured with retinoic acid and

stained with biotinylated gp120, with the inclusion of the cyclic peptide

CWLDVC (0.19–12.5 mM) or a ‘scrambled’ control peptide (Ctrl; 3.1 mM).

(b) Flow cytometry of CD4+ or CD8+ T cells treated with retinoic acid and

stained with wild-type (WT gp120) or mutant (gp120 L182A,D183A)

biotinylated AN1 gp120. + H (above bar, a,b), inclusion of HP2/1; + Leu3A

(horizontal axis, b), inclusion of Leu3A. Data are representative of three or

more independent experiments.

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the HIV-1 envelope mimics the tripeptide loop present in each of thenatural ligands of a4b7 and that this tripeptide, in large measure,mediates a4b7 binding. Notably, gp120 proteins from HIV-1 subtypeC typically contain the amino acid sequence Leu-Asp-Ile in place ofLeu-Asp-Val (Supplementary Fig. 8 online). Although Leu-Asp-Iledoes not appear in any of the natural ligands of a4b7, it is recognizedas an alternative minimal essential epitope for a4 binding18. Envelopeproteins of simian immunodeficiency virus from the sooty mangabey(SIVsmm) contain the tripeptide Asp-Leu-Val in place of theLeu-Asp-Val of HIV-1. We determined that a gp120 derived froman SIVsmm strain bound a4b7 on rhesus macaque PBMCs, as did anHIV-1 gp120 trimer (Fig. 4b).

Variable a4b7 reactivities of chimeric gp120 proteins

As noted above, gp120 proteins from various human and simianimmunodeficiency viruses vary substantially in their ability to binda4b7. We thus questioned whether inefficient a4b7 reactivity mightcorrelate with inefficient replication in GALT. Chemokine receptorCCR5–tropic HIV shows a greater propensity for substantial replica-tion in GALT than does chemokine receptor CXCR4–tropic HIV8. In a

series of comprehensive studies, two serially passaged pathogenicsimian-human immunodeficiency virus (SHIV) chimeric viruses,SF162P3, a CCR5-tropic SHIV, and SF33A2, a CXCR4-tropic SHIV,were compared in rhesus macaques19–21. Analysis of lymphoid tissues,including GALT, showed that both viruses undergo substantial repli-cation in peripheral tissues but the CCR5-tropic SF162P3 virus has amuch greater capacity to replicate in GALT. Moreover, the CCR5-tropic SHIV mediates considerable depletion of GALT CD4+ T cells,whereas the CXCR4-tropic SHIV does not.

To compare the a4b7 reactivity of these two viruses, we expressedand analyzed recombinant gp120 proteins corresponding to SF33A2and SF162P3. We designated the SHIV gp120 proteins SF33A2¢ andSF162P3¢, respectively (Fig. 4c). In the absence of the CD4-blockingantibody Leu3A, both SF33A2¢ and SF162P3¢ stained cells brightly.Preincubation of cells with Leu3A before the addition of SF33A2¢decreased binding to an amount close to background, which indicatedlittle measurable a4b7 binding. In contrast, although Leu3A substan-tially decreased the staining of SF162P3¢, measurable CD4-indepen-dent binding remained. When we preincubated cells with both Leu3Aand the a4-specific mAb HP2/1, SF162P3¢ binding was decreased to

CSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYSLTSCVPID–DNNSYRLINCCSFNITTEIRDKKKKVYALFYKLDV

LDVLDVLDVLDILDV

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Consensus:Consensus A1Consensus A2Consensus BConsensus CConsensus D

Consensus SIVsmmConsensus HIV-2

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Figure 4 Sequence conservation of the a4b7-binding motif in the V2 loop of HIV-1. (a) V2 loops of HXB2 and

a consensus sequence (consensus of all HIV-1 subtype consensus sequences). Aligned residues 182–184

(HXB2 numbering) are presented for each of the main subtypes of HIV-1 and the corresponding residues in

the ancestral sequences for HIV-1 subtypes A1, B and C. Below, corresponding sequences for consensus HIV-1 group O virus, SIVsmm and HIV-2. ‘LDV’

in red indicates the Leu-Asp-Val sequence. (b) Flow cytometry of rhesus macaque PBMCs cultured with retinoic acid and incubated with SIVsmm gp120

(left) or gp120 trimer (right) in the presence of Leu3A or Leu3A plus MadCAM-Ig. (c) Flow cytometry of the binding of a4b7 to gp120 proteins SF162P3¢(derived from gut-tropic SHIV) and SF33A2¢ (derived from non-gut-tropic SHIV), incubated with retinoic acid–cultured PBMCs in the presence or absence of

Leu3A with or without HP2/1. (d) Flow cytometry of retinoic acid–cultured PBMCs incubated with the L182H AN1 gp120 mutant with or without Leu3A.

