genetic signatures of hiv-1 envelope-mediated bystander apoptosis

Genetic Signatures of HIV-1 Envelope-mediated BystanderApoptosis□S

Received for publication, August 28, 2013, and in revised form, November 21, 2013 Published, JBC Papers in Press, November 21, 2013, DOI 10.1074/jbc.M113.514018

Anjali Joshi‡, Raphael T. C. Lee§¶, Jonathan Mohl‡, Melina Sedano‡, Wei Xin Khong¶, Oon Tek Ng¶,Sebastian Maurer-Stroh§�, and Himanshu Garg‡1

From the ‡Center of Excellence for Infectious Diseases, Department of Biomedical Sciences, Texas Tech University Health SciencesCenter, El Paso, Texas 79905, the §Bioinformatics Institute, Agency for Science, Technology and Research, Singapore 138671,Singapore, the ¶Department of Infectious Disease, Tan Tock Seng Hospital, Singapore 308433, Singapore, and the �School ofBiological Sciences, Nanyang Technological University, Singapore 637551, Singapore

Background: Determinants of HIV-1 Env-mediated apoptosis remain poorly understood.Results: We studied the bystander apoptosis-inducing activity of a panel of primary HIV Envs.Conclusion: Residues Arg-476 and Asn-425 are associated with differences in HIV-1 Env-mediated bystander apoptosis induction.Significance: We identified specific genetic signatures within the HIV-1 Env that are associated with the bystander apoptosis-inducing phenotype.

The envelope (Env) glycoprotein of HIV is an importantdeterminant of viral pathogenesis. Several lines of evidence sup-port the role of HIV-1 Env in inducing bystander apoptosis thatmay be a contributing factor in CD4� T cell loss. However, mostof the studies testing this phenomenon have been conductedwith laboratory-adapted HIV-1 isolates. This raises the questionof whether primary Envs derived from HIV-infected patients arecapable of inducing bystander apoptosis and whether specificEnv signatures are associated with this phenomenon. We devel-oped a high throughput assay to determine the bystander apo-ptosis inducing activity of a panel of primary Envs. We tested 38different Envs for bystander apoptosis, virion infectivity, neutraliz-ing antibody sensitivity, and putative N-linked glycosylation sitesalong with a comprehensive sequence analysis to determine if spe-cific sequence signatures within the viral Env are associated withbystander apoptosis. Our studies show that primary Envs vary con-siderably in their bystander apoptosis-inducing potential, a phe-nomenon that correlates inversely with putative N-linked glycosyl-ation sites and positively with virion infectivity. By use of a novelphylogenetic analysis that avoids subtype bias coupled with struc-tural considerations, we found specific residues like Arg-476 andAsn-425 that were associated with differences in bystander apopto-sis induction. A specific role of these residues was also confirmedexperimentally. These data demonstrate for the first time thepotential of primary R5 Envs to mediate bystander apoptosis inCD4� T cells. Furthermore, we identify specific genetic signatureswithin the Env that may be associated with the bystander apopto-sis-inducing phenotype.

The mechanism behind the slow but progressive depletion ofCD4� T cells in HIV infection remains a pertinent yet unan-swered question. Various hypotheses have been proposed for

this HIV-induced CD4� T cell depletion like direct cell killingdue to infection (1), bystander apoptosis (2), immune activation(3), immune exhaustion (4, 5), etc. Several lines of evidencesuggest that CD4� T cells undergo apoptosis in HIV infections(6 – 8). A majority of these cells are shown to be uninfected,lying in close proximity to HIV-infected cells (9), suggesting arole for bystander apoptosis in this pathology. The phenome-non of bystander apoptosis has been demonstrated in both SIV2

(4, 10) and the humanized mouse model (11) of HIV infection,and a role of viral proteins from infected cells mediating apo-ptosis in bystander cells has been proposed. Among the viralproteins, the Env glycoprotein remains as the frontrunner formediating bystander apoptosis because it is expressed on thesurface of infected cells and can interact selectively withbystander CD4� T cells (2, 12, 13).

HIV Env glycoprotein is the most dynamic viral protein thatconstantly evolves throughout the course of the disease (5,14 –16). The high rate of Env evolution and variability withinthe HIV-infected population (17) has stimulated a number ofstudies aimed at understanding the polymorphic nature of theEnv glycoprotein (5, 18 –20). One of the best studied phenom-ena associated with HIV Env is the evolution of virus from aCCR5-utilizing phenotype (non-syncytia-inducing) early dur-ing the infection to a CXCR4-utilizing phenotype later duringthe disease (15, 21, 22). This change in co-receptor use has beenassociated with evolution of the virus to a syncytia-inducingphenotype, leading to rapid decline in CD4 counts and acceler-ated disease progression (23). However, co-receptor switch isnot an absolute requirement for disease progression, and in�50% of the patients evolution of virus to X4 usage does notoccur prior to AIDS development or terminal disease (24, 25).Interestingly, these AIDS-associated R5 viruses have beenshown to be more fusogenic than early asymptomatic phaseviruses (26, 27). Furthermore, the evolution of virus toward X4

□S This article contains supplemental Table 1 and Fig. 1.1 To whom correspondence should be addressed: Dept. of Biomedical

Sciences, Texas Tech University Health Sciences Center, 5001 El PasoDr., El Paso, TX 79905. Tel.: 915-215-4271; Fax: 915-783-1253; E-mail:[email protected].

2 The abbreviations used are: SIV, simian immunodeficiency virus; PNGS,putative N-glycosylation site(s); Env, envelope protein; PBMC, peripheralblood mononuclear cell.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 5, pp. 2497–2514, January 31, 2014© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

JANUARY 31, 2014 • VOLUME 289 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 2497

by guest on February 11, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

usage has also been variable between different subtypes of HIVwith lower rates of X4 tropism seen in subtype C viruses (28).These findings emphasize the importance of the viral Env inregulating the course of disease with more fusogenic Envs beingpotentially more deleterious to CD4� T cells versus the lessfusogenic ones.