Data are representative of three or more independent experiments.

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Figure 5 Binding of gp120 to a4b7 activates

LFA-1. (a–e) Flow cytometry of CD4+ T cells

cultured for over 7 d with IL-2 plus retinoic acid,

then allowed to ‘rest’ for about 18 h in media

without IL-2 or retinoic acid. (a) Cells stained

with MEM-148 or a b2-nonspecific mAb.

(b) Cells treated with unlabeled HP2/1, then

stained with MEM-148. (c) Cells treated for

10 min with gp120 trimer, then stained with

MEM-148. (d) Cells treated with HP2/1 and

gp120 trimer. (e) Cells treated for 10 min with

gp120 trimer, gp120 monomer or mutant gp120

(L182A-D183A), then stained with MEM-148.(f) Flow cytometry of CD8+ T cells cultured as

described in a–e, then treated for 10 min with

gp120 trimer or HP2/1 alone, or gp120 trimer

plus HP2/1, and then stained with MEM-148.

FSH-H, forward scatter; FL2-H, fluorescence

channel 2. Mock, protein preparation without

gp120. Data are representative of three or more

independent experiments.

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an amount close to background. Thus, although we found reactivityof a4b7 with gp120 derived from the gut-tropic SHIV SF162P3,we were unable to detect similar reactivity with gp120 derived fromSHIV SF33A2.

Inspection of the SF33A2 V2-loop sequence shows that the con-sensus Leu-Asp-Val tripeptide is replaced with His-Asp-Val22. Histi-dine at position 182 is rare, appearing in only 7 of the 976 gp120proteins in the HIV database. To determine whether this divergencefrom the consensus sequence could explain the poor a4b7-SF33A2¢interaction, we created the same substitution in AN1 gp120 of HIV-1.Unlike the wild-type AN1 gp120, which bound readily to a4b7

(Fig. 3), the L182H AN1 gp120 mutant showed little if any interactionwith a4b7 (Fig. 4d). Because both of the SHIV envelopes we analyzedwere derived from molecularly cloned replication-competent pro-viruses with established phenotypes, we concluded that efficientrecognition of a4b7 is not a prerequisite for substantial replicationin vivo. However, the different interactions of a4b7 with SF162P3¢ andSF33A2¢ correlated with the capacity of the two viruses to replicate inand deplete gut CD4+ T cells. The relationship between CCR5 tropismand gut tropism is well established; therefore, we suggest that theretention of a4b7 reactivity by the CCR5-tropic SF162P3¢ reflects thepropensity of SF162P3 virus to replicate in a tissue in which a4b7 ispresented in an active conformation, whereas SF33A2¢, because theSF33A2 virus replicates in tissues in which a4b7 is present mostly in aninactive conformation, has lost this reactivity.

The gp120-a4b7 complex mediates LFA-1 activation on T cells

To identify the selective advantage that a4b7 reactivity confers on gut-tropic HIV, we considered the function of this integrin in GALT.Integrin a4b7 acts in concert with L-selectin, chemokine receptors andintegrin LFA-1 (aLb2) in a highly coordinated ‘multistep adhesioncascade’ that mediates the extravasation of leukocytes into the gut23.Of note, LFA-1 activation is linked directly to a4 (ref. 24).LFA-1 provides a second important function for T cells: it is centralto the formation of immunological synapses25. Viruses including

HIV-1 use structures called ‘virologicalsynapses’, which approximate immunological

synapses, to increase the efficiency of cell-to-cell transmission26,27.Notably, only intermediate and/or active conformations of LFA-1promote synapse formation and HIV-1 infection28. Thus, gp120-mediated activation of LFA-1 could increase the efficiency of infectionby promoting synapse formation.

We sought to determine whether gp120 could activate LFA-1. Tomeasure LFA-1 activation, we used the integrin b2–specific mAbMEM-148 (ref. 29). This mAb recognizes only active forms ofLFA-1 and, once bound, ‘locks’ the active conformation in place.We cultured retinoic acid–treated CD4+ T cells overnight in theabsence of interleukin 2 (IL-2) to decrease basal LFA-1 activation.These suboptimally activated cells readily bound an integrin b2–nonspecific mAb that recognizes both low- and high-affinity con-formations of b2. In contrast, little MEM-148 reactivity was evident(Fig. 5a), which indicated that most of the LFA-1 was present in a low-affinity conformation. When we treated these cells with HP2/1, theintegrin a4–specific mAb that blocks gp120 binding, MEM-148reactivity remained unchanged (Fig. 5b). Cells treated for 10 minwith trimeric gp120 showed an increase in LFA-1 activation (Fig. 5c),whereas cells treated with the same gp120 trimer in the presence ofHP2/1 showed no increase (Fig. 5d). Thus, trimeric gp120 inducesactivation of LFA-1 in a way that can be inhibited by an a4b7-specificmAb that blocks gp120 binding.

We next compared wild-type AN1 gp120 and AN1 gp120 with amutant V2 loop. Relative to the gp120 trimer, wild-type monomericAN1 gp120 induced less MEM-148 reactivity, but well above back-ground (Fig. 5e). In contrast, AN1 gp120 with a mutant V2 loop,which bound poorly to a4b7, induced only slightly more MEM-148reactivity (Fig. 5e). To demonstrate that this response occurred in theabsence of CD4 ligation, we determined that a gp120 trimer alsoinduced LFA-1 activation on retinoic acid–cultured CD8+ T cellsin a way that could be inhibited by HP2/1 (Fig. 5f). We alsocharacterized gp120-induced LFA-1 activation in greater detailand found that gp120 treatment increased the binding of ICAM-1–Ig to memory CD4+ T cells (Supplementary Figs. 9 and 10 online).

a

b Series 1

Series 2

Series 3

p24, α4

gp120 α4 LFA-1 CD4 α4, LFA-1

gp120 α4 LFA-1 CD4 α4, LFA-1, gp120

gp120, α4, LFA-1 gp120, α4 LFA-1, α4 LFA-1, gp120 gp120, α4, LFA-1

α4, LFA-1 α4, CD4LFA-1

Figure 6 Integrin a4b7 localizes together with

active LFA-1 and CD4 at the interface of adhesive

junctions. (a) Fluorescence microscopy of

uninfected CD4+ T cells cultured together with

CD4+ T cells infected with HIV-1, cultured with

retinoic acid and stained with antibody specific

for HIV-1 p24 (blue), a4 (green), active LFA-1

(red) or CD4 (purple). Yellow, colocalization of a4

and LFA-1; white, colocalization of a4 and CD4.

(b) Fluorescence microscopy of CD4+ T cell

conjugates treated with gp120 and stained with

mAb specific for gp120, a4, LFA-1 or CD4 (Series

1 and Series 2); far right (yellow), colocalization

of a4 and LFA-1 (far right, series 2, also includes

gp120 (blue)). Series 1, z-axis reconstruction;

series 2, single xy section. Series 3, magnifi-

cation of the series 2 conjugate, visualized

with the a4-specific mAb 7.2R, the MEM-148

mAb to LFA-1 (integrin b2 chain), and mAb

specific for gp120, and z-axis reconstructions of

the cell-cell interface visualized with various

combinations of mAbs. Yellow, colocalization of

a4 (green) and LFA-1 (red). Original

magnification, �4 (series 1 and 2) or �9 (series

3). Data are representative of three or more

independent experiments.

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These results collectively demonstrate that gp120 (particularly trimericgp120) rapidly induces the activation of LFA-1 on suboptimallyactivated memory CD4+ T cells and that this activity requires directinteraction between gp120 and a4b7.