HIV disease progression is a complex interplay between sev-eral viral and host factors. The host immune system in mostcases can suppress the virus to undetectable set point/levelsafter an acute phase. The CD8 cytotoxic T cell response (29, 30)and the neutralizing antibody production are largely responsi-ble for the viral control leading into the chronic phase. Duringthe chronic phase, the highest selection pressure due to neu-tralizing antibodies is on the Env glycoprotein of the virus (31),which undergoes rapid evolution via changes in the pattern ofN-glycosylation and charge on the Env glycoprotein (32).Recently, several genetic signatures have been identified thatcan distinguish viruses from different stages of the disease thatinclude but are not limited to changes in putative N-glycosyla-tion sites (PNGS) (18, 32–35). Moreover, during the course ofintrapatient HIV-1 evolution, it is also documented that theviral Env acquires mutations that alter CD4 binding and/or Envfusogenicity (36 –39). However, how this variability in HIV Envaffects bystander apoptosis and virus pathogenesis remainsundetermined.

We and others have extensively studied the phenomenon ofbystander apoptosis mediated by HIV Env both in vitro (40 –43) and in vivo (11) in a humanized mouse model of HIV infec-tion. Our studies demonstrate that Env fusogenic activity deter-mines bystander apoptosis and CD4 decline but not virusreplication (11). Moreover, with respect to R5 viruses, using celllines that express different levels of CCR5, we have shown thatcell surface CCR5 expression levels determine Env-mediatedbystander apoptosis (44). Although experimental data from usand others demonstrate that bystander apoptosis can beinduced by HIV Env in vitro, the clinical implication of thesefindings is limited due to a lack of data from primary HIV iso-lates and patient-derived Env clones.

In this study, we developed a high throughput assay to studya panel of primary patient-derived HIV Envs for their bystanderapoptosis-inducing activity and determined its correlation withboth phenotypic and genotypic characteristics of the Envs.These included virus infectivity, sensitivity to neutralizing anti-bodies, putative N-glycosylation sites, Env charge, and associa-tion of specific residues in the Env with bystander apoptosis.Our study demonstrates that although primary R5 Envs varyconsiderably in their bystander apoptosis-inducing potential,the phenomenon correlates inversely with PNGS and to someextent neutralizing antibody sensitivity. Moreover, using com-putational sequence and structure analysis, we identified spe-cific Env signatures associated with differential bystander apo-ptosis-inducing phenotypes. Specific residues associated withthese signatures were confirmed by site-directed mutagenesisexperiments. These findings not only corroborate the role ofviral Env in bystander apoptosis but take our previous studies astep further in attempting to identify specific Env signaturesassociated with bystander apoptosis. To the best of our knowl-edge, this is the first study aimed at studying the bystander

apoptosis-inducing activity of a large panel of primary Envsutilizing a high throughput assay and correlating this phenom-enon with specific Env signatures.

EXPERIMENTAL PROCEDURES

Cell Lines and Transfections—SupT1 cells were maintainedin RPMI medium supplemented with 10% FBS and penicillinstreptomycin (5000 units/ml). SupT1 cells expressing CCR5were generated by transduction of wild type SupT1 cells with alentiviral vector expressing the CCR5 gene and have beendescribed previously (44). These cells were maintained in RPMImedium supplemented with 10% FBS and penicillin streptomy-cin (5000 units/ml) and blasticidin at a concentration of 3�g/ml. 293T, HeLa, and TZM-bl cells (National Institutes ofHealth AIDS Research and Reference Reagent Program) weremaintained in Dulbecco’s modified Eagle’s medium supple-mented with 10% FBS and penicillin/streptomycin (5000 units/ml). TZM-bl are HeLa-derived cells that express the HIV recep-tor CD4 and the co-receptors CXCR4 and CCR5 along with theluciferase and �-galactosidase genes under the control of HIVLTR (45). These cells thus readily support HIV infection/repli-cation. All transfections were conducted using the Exgen 500transfection reagent (Fermentas) following the manufacturer’sinstructions.

Env Constructs—Reference panels for different subtype Envclones were obtained from the NIH AIDS research and refer-ence reagent program (supplemental Table 1). These includethe HIV-1 subtype A/G Env clones (catalog no. 11673), (46) thereference panel for subtype B HIV-1 Env clones (catalog no.11227) (35, 47, 48), and clade C HIV-1 reference panel of Envclones (catalog no. 11326) (49, 50). These constructs includedifferent patient-derived primary Envs as insert in thepcDNA3.1 expression vector (Invitrogen). For the purpose ofcomparison and to be used as normalizing control, we used alaboratory-adapted R5-utilizing HIV-1 clone, YU-2. The YU-2Env was thus similarly cloned into the pcDNA3.1 directionalTopo expression vector (Invitrogen) and contains the openreading frames for the env and rev genes. Point mutations wereintroduced into different Env constructs using specific muta-genic primers and the QuikChange site-directed mutagenesiskit (Stratagene). The introduced mutations were confirmed bysequencing. For detection of Env expression, HeLa cells trans-fected with the various Envs were cultured in growth mediumdevoid of methionine and cysteine and supplemented with[35S]Met/Cys. Thereafter, cell lysates were immunoprecipi-tated with HIV-Ig (kindly provided by the NIH AIDS Researchand Reference Reagent Program) resolved by SDS-PAGE fol-lowed by PhosphorImager analysis.

High Throughput Assay for Bystander Apoptosis—HeLa cellswere seeded in 96-well plates at 7500 cells/well and transfectedwith various Env constructs using the Exgen 500 transfectionreagent. The next day, SupT-R5-H6 cells were added at 40,000cells/well. The cells were co-cultured for 24 h, following whichcaspase-3 substrate was added directly to the wells (Caspase-Glo 3/7 assay, Promega). The plates were incubated for 30 minat 37 °C and read on a luminescence plate reader (FluostarOmega, BMG LabScience). The Caspase-Glo 3/7 assay is aluminescence-based assay that directly measures caspase-3/7

HIV Envelope-mediated Apoptosis

2498 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 5 • JANUARY 31, 2014


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

activity in cultures. Apoptosis for each Env was calculated as thepercentage of YU-2 Env-mediated apoptosis after subtractionof the background derived from pcDNA3.1 empty vector-transfected cells.

To determine if bystander apoptosis induction by primaryEnvs requires CCR5 binding or gp41-mediated membranefusion, the gp41 inhibitor enfuvirtide (used at a concentrationof 2 �M) or CCR5 inhibitor maraviroc (used at a concentrationof 1 �M) was added at the time when SupT-R5-H6 were addedto each well. After an overnight incubation, the cultures wereassayed for caspase-3 activity as indicated above.

Apoptosis Assay—For the standard flow cytometry-basedapoptosis assay, Env-transfected HeLa cells were co-culturedwith CCR5-expressing SupT-R5-H6 cells followed by the clas-sical method of apoptosis detection via annexin V staining.HeLa cells transfected with different Env constructs wereseeded in 24-well plates at a concentration of 105 cells/well. Thecells were allowed to adhere for 4 – 6 h. Subsequently, themedium was removed, and SupT-R5-H6 cells were added at aconcentration of 0.5–1 � 106 cells/well. The cells were co-cul-tured for 24 h, following which the suspension cells were care-fully collected, stained with annexin V (BD Biosciences), andanalyzed by flow cytometry using a Gallios flow cytometer(Beckman Coulter). At least 10,000 events were collected andanalyzed using Flow Jo software (Tree Star).

Virus Infectivity—293T cells were transfected with thepNLLuc-R�/E� HIV backbone along with different Env con-structs. Virus stocks were harvested 48 h post-transfection andused to infect the indicator TZM-bl cell line in the presence of20 �g/ml DEAE-dextran (Sigma). Luciferase activity was deter-mined 72 h postinfection using the BriteLite plus luciferaseassay substrate (PerkinElmer Life Sciences). Infectivity for eachEnv was calculated as a percentage of YU-2 Env control aftersubtraction of the background derived from pcDNA3.1 emptyvector-transfected cells.

Neutralizing Antibody Sensitivity—pNLLuc-R�/E� virusstocks pseudotyped with different envelopes were prepared asdescribed above. Virus stocks were then added to different con-centrations of the TriMab antibody mix (IgGb12, 2F5, and2G12) starting with a concentration of 25 �g/ml. The virusantibody mix was incubated at 37 °C 1 h and subsequentlyadded to TZM-bl cells. Infection was determined 48 h postin-fection by measuring luciferase activity in the cultures. Infec-tivity curves of each Env were fitted using Sigma Plot software,and IC50 values were calculated from the curves.

Identification of Bystander Apoptosis Determinants in gp120—Nine pairs of genetically similar high-low apoptosis strainswere chosen based on a phylogenetic tree generated using themaximum likelihood method with �-distributed evolutionaryrates (shape parameter, 0.4327) using MEGA5 (51). Mutationsbetween these nine pairs were compared for distinctive changesin their physiochemical properties (positive charge, negativecharge, aromatic groups, aliphatic groups, prolination, glyco-sylation, size) between the two groups. Sites that were found inat least three pairs of high-low apoptosis strains to possess dis-tinctive differences between the two groups were considered ascandidates for determinants of bystander apoptosis. We exam-ined each of these sites in crystal structures (Protein Data Bank

entries 3U2S, 2QAD, and 2B4C) (52–54) for possible structuraleffects that they may have in altering the function of gp120.Protein Data Bank structure 3U2S is the crystal structure of anantibody (PG9 Fab) in complex with the V1V2 region fromHIV-1 strain ZM109 (54). 2QAD is the structure of tyrosine-sulfated 412d antibody in complex with HIV-1 YU2 strainGP120 and CD4 (52). The structure of 2B4C is that of HIV-1JR-FL strain gp120 containing the V3 loop region in complexwith CD4 and the X5 antibody (53). Sites where the mutationwas predicted to affect CD4 binding were selected for mutagen-esis experiments. All crystal structures were rendered usingYasara (55).

Analysis of Charge, PNGS, and Variable Loop Lengths—Thevariable loop regions of gp120 are V1(6615– 6692), V2(6693–6812), V3(7110 –7217), V4(7377–7478), and V5(7596 –7637),numbered according to the HxB2 nucleotide sequence. V1V2length is the combined peptide length of the V1 and V2 loopregions. A site with the sequence motif NX(S/T)X, where Xrepresents a non-proline amino acid, is used to predict PNGS.Molecular models of gp120 were generated with MODELLER(56), using Protein Data Bank entry 2B4C (53) as a template.These models were used as inputs for the PROPKA server (ver-sion 3.1) (57) to estimate the pKa values of the amino acids forthe calculation of charges at pH 7.0. Sequences were alignedusing MAFFT, and the resulting alignment was used for thePNGS prediction, charge, and loop length calculations and thesearch for molecular determinants of bystander apoptosis (58).