Localization of a4b7 and LFA-1 together at adhesive junctions

In published investigations of the transmission of HIV-1 acrossvirological synapses, viral antigens have been visualized aggregatingon the inner side of the infected cell membrane at sites of cell-cellcontact that ultimately form stable synapses27. We sought to deter-mine whether a4b7 could be detected by high-resolution confocalmicroscopy at these junctions between cells where synapses areestablished. We incubated HIV-1-infected CD4+ T cells for 2 h withautologous uninfected cells that had been cultured overnight in theabsence of IL-2. We then stained cells with mAb specific for the HIV-1group-associated antigen protein p24, along with the a4-specific mAb7.2R and the CD4-specific mAb OKT4, neither of which interfereswith the binding of gp120 to their respective receptors, and MEM-148,which recognizes the active form of LFA-1. We noted considerableaggregation of a4 on uninfected cells at the interface with infected cells(Fig. 6a). Closer inspection showed that activated LFA-1 could also bevisualized at this interface on both infected and uninfected cells.Moreover, colocalization of a4 and LFA-1 was evident, as wascolocalization of a4 and CD4. We then sought to determine whethergp120 alone, in the absence of other viral proteins, could by itself drivethe clustering of a4, active LFA-1 and CD4 at sites of cell-cell adhesion.Examination of the interface of gp120-treated cells undergoing homo-typic aggregation showed considerable colocalization of activated LFA-1 and a4 (Fig. 6b). Moreover, at the site of cell-cell contact we notedaggregation of CD4 and gp120 as well as of LFA-1 and a4 (Fig. 6b).Thus, the two principal cell surface components of an HIV-1-mediated virological synapse, CD4 and activated LFA-1, cluster

together with a4 and gp120 at sites of cell-cell contact. Theseobservations support a model in which gp120-a4b7–mediated activa-tion of LFA-1 facilitates the adhesive junctions that are a prerequisitefor the formation of a virological synapse.

A Leu-Asp-Val substitution decreases HIV-1 replication

Replication of HIV-1 in vitro occurs mainly by cell-to-cell spread30 andthus probably involves transfer through virological synapses. Toaddress the function of gp120-a4b7 interactions in viral replicationdirectly, we substituted the critical aspartic acid residue at position 183with alanine in the gp120 of a CCR5-tropic HIV-1 provirus. Replica-tion of this provirus in retinoic acid–treated CD4+ T cells was muchlower than that of its ‘parent’ provirus (Fig. 7a). This lower replicationcannot necessarily be ascribed to a loss of a4b7 reactvity, becausereplacement of Asp183 might disrupt other functions of the envelope.We passaged the mutant virus in retinoic acid–treated CD4+ T cells;after about 56 d, a highly replication competent virus appeared.Sequence analysis showed a limited number of amino acid substitu-tions in gp160 (data not shown), including a reversion of the aminoacid at position 183 back to aspartic acid, which restored the Leu-Asp-Val sequence. We tested the sensitivity of this revertant virus to mAbsthat antagonize gp120-a4b7. We found that mAbs HP2/1, 2B4 andAct-1, all of which disrupted the binding of gp120 to a4b7 (Fig. 2c),suppressed viral replication relative to treatment with isotype controls(Fig. 7b). In contrast, the a4-specific mAb 7.2R, which does notinterfere with gp120-a4b7 interactions, had almost no measurableeffect on viral replication.

In CD4+ T cells from six donors, HP2/1, the most potent gp120-a4b7 antagonist, suppressed viral replication by an average of 71%(range, 43–90%; P ¼ 0.0046; Fig. 7b and data not shown). This was incontrast to T-20, which neutralized all measurable viral replication.The range in variation of the inhibitory effect of HP2/1 suggested thatculture conditions or some as-yet-undefined variables influence theeffectiveness with which gp120-a4b7 antagonists suppress viral repli-cation. Of note, the parental virus from which the revertant virus wasderived was less sensitive to gp120-a4b7 antagonists (data not shown).The finding that HIV-1 showed greater sensitivity to a receptorantagonist after tissue culture adaptation is not new. Soluble CD4potently inhibits some laboratory-adapted isolates of HIV-1 whileshowing little or no inhibition of many primary patient isolates31. Weconclude that HIV-1 is able to use a4b7 to increase its efficiency ofviral replication.

DISCUSSION

A gradual decrease in CD4+ T cells is the defining characteristic ofAIDS, yet many questions relating to the mechanisms underlying this

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%)

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b Figure 7 Reversion of a Leu-Asp-Val–mutant HIV-1 provirus results in

enhanced sensitivity to gp120-a4b7 antagonists. (a) Flow cytometry of

retinoic acid–cultured, purified CD4+ T cells 7 d after infection, analyzing

intracellular p24 staining of wild-type provirus NL43-SF162 (WT LDV),

mutant NL43-SF162 with aspartic acid residue at position 183 replaced

with alanine (D183A) and the NL43-SF162 revertant (Revertant). Numbers

in outlined areas indicate percent p24+ cells; red arrow indicates p24+ cells

for the mutant infection. Data are representative of three or moreindependent experiments. (b) Replication of wild-type NL43-SF162

(SF162 WT), the NL43-SF162 D183A mutant (SF162 D183A) and the

NL43-SF162 revertant (SF162 rev) in retinoic acid–cultured, purified

CD4+ T cells in the presence of various mAbs (horizontal axis), including

HP2/1, 2B4 and Act-1, which inhibit gp120-a4b7 binding (antag); 7.2R,

which does not; FIB504, a mAb to b7 that partially inhibits gp120-a4b7

binding; and T-20, a control inhibitor. Experiments were repeated two to

six times (error bars, s.d. of two replicate experiments).