RESULTS

Development of a High Throughput Assay for HIV-1 Env-me-diated Bystander Apoptosis—Several groups, including ours,have demonstrated unequivocally that the Env glycoproteinexpressed on the surface of infected cells can induce apopto-sis in uninfected bystander CD4� T cells (41, 59, 60). Clas-sical methods of apoptosis detection like annexin V staining,TUNEL assay, etc. have been used for studying this phenom-enon. Previously, we have shown that determination ofcaspase-3 activity in cells correlates well with bystander apo-ptosis induction by HIV Env (41, 44). Building on this informa-tion, we asked whether this phenomenon could be developed asa high throughput assay for determination of bystander apo-ptosis-inducing activity of a panel of primary HIV Envs derivedfrom patients. For this, HeLa cells were transfected with variousprimary HIV Envs in a 96-well format. The next day, SupT-R5-H6 cells expressing CD4 and CCR5 (44) were added to thetransfected wells. After an overnight incubation, the cells wereanalyzed for caspase-3 activity using a luminescence substrate-based assay (Caspase-Glo 3/7 assay, Promega). A schematicrepresentation of the assay is shown in Fig. 1A. Because allmanipulations are done in a single 96-well plate, the assay pro-vides a rapid and easy method for analysis of bystander apo-ptosis-inducing activity of different Envs. We optimized theassay for maximum signal/noise ratio by using different ratiosof effector (HeLa cells transfected with HIV-1 YU-2 Env) andtarget (SupT-R5-H6) cells. As seen in Fig. 1B, the highest signal/noise ratio was seen for 7500 effector and 40,000 target cells/well. We next asked whether the assay can accurately reproducevariability in bystander apoptosis-inducing potential of various




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

HIV-1 Envs from laboratory-adapted strains. We cloned theEnv and Rev region of a number of laboratory strains of HIV likeYU-2, AD-8, JRCSF, Bal, and 89.6. We found that the assayshowed differences in bystander apoptosis-inducing activity ofthese Envs with high level of reproducibility (Fig. 1C). Further-more, the high throughput assay correlated well with the clas-sical method of apoptosis detection via annexin V or 7-amino-actinomycin D staining followed by flow cytometry (Fig. 1C)used by us in the past. Interestingly, the caspase assay was moresensitive at detecting bystander apoptosis of Env variants thatare low inducers of apoptosis. This is most likely a differencedue to measurement of the percentage of apoptotic cells inannexin V and 7-aminoactinomycin D staining compared withtotal caspase-3 activity in the high throughput assay. Moreover,H6 cells but not peripheral blood mononuclear cells (PBMCs)are best suited for this high throughput assay due to a limitedavailability of CD4�CCR5� cells in the peripheral blood, whichmakes apoptosis detection via R5 isolates like YU-2 difficult(Fig. 2). Hence, our optimized assay could be used for studying

a large panel of primary HIV-1 Envs with a high degree ofreproducibility.

Primary Envs Vary Considerably in Their Bystander Apopto-sis-inducing Activity—Using the assay developed above, wenext asked whether there were differences in the bystanderapoptosis-inducing activity of a panel of primary patient-de-rived HIV-1 Envs. We obtained the reference panel of HIV Envsfrom the National Institutes of Health AIDS Reference ReagentProgram, representing subtypes B, C, and AG. A total of 37primary Envs were tested with YU-2 Env, a laboratory-adaptedR5 strain, as a normalizing control and the parent vectorpcDNA3.1 as the negative control. Apoptosis was referenced asthe percentage of WT or YU-2 after subtracting vector controlfrom each sample. As seen in Fig. 3A, there was a wide variationin the bystander apoptosis-inducing activity of the primaryHIV-1 Envs. Based on this variability, we classified the Envs intothree groups: high (H) apoptosis-inducing, with the percentageof apoptosis more than 40; low (L) apoptosis-inducing, with thepercentage of apoptosis less than 20; and medium (M) apopto-

FIGURE 1. Development and validation of high throughput assay for HIV-1 Env-mediated bystander apoptosis. A, flow chart depicting the procedure fordetection of apoptosis mediated by different HIV Envs via the caspase-3-based apoptosis detection assay. B, optimization of the high throughput assay forbystander apoptosis. Different numbers of HeLa cells were transfected with the YU2-Env clone expressing the Env and Rev proteins in a 96-well plate. The nextday, the indicated numbers of SupT-R5-H6 were added to the transfected wells. After an overnight incubation, the cells were analyzed for caspase-3 activityusing a luminescence substrate-based assay (Caspase-Glo 3/7 assay, Promega). Bars represent means of duplicate wells. One representative of three inde-pendent experiments is shown. C, comparison of the high throughput caspase-3 based assay for bystander apoptosis detection with the classical method ofapoptosis detection via annexin V staining or by 7-aminoactinomycin D staining. 7500 HeLa cells were transfected with HIV-1 Envs from different laboratory-adapted strains of HIV-1 like YU-2, AD-8, JRCSF, Bal, and 89.6. The next day, 40,000 SupT-R5-H6 cells were added to each well. After an overnight incubation, thecells were analyzed for caspase-3 activity using the Caspase-Glo 3/7 assay. In parallel, HeLa cells were transfected with the above Envs in a 6-well plate. After anovernight incubation, the target cells were harvested and stained for surface phosphatidylserine expression using the annexin V staining kit (BD Biosciences)or by use of cell viability stain 7-aminoactinomycin D (7AAD) (BD Biosciences) followed by flow cytometry. Data are mean � S.D. (error bars) from triplicateobservations.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

sis-inducing, between 20 and 40% apoptosis (Fig. 3B). Whenclassified based on subtypes, there was no statistically signifi-cant difference between the bystander apoptosis-inducingactivity of different Envs (Fig. 3C). All of the Envs used in thestudy were expressed as detected after immunoprecipitationusing pooled sera from HIV-infected individuals (Fig. 3D). Thissuggests that although primary Envs vary in their bystanderapoptosis-inducing activity, this phenomenon may be universalfor the subtypes tested. The lack of differences between thesubtypes may be due to the selection of the reference panel torepresent the most diverse Envs from each subtype. Hence,overrepresentation of certain phenotypes in the populationcannot be ruled out.

Bystander Apoptosis-inducing Activity of Primary Envs Cor-relates with Virion Infectivity—Because both virion infectivityand apoptosis induction are dependent on Env function, wenext asked whether there were differences in the virion infec-tivity of the Envs studied. We used a pseudotyped virus particleapproach to determine the single round infectivity of the Envstested. Here again we found that there was a huge variation inthe infectivity of the Envs tested (Fig. 4A). We found that highapoptosis-inducing Envs were more infectious compared withlow apoptosis (p � 0.05) (Fig. 4B), although subtype-based clas-sification did not show significant differences between the Envs(Fig. 4C). This is not surprising because both virion infectivityand bystander apoptosis are dependent on the Env fusion activ-ity. Although as a group, virus infectivity correlated with

bystander apoptosis, this was not necessarily true for each Env.For instance, we found a number of Envs that showed relativelylow apoptosis but very high infectivity and vice versa (Fig. 4D).Hence, the spectrum of Envs studied here also includes a num-ber of Envs that may be highly infectious without causing sig-nificant apoptosis. This is consistent with several mutagenesisstudies whereby point mutations in HIV Env significantly alterbystander apoptosis while having a limited effect on virioninfectivity (40, 61).

Bystander Apoptosis-inducing Activity of Primary Envs Cor-relates Negatively with PNGS—One of the characteristics ofHIV evolution in patients over the course of the infection isacquisition of variations in PNGS (35). It has been demon-strated that PNGS tend to increase during the course of infec-tion, with the highest PNGS seen during chronic infection (33)associated with a minimal decline in CD4 T cell counts. ThePNGS have been shown to decrease at the late stages of thedisease, where there is an accelerated CD4 cell decline (32).Because we hypothesize that bystander apoptosis mediatedby Env may be one of the contributing factors in CD4 T celldecline, we asked whether PNGS in Env correlate withbystander apoptosis. PNGS were estimated using the sequenceinformation for each Env and the N-Glycosite software avail-able at the LANL database (Fig. 5A). When the Envs were clas-sified based on high, medium, or low bystander apoptosis activ-ity and PNGS were determined for the Env region (gp160) ineach group, there was a significant difference in PNGS between

FIGURE 2. Suitability of H6 cells for the high throughput assay. A, PBMCs were obtained from Stem Cell Technologies, and CD4� T cells were isolated usingthe Human CD4� T Cell Enrichment Kit (Stem Cell Technologies). HeLa cells were transfected with the indicated Env clones or pcDNA3.1 control vector. Thenext day, phytohemagglutinin-activated purified CD4� PBMCs were added to the transfected wells, incubated overnight, and analyzed for caspase-3 activityusing the Caspase-Glo 3/7 assay as described in the legend to Fig. 1. B, in parallel, the assay described in A was also conducted with the H6 cell line. C, bright fieldanalysis of syncytia formation in cultures in A and its inhibition via use of AMD-3100 or maraviroc. D, purified CD4� T cells isolated from PBMCs were stainedusing anti-CXCR4 and anti-CCR5 antibodies followed by flow cytometry. RLU, relative light units. Error bars, S.D.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

the high and low apoptosis groups (Fig. 5B). The high apoptosisgroup shows significantly lower PNGS compared with the lowapoptosis group (p � 0.05). We also determined whether therewere differences between PNGS in the gp120 and gp41 region.Interestingly, most of the differences in PNGS were seen in thegp120 region (Fig. 5C) and not gp41 (Fig. 5D). This suggests thatadaptation of the virus via an increase in PNGS may be coun-terproductive to bystander apoptosis induction. However,when the Envs were grouped on the basis of subtype, no signif-icant differences were seen (Fig. 5, E–G). Once again, this maybe due to the diverse set of Envs selected for the reference panel.

Neutralizing Antibody Sensitivity of Primary Envs Correlateswith Bystander Apoptosis Induction—Primary Envs derivedfrom HIV-infected patients are known to vary considerably intheir sensitivity to neutralizing antibodies (62). Interestingly,the sensitivity to broadly neutralizing antibodies also varieswith the stage of the disease as well as PNGS, with end stageviruses being more sensitive and having lower PNGS (32).Because we saw a strong correlation between PNGS and apo-ptosis induction, we next asked whether there was a correlationbetween bystander apoptosis activity of primary Envs and sen-sitivity to broadly neutralizing antibodies. For this purpose, we

FIGURE 3. Analysis of bystander apoptosis-inducing activity of primary Envs derived from HIV-infected patients. A high throughput assay was used todetermine bystander apoptosis induction of a panel of HIV-1 Envs as described in the legend to Fig. 1. The YU-2 Env (100%) was used as a normalizing control,whereas cells transfected with empty pcDNA3.1 vector were used as background, which was subtracted from all wells for data analysis. A, the graph representspooled data from three independent experiments with each well assayed in triplicate in each experiment. B, classification of the apoptotic potential of eachprimary Env into high, medium, or low apoptosis inducers based on the caspase assay data from A. C, classification of the apoptotic potential of each primaryEnv based on the virus subtype. D, detection of Env expression via HIV Ig-mediated immunoprecipitation. Error bars, S.D.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

used a mixture of the three well characterized broadly neutral-izing antibodies 2F5, 2G12, and IgGb12 commonly referred toas TriMab mix. We utilized HIV particles pseudotyped with thepanel of HIV Envs to infect the TZM-bl target cell line in thepresence of serial dilutions of the TriMab antibody mix (Table1). Interestingly, the low apoptosis-inducing Envs showed atrend toward being more resistant to inhibition via neutralizingantibodies (Table 1 and Fig. 6A). In this analysis, the Envs thatshowed no inhibition to the TriMab mix were excluded becausean IC50 could not be calculated. Interestingly, when classifiedbased on virus subtype, the non-BC (comprising largely of AGsubtype) type isolates showed higher resistance to neutraliza-tion when compared with the B subtype (Fig. 6B). It is worthnoting that subtype B has been shown to be more susceptible tobroadly neutralizing antibodies like b12 (62) used in our study,and hence it is not surprising that the IC50 values for this sub-type were low. Several previous studies have reported a corre-lation between PNGS and neutralizing sensitivity, wherebyhigher PNGS in Env correlated with increased resistance toneutralizing antibodies (32–34, 63). Although the most signifi-cant association of bystander apoptosis was found to be withPNGS, the indirect correlation of PNGS and neutralizing anti-

body sensitivity reported by others (32–34, 63) is indicative ofpossible selective pressure on the virus altering its bystanderapoptosis-inducing phenotype. However, this is largely specu-lative because data are limited in this regard in our study.