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decrease remain unanswered. It has become apparent that replicationof HIV-1 in CD4+ T cells in the gut has a prominent function in AIDSpathogenesis. It has been suggested that the massive depletion ofmemory CD4+ T cells from the gut, soon after transmission, is centralto HIV-1 disease. It is in this context that we report that the HIV-1envelope binds to and signals through a4b7, the gut-homing receptor.Our observations raise a host of new questions; however, the specificinteraction of the HIV-1 envelope with a4b7 establishes a new linkbetween GALT and HIV-1 that should help to elucidate the funda-mental mechanisms that underlie AIDS.

Three lines of evidence suggest that gp120-a4b7 interactions arebiologically relevant. The highest frequencies of HIV-1 infection occurin the memory CD4+ T cell compartment in the gut, in which, unlikein almost all other tissue compartments, memory CD4+ T cells expressactivated a4b7. Second, this interaction is well conserved. We haveshown that gp120 proteins derived from HIV-1 subtypes A, B, C andD, as well a simian immunodeficiency virus (SIVsmm) gp120, boundto a4b7. The critical Leu-Asp-Val tripeptide in the V2 loop of gp120that mediates a4b7 reactivity effectively mimics a similar epitope onthe natural ligands of a4b7. Moreover, this epitope is highly conserveddespite the fact that it resides in one of the most variable domainsof gp120. Finally, we have shown that gp120 activated LFA-1 in ana4b7-dependent way. The capacity of LFA-1 to increase the efficiencyof HIV-1 infection is well established28,32. These observations collec-tively lead us to the conclusion that a4b7 reactivity provides a selectiveadvantage to HIV-1.

Interactions between LFA-1 and its ligand ICAM-1 stabilize immu-nological synapses25. Viruses, including HTLV-1 and HIV-1, coopt thecomponents of synapses to generate virological synapses to facilitatethe efficient cell-to-cell transfer of virions26. Although we haveidentified LFA-1 activation as one consequence of gp120-a4b7 inter-actions, it is likely that the response of CD4+ T cells to this stimulusinvolves additional cellular components associated with cytoskeletalrestructuring and synapse formation. Moreover, gp120 signalingthrough interactions with host cell CD4 and CCR5 receptors mayalso be involved. The delivery of signals to CD4+ T cells by gp120 wasfirst demonstrated in a description of gp120-CD4–mediated mobiliza-tion of the tyrosine kinase Lck33. It has been reported that minutesafter being treated with gp120, CD4+ T cells flux Ca2+ in a CCR5-dependent way34 and phosphorylate focal adhesion kinase35. Yet thevalue of such responses to HIV-1 is unclear. In retrospect, we nowrecognize that Lck mobilization, chemokine receptor–mediated Ca2+

flux and the phosphorylation of focal adhesion kinase can be linkeddirectly to the cytoskeletal restructuring that is associated with theformation of immunological synapses25. Along with the data we havepresented here, such observations lead to the hypothesis that gp120-mediated signaling through host cell–expressed a4b7, CD4 and CCR5works together to promote infection by reiterating events that areassociated with the formation of an immunological synapse.

Substitution of the Leu-Asp-Val epitope on gp120 lowered thereplicative capacity of an HIV-1 provirus. Serial passage resulted inrestoration of the Leu-Asp-Val epitope and in the replicative capacityof the virus. This revertant virus showed greater sensitivity to gp120-a4b7 antagonists, reminiscent of the effect that tissue culture passageexerts on the sensitivity of HIV-1 to soluble CD4 (ref. 31). Indeed,binding of the V2 loop to a4b7 may facilitate viral entry by loweringthe entropic barrier that slows the ligation of envelope spikes to cellsurface CD4 (refs. 36,37).