Sequence Analysis of Primary Envs Reveals Several ResiduesAssociated with Bystander Apoptosis-inducing Activity—Usinggenotypic information to predict the co-receptor usage ofprimary HIV Envs is a routine practice (64). The co-receptorprediction software programs utilize sequence informationgathered from numerous laboratory experiments using pseu-dotyped virus-based infection assays as a standard. Based onearly findings, a set of rules has been established for predictionof CXCR4/CCR5 co-receptor usage by HIV isolates. Theserules include the V3 loop sequence and charge rules (65). How-ever, there is very limited information on the Env sequencechanges that can predict or may be associated with pathogenic-ity of primary Envs. Recently, Sterjovski et al. (66) demon-strated that the presence of Asn-362 in the HIV gp120 regionwas associated with increased cell-to-cell fusion activity. Wehence asked whether there were specific amino acids in the HIVEnv that could be associated with bystander apoptosis induc-tion. A search for distinctive physicochemical properties

FIGURE 4. Single cycle infectivity of primary Envs derived from HIV-infected patients. Different Envs were used to generate pseudotyped virus particlesthat were used to determine relative infectivity in the TZM-bl cell line. A, bars represent pooled data (mean � S.E. (error bars)) of duplicate or triplicateobservations from three independent experiments. B, classification of infectivity of each primary Env based on high, medium, or low apoptosis inducers (*, p �0.05). C, classification of infectivity of each primary Env based on the virus subtype. D, graph depicting variation in apoptosis and infectivity of selected Envs.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

between pairs of phylogenetically close high-low apoptosisstrains (Fig. 7 and Supplemental Fig. 1) revealed several impor-tant residues that may be relevant to the bystander apoptosis-inducing phenotype. Twelve sites (134, 139, and 189 (in the V1loop); 328, 330, 332, and 334 (V3 loop and vicinity); and 362,

363, 425, 462, and 476 (adjacent to the CD4 binding site) (HXB2numbering)) were found (Table 2) in at least three pairs of high-low apoptosis strains with distinctive differences in amino acidresidues in the above positions. Notably, we found that Asn-362, which has previously been shown to be associated with

FIGURE 5. Bystander apoptosis-inducing activity of primary Envs correlates negatively with PNGS. PNGs were estimated using the sequence informationfor each Env and the N-Glycosite software available at the LANL database. The variable loop regions of gp120 are V1(6615– 6692), V2(6693– 6812), V3(7110 –7217), V4(7377–7478), and V5(7596 –7637), numbered according to the HxB2 nucleotide sequence. V1V2 length is the combined peptide length of the V1 andV2 loop regions. A site with the sequence motif NX(S/T)X, where X represents a non-proline amino acid, is used to predict PNGS. A, representation of PNGS ofall of the Envs used in the study. Distribution of PNGS in gp160 (B and E), gp120 (C and F), or gp41 (D and G) is based on low, medium, or high apoptosis-inducinggroups (B–D) or virus subtype (E–G) (*, p � 0.05).




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

increased fusogenicity of the Env glycoproteins (66), was pres-ent in a number of high apoptosis inducers in our analysis (Fig.7 and supplemental Fig. 1).

To further our analysis and to avoid complications fromfounder effects through amino acid differences that may ariseas a result of separate evolution of the different subtypes, weconsidered the relative phylogenetic positions of all analyzedstrains. Thereafter, we selected genetically closely related pairswith differing apoptosis-inducing results to increase the speci-ficity of identifying mutations directly related to this phenotype(Fig. 8A). In essence, this approach identifies Envs that are mostclosely related in sequence but varied in phenotypic character-istics, thus classifying them as nearest pair neighbors. Forinstance, using this approach, Envs 11034 and 11035 werefound to be closely related in sequence but widely varied inapoptosis induction. This novel analysis revealed a number ofsites that may be associated with bystander apoptosis inductionwith two sites that stood out in particular. These include aminoacid positions 425 and 476. Interestingly, four of the nine highapoptosis inducers selectively showed the presence of an aspar-agine at position 425 by our pairwise analysis approach (Fig.8B). Additionally, position 476 was of particular interest (Fig.8C) because five of nine high-low apoptosis pairs possessed alysine in the low apoptosis inducers that was not found in any ofthe high apoptosis strains examined. In all nine pairs, the high

apoptosis group had an arginine at this position. In addition toour semimanual phylogeny and structure-aware candidateselection procedure, we also compared the results with therelated sequence harmony approach (67), which confirms ourtwo studied candidates as being highly significant (Fig. 8D andsupplemental Fig. 2). Using this comprehensive analysis, wewere able to identify several potential residues important forbystander apoptosis, of which residues 425 and 476 were mostpromising due to the reasons described below and hence cho-sen for further in depth analysis.