Interaction between gp120 and a4b7 may be particularly relevant inthe earliest stages of HIV-1 infection. Studies of SIV-infected maca-ques indicate that suboptimally activated memory CD4+ T cells are

among the first cells infected4. Consistent with that observation, inhumans, nonactivated cells in the gut show evidence of productiveinfection38. In resting cells, LFA-1 is present mainly in an inactiveconformation39 and therefore represents one target by which gp120-mediated activation, through gp120–a4b7 interaction, could facilitateinfection. We note that the efficiency with which gp120 proteinsbound a4b7 varied considerably, even in comparisons of gp120proteins derived from a single patient. This raises the possibilitythat viral isolates with optimal binding to a4b7 might establishinfection more efficiently. Several a4b7 antagonists in clinical devel-opment for the treatment of immune disorders40 may thus proveuseful in the context of HIV-1 infection.

In conclusion, here we have identified a4b7 as an additionalreceptor that the HIV-1 envelope binds to and signals through. Thisis the first demonstration to our knowledge of direct interactionbetween the HIV-1 envelope and an integrin receptor. An epitope inthe V2 loop of gp120 mediated this interaction. Signaling produced bygp120-a4b7 interaction led to LFA-1 activation, an event that is centralto the formation of virological synapses. Future studies shoulddetermine whether targeting gp120-a4b7 interactions affects HIV-1replication in vivo.

METHODSCells and reagents. Freshly isolated PBMCs were obtained from healthy donors

after they provided informed consent approved by the institutional review

board. Cells were separated by Ficoll-Hypaque, and purified CD4+ T cells and

NK cells were obtained by negative selection with magnetic beads (StemCell

Technologies). Cultured PBMCs were activated with the OKT3 mAb to CD3,

IL-2 (20 IU/ml) and retinoic acid (10 nM) unless otherwise specified. Retinoic

acid was obtained from Sigma. Recombinant fusion proteins of immuno-

globulin and MadCAM, VCAM, E-cadherin or LFA-1 were from R&D Systems.

Antibodies to integrins were from Serotech and Chemicon; antibodies to

surface markers labeled with fluorescein isothiocyanate, phycoerythrin and

allophycocyanin were from Becton Dickinson. Act-1 was provided by S. Shaw.

MEM-148 was from Serotec. CWLDVC and the ‘scrambled’ control peptide

CDLWC were from NeoMPS. Integrin cDNA expression vectors were from

Origene. Serum from two HIV-1 viremic patients was obtained for binding

studies after they provided informed consent approved by the institutional

review board. Recombinant envelope proteins are described in the Supple-

mentary Methods online. The full-length chimeric proviral clone NL43-SF162

was provided by R.L. Willey through the National Institute of Allergy and

Infectious Diseases AIDS reagent repository. Amino acid positions for SF162

gp120 are numbered according to the HIV-1 clone HXB2.

Recombinant gp120 protein labeling. Purified recombinant gp120 proteins

were biotinylated by amine-coupling chemistry. Proteins were allowed to react

for 30 min with a 20-fold molar excess of EZ-Link NHS-Biotin (Pierce) and

reactions were ‘quenched’ by rapid buffer exchange into HEPES-buffered saline

(10 mM Hepes and 150 mM NaCl). Biotin incorporation was assessed by the

reaction of gp120 proteins with HABA (4¢-hydroxyazobenzene-2-carboxylic

acid–avidin conjugates) according to the manufacturer’s instructions (Pierce).

Matched protein preparations showing a incorporation of biotin and gp120 at a

ratio of 1.0–1.2 mol/mol were used in comparative semiquantitative flow

cytometry binding assays.

Tandem mass spectrometry. For in-gel digestion, eluant fractions collected

from the gp120 affinity column were resolved by SDS-PAGE through a 4–12%

gradient gel. Proteins were visualized by silver staining (Invitrogen). Protein

bands were excised individually. Gel bands were destained in 50% (vol/vol)

acetonitrile in 25 mM NH4HCO3, pH 8.4, and were vacuum-dried. Trypsin

solution (20 mg/ml in 25 mM NH4HCO3, pH 8.4) was added to the destained

and dried bands. After incubation on ice for 1 h, excess trypsin solution was

removed and the gel bands were covered with 25 mM NH4HCO3, pH 8.4. After

overnight incubation at 37 1C, tryptic peptides were extracted with 70% (vol/

vol) acetonitrile and 5% (vol/vol) formic acid, then were lyophilized and

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desalted with ZipTips (Millipore). For nanoflow reversed-phase liquid chro-