Structural Analysis of Candidates for Determinants of HIVEnv-mediated Bystander Apoptosis—Having identified specificamino acid residues that may be involved in bystander apopto-sis induction, we next conducted structural analysis to deter-mine the location of these residues with respect to the viral Env.Structural mapping of the above identified 12 candidate sitesonto Protein Data Bank structure 2QAD (52) revealed thatthese sites could be found in the V1 loop (positions 134, 139,and 189), V3 loop and its vicinity (positions 328, 330, 332, and334), and the CD4 binding site region (positions 362, 363, 425,462, and 476). For this study, we chose candidate sites thatcould play a role in directly affecting CD4 binding (positions362, 363, 425, and 476) for further experimental analysis.Because position 362 has already been shown to affect HIV cellfusion (66) and position 363 may possibly play a similar rolebecause it lies next to residue 362, we tested the other promis-ing positions, 425 and 476, for association with bystander apo-ptosis. Because both of these positions are within 5 Å of theCD4 binding site, residue changes R476K and N425R in thehigh apoptosis-inducing strains, converting them to low apo-ptosis inducers, were also expected to affect CD4 binding viasteric hindrance and also lead to charge alterations in the Env(Fig. 9).

Mutational Analysis Shows That R476K and N425R May BeRelevant to Bystander Apoptosis-inducing Activity of EnvGlycoprotein—Our sequence analysis data and molecular mod-eling identified specific amino acid residues that may be linkedto apoptosis induction by the primary HIV Env used in thisstudy. Of those, the most noticeable ones were Asn-425 andArg-476 because these sites are probably involved in CD4 bind-ing based on our structure analysis. To confirm the involve-ment of these residues in apoptosis induction, we used a site-directed mutagenesis approach to induce R476K and N425Rmutations in the high apoptosis-inducing Envs. As seen in Fig.9, A and B, the N425R and R476K mutations resulted in a loss offunction in some of the Envs tested. Whereas N425R showedthe most significant loss in two of the four Envs tested (Fig.10A), R476K was found to result in loss of function (apoptosis)in three of five Envs tested (Fig. 10B). These findings corrobo-rate the potential role of these two sites in determiningbystander apoptosis potential of Envs. To further corroborateour findings, we constructed a gain of function mutation in thecorresponding low apoptosis-inducing variant. Because Env11035 R476K showed the most significant loss of function in theexperiments above, we selected its low apoptosis-inducing coun-terpart (11034) to induce a K476R mutation. Interestingly, we sawa gain in apoptosis-inducing activity for 11034 K476R comparedwith the wild type counterpart 11034 (Fig. 10C). Although both

TABLE 1Concentration of TriMab antibody required for 50% infectivity inhibi-tion (IC50) of pseudotyped virus stocksData are the mean of duplicate observations. Name, classification of Envs as highmedium or low apoptosis inducers; Env. no, NIH-ARP designated catalog numberfor the Envs used in the study; Clade, virus subtype; NA, not available.

ID Name Env. no. Clade IC50

1 H1 11018 B 0.31232 H2 11314 C �253 H3 11605 AG 0.96854 H4 11604 AG 4.52555 H5 11310 C 1.14416 H6 11313 C �257 H7 11608 AG 1.06958 H8 11035 B 0.38389 H9 11600 CRF13_cpx �2510 M1 11312 C 1.393211 M2 11058 B 0.181312 M3 11601 AG 2.880913 M4 11023 B 0.11714 M5 11606 AG/A1 4.228715 M6 11024 B 0.186616 M7 11317 C 1.330717 M8 11037 B 0.482418 M9 11315 C NA19 L1 11602 AG 6.50E�0520 L2 11017 B 0.406721 L3 11033 B 0.636322 L4 11597 AG 1.470323 L5 11038 B 2.218824 L6 11022 B 0.7125 L7 11596 G 5.913526 L8 11034 B �2527 L9 11316 C 0.56928 L10 11307 C 0.360329 L11 11595 AG 2.542130 L12 11599 AG 2.000431 L13 11311 C �2532 L14 11306 C 0.336933 L15 11309 C 5.5934 L16 11036 B 0.168935 L17 11603 AE �2536 L18 11591 AG 1.251237 L19 11592 AG 0.200938 C YU-2 B 1.3525




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

N425R and R476K are associated with bystander apoptosis in ourstudy, R476K was also confirmed to be a bystander apoptosis-reg-ulating site via gain of function mutations. Env expression between

each pair was found to be identical (Fig. 10H), confirming thatchanges in Env bystander apoptosis are not likely to be due todifferences in expression levels.

FIGURE 6. Determination of neutralizing antibody sensitivity of primary Envs. Virus stocks pseudotyped with various Envs were used to infect TZM-bl cellsin the presence of different concentrations of the TriMab antibody mix as described under “Experimental Procedures.” Infection was determined 48 hpostinfection by measuring luciferase activity. A, classification of IC50 concentrations of the TriMab antibody for different Envs based on high, medium, or lowapoptosis inducers. B, classification of IC50 concentrations of the TriMab antibody for different Envs based on virus subtype. Due to availability of the TriMabantibody mix in limited amounts, the experiment was conducted once. For analysis based on apoptosis groups (A) and virus subtypes (B), Envs with an IC50 of�25 were excluded.

FIGURE 7. Sequence analysis of primary Envs. Sequences for the HIV Env proteins used in the study were downloaded from NCBI and aligned with MAFFT,and the respective motif regions were visualized in Jalview using ClustalX-like coloring based on physicochemical properties and conservation. Virus names areshown on the left with the NCBI accession numbers. This analysis revealed a number of amino acid differences between the high and low apoptosis inducers,which led to apoptosis-determining sites like those at positions 425, 462, and 476.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Because our approach involves a pairwise analysis of thenearest neighbors that are genetically similar but with differingapoptosis induction capability, we tested the various character-istics of 11034 and 11035 (R476K pair). A side by side compar-ison of this pair revealed maximum differences with respect toall of the characteristics studied like apoptosis (Fig. 10D), virusinfectivity (Fig. 10E), antibody neutralization (Fig. 10F), andPNGS (Fig. 10G). Thus, this pair represents the perfect scenariofor identification of characteristics associated with low versushigh apoptosis-inducing Envs. Also this pair corroborates manyof the associations revealed in our study, including virion infec-tivity, PNGS, neutralizing antibody sensitivity, and specificamino acids, in determining bystander apoptosis activity. Thisapproach also reveals that the Env phenotype in terms ofbystander apoptosis is probably quite complex, and multipleEnv regions and characteristics may collectively determinethe apoptosis outcome. Thus, studying genotypically closelyrelated Envs that show a diverse apoptosis phenotype is proba-bly the best approach for identifying the residues/genetic sig-natures important for this phenomenon.