matography–tandem mass spectrometry, a microcapillary column with an

integrated electrospray ionization emitter was used for chromatographic

separation of the tryptic peptides. A fused silica capillary column (75 mm

(inner diameter) � 360 mm (outer diameter) � 10 cm (length)) with a fine

flame-pulled tip (orifice, about 5–7 mm; Polymicro Technologies) was packed

with slurry ‘in-house’ with a 5-mm C-18 stationary phase 300 A in pore size

(Jupiter; Phenomenex). The column was connected to an Agilent 1100 nano-

flow LC system (Agilent Technologies) and then was coupled with a linear ion-

trap mass spectrometer by a nanoelectrospray source (LTQ; ThermoElectron)

operated with Xcalibur 1.4 SR1 software. Capillary temperature and electro-

spray voltage were set to 160 1C and 1.5 kV, respectively. Mobile phase A was

0.1% (vol/vol) formic acid in water and phase B was 0.1% (vol/vol) formic acid

in acetonitrile. After peptide injection, gradient elution was done in the

following conditions: 2% phase B at a rate of 500 nl/min in 20 min; a linear

increase of 2–42% phase B at a rate of 250 nl/min in 40 min; 42–98% phase B at

a rate of 250 nl/min in 10 min; 98% phase B at 500 nl/min for 18 min.

The column was then re-equilibrated with 98% to 2% phase B at a rate of

500 nl/min for 12 min and then was washed with 2% phase B at a rate of

500 nl/min for 20 min before subsequent sample injection. The linear ion-trap

mass spectrometer was operated in a data-dependent tandem mass spectro-

metry mode in which the five most abundant peptide molecular ions in every

mass spectrometry scan were sequentially selected for collision-induced dis-

sociation with a normalized collision energy of 35%. Dynamic exclusion was

applied to minimize redundant acquisition of peptides selected before for

collision-induced dissociation.

Bioinformatic analysis. Tandem mass spectra were used to search the UniProt

Homo sapiens proteomic database from the European Bioinformatics Institute

(March 2006 UniProt release; 37,542 accession entries) with SEQUEST operat-

ing on a 40-node Beowulf cluster (SEQUEST Cluster version 3.1 SR1; Bioworks

Browser 3.2). Peptides were searched with fully tryptic cleavage constraints. For

a peptide to be considered legitimately identified, it had to achieve a minimal

delta correlation of 0.08 and stringent charge state–dependent cross-correlation

scores of 1.9 for [M + H]1+, 2.2 for [M + 2H]2+ and 3.1 for [M + 3H]3+

peptide molecular ions. From the list of proteins identified, a subset was

derived that included only known membrane proteins. This list was then

further subdivided into functional categories that included metal-binding

proteins with the bioinformatic analysis resource DAVID (Database for

Annotation, Visualization and Integrated Discovery) of the Advanced Biome-

dical Computing Center (National Cancer Institute–Frederick).

Flow cytometry binding assays. Cells were stained with fluorescein-conjugated

mAbs with standard procedures, preceded by blocking of Fc receptors

with human IgG. The buffer was HEPES-buffered saline with 100 mM CaCl2and 1 mM MnCl2 unless otherwise specified. Buffer without divalent cations

included 10 mM EDTA. Staining with biotinylated gp120 (E-Z Link bio-

tinylation reagent; Pierce) followed by phycoerythrin conjugated neutravidin

(Pierce) was used for analysis of gp120 expression. Data were acquired with a

BD FACSCalibur.

Transient transfection of 293T human fibroblasts. The 293T human fibro-

blasts were transfected with 2 mg of each integrin expression vector plasmid

DNA (total, 4 mg) or an empty control vector with Polyfect (Qiagen) according

to the manufacturer’s instructions. After 48 h, cells were collected with versene,

were rinsed thoroughly and were stained as described above.

LFA-1-activation assays. Highly purified (over 95%) CD4+ T cells were

cultured in retinoic acid and IL-2 for a minimum of 7 d before use. Unless

otherwise specified, IL-2 and retinoic acid were removed about 18 h before the

start of each assay. Cells were incubated on ice for 20 min before gp120

treatment. Cells were treated for 10 min at 37 1C with 50 nM trimeric gp120 in

HEPES-buffered saline plus 100 nM Ca2+, 1 mM Mn2+ and 0.5% (wt/vol) BSA,

then were placed on ice and incubated with phycoerythrin-conjugated MEM-

148 or biotin-conjugated ICAM-1–Ig, without removal of the envelope protein.

Cells were rinsed twice with staining buffer. For biotinylated ICAM-1–Ig, a

second NeutrAvidin-phycoerythrin incubation step was added, followed by

additional rinses. Samples were analyzed by flow cytometry as described above.

Confocal microscopy. Retinoic acid–stimulated CD4+ T cells were infected

with HIV-1 NL43-SF162 and cultured for 5 d, then were incubated for 2 h

together with uninfected CD4+ T cells from the same donor. Cell surfaces were

stained with a4-specific antibody (mAb 7.2R), antibody to CD4 (anti-CD4;

OKT4) and anti-LFA-1 (MEM-148). Cells were fixed and made permeable with

Cytofix/Cytoperm (BD Biosciences) and then were stained with phycoerythrin-

conjugated mAb to HIV-1 p24 (KC57-RD1; Beckman Coulter) followed by

biotin-conjugated anti-phycoerythrin (E31-1459; BD Biosciences) and strepta-

vidin–Pacific blue (Invitrogen). Stained cells were loaded on to slides, were

stabilized with ProLong Gold (Invitrogen) and were viewed with a Leica SP5

confocal microscope. For gp120-treated cells, retinoic acid–stimulated CD4+

T cells were treated for 10 min at 4 1C with biotin-conjugated AN1 gp120

(5 mg/ml), followed by staining with streptavidin–Pacific blue (Invitrogen),

anti-a4 (7.2R), anti-CD4 (OKT4) and anti-LFA-1 (MEM-148). Some anti-

bodies were conjugated directly with Alexa Fluor 488 and Alexa Fluor 564

(Invitrogen). Stained cells were made to adhere to coverslips (BD Biosciences)

and were fixed in 2% (vol/vol) paraformaldehyde, then were viewed with a

Leica SP5 confocal microscope. All confocal images were processed and

cropped with Imaris imaging software.

HIV-1 infection. A single base substitution was introduced into a full-length

proviral clone of NL43-SF162 at position 183, resulting in a substitution of

aspartic acid with alanine, by a standard site-directed mutagenesis protocol

(Quikchange XL; Stratagene). Wild-type and mutant viruses were produced by

transient transfection into 293T fibroblasts. Viral stocks were normalized by

p24 antigen concentration with an enzyme-linked immunoassay of p24 antigen

(Perkin-Elmer), then were used to infect CD4+ T cells cultured for at least 3 d

in IL-2, OKT3 and retinoic acid. Viral replication was measured by intracellular

p24 staining (mAb RD1; Beckman Coulter), and results are reported for the

day of or the day before peak replication for wild-type virus, typically 6–9 d

after infection. Cells infected with the mutant virus were passaged every 14 d

for about 56 d with the addition of fresh uninfected CD4+ T cells, after which a

revertant virus stock was collected from the culture supernatant. Cells were

lysed, and total high-molecular-weight DNA was used to obtain the gp160

sequence of the revertant virus by standard DNA sequencing (Genewiz). For

mAb inhibition of viral replication, 33 nM mAb was added at the time of

infection and each time culture media was changed. Infections were done in

duplicate. For inhibition assays with HP2/1, six independent infections were

done and the significance of inhibition was calculated relative to that achieved

with IgG1 with a two-tailed Student’s t-test (a-significance, 0.05; P Z a).

Additional methods. Information on recombinant envelope proteins, gp120-

induced p38 phosphorylation in NK cells, virion capture experiments, surface

plasmon resonance spectroscopy and the binding of ICAM-1–Ig to LFA-1 on

CD4+ T cells is available in the Supplementary Methods online.

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSWe thank S. Shaw, G. Snyder, T.-W. Chun, A. Kinter and S. Moir for discussions;J.I. Mullins for providing the AN1 coding sequence; J. Weddle for figurepreparation; and the National Institutes of Health AIDS Research and ReferenceReagent Program for many reagents. Act-1 was provided by S. Shaw (NationalCancer Institute), and NL43-SF162 was provided by R.L. Willey (National Instituteof Allergy and Infectious Diseases). Supported by the National Institutes ofHealth (AI058894 and AI047734 to S.M.; and the Intramural Research Program).

AUTHOR CONTRIBUTIONSD.J.G., K.M., E.M., C.C., J.M., M.P., D.W., S.K., C.C.C., N.C., E.C., K.N.R. andD.V.R. did biological experiments; Z.X., T.D.V., T.P.C. and R.A.L. did proteomicand bioinformatics analyses; J.A., C.C., E.M. and A.S.F. conceived and designedexperiments; and all authors participated in manuscript preparation.

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