Bystander Apoptosis Induction by Primary Envs RequiresCCR5 Binding as Well as gp41-mediated Membrane Fusion—The membrane fusing activity of HIV Env has long been sus-pected to have a role in HIV pathogenesis (68). Specifically, therole of the gp41 subunit in mediating bystander apoptosis hasonly recently been demonstrated (41, 59). Studies by our group

and others have shown that gp41-mediated fusion/hemifusionis critical for apoptosis induction, and binding of gp120,although required, cannot alone induce apoptotic signaling byHIV Env (40, 41, 59). Although studies with laboratory viruseshave provided proof of principle for this hypothesis (69), thereis a lack of data from primary Envs derived directly frompatients to corroborate the physiological relevance of thesefindings. We therefore asked whether bystander apoptosisinduction by primary Envs is also gp41-dependent. To answerthis question, we selected the highest apoptosis-inducing Envsfrom our panel and tested bystander apoptosis induction in thepresence or absence of CCR5 antagonist maraviroc and gp41inhibitor enfuvirtide. As seen in Fig. 11, both maraviroc andenfuvirtide inhibited bystander apoptosis by all of the primaryEnvs. Inhibition by CCR5 inhibitor confirms a role of gp120binding to the co-receptor as a requirement for apoptosisinduction, whereas inhibition by enfuvirtide is indicative of acritical role played by the gp41 unit in this phenomenon. Thesefindings are consistent with previous studies and further sup-port the role of HIV gp41-mediated fusion in apoptosis induc-tion not only with laboratory isolates but also primary viruses.Furthermore, this also suggests that the mechanism ofbystander apoptosis by primary and laboratory variants of HIVis probably the same.

DISCUSSION

HIV infection leads to a selective depletion of CD4� T helpercells, resulting in immunodeficiency. However, the mechanismby which HIV mediates this process remains highly debated (2,6, 12, 70 –73) and forms the basis of our study. It is proposedthat the loss of CD4 cells during HIV infection is due to theprocess of bystander apoptosis (2). This is supported by earlystudies conducted by Finkel et al. (9), who demonstrated thatthe majority of cells undergoing apoptosis during HIV infectionare not infected but are in close proximity to infected cells. Arole of direct infection in the loss of CD4 cells is also refuted bynatural infections in sooty mangabeys with the simian SIVsm,where high levels of infection and viremia do not result in CD4loss or AIDS development (74). Hence, bystander apoptosis isbelieved to be one of the major causes of CD4 loss leading toAIDS (6, 70, 75). Bystander apoptosis is a phenomenon that hasbeen demonstrated in various animal models of HIV, includingthe SIV model in rhesus macaques (4, 10) and more recently inthe humanized mouse model (11). Interestingly, bystander apo-ptosis induction in these models correlates with CD4 loss, fur-ther supporting the role of this phenomenon in diseaseprogression.

The mechanism of bystander apoptosis of CD4 T cells in HIVinfection remains unresolved. Interestingly, the role of the Envglycoprotein in this process is becoming increasingly evident(12, 13). This is largely supported by the arguments that 1) ascell death in HIV infection outnumbers the infected cell popu-lation, a role of bystander cell death in the progression to AIDSis likely; 2) as the depletion of immune cells is restricted toCD4� helper phenotype and as the Env glycoprotein of HIVbinds to CD4, it probably plays a role either directly or indi-rectly in CD4 T cell death; and 3) the Env glycoprotein is theonly viral protein expressed on the surface of infected cells and

TABLE 2Amino acid residues that may be associated with bystander apoptosisinduction using the approach delineated in Figs. 7 and 8The region of those residues identified, their amino acid phenotype, and their struc-tural attributes are listed. Position, amino acid position based on HXB2 proteinsequence starting from the first methionine residue of GP120; 3U2S, crystal struc-ture of PG9 Fab in complex with the V1V2 region from HIV-1 strain ZM109 (52);2QAD, structure of tyrosine-sulfated 412d antibody complexed with HIV-1 YU2gp120 and CD4 (53); 2B4C, crystal structure of HIV-1 JR-FL gp120 core proteincontaining the third variable region (V3) complexed with CD4 and the X5 antibody(54); NAG, N-acetylglucosamine; AI, apoptosis inducers.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

has been shown to interact with and mediate apoptosis inbystander cells, albeit only in in vitro studies (41, 59, 60, 76).

With support from in vitro studies, understanding thegenetic signatures associated with the bystander apoptosis-in-ducing phenotype of the Env glycoprotein is of increasingimportance. Although the phenotypic and genotypic character-ization of HIV Env has been well documented for co-receptorusage and neutralizing antibody sensitivity, there is virtually noinformation on the phenotypic characterization of HIV Env forthe phenomenon of bystander apoptosis induction. We havepreviously developed cell lines that express varying levels of theHIV co-receptor CCR5 (44). Using these cell lines and a labo-ratory-adapted YU-2 clone of HIV, we studied the bystanderapoptosis-inducing activity of HIV Env and the role of CCR5co-receptor levels on this phenomenon. Our findings suggestthat the bystander apoptosis induction is dependent on the Envfusogenic activity as well as the surface CCR5 expression levelson cells (44). Although this study provided valuable insightsinto the mechanism of R5 Env-mediated apoptosis, the physio-logical relevance of these findings remains untested. Hence, weundertook this study with the objectives to understand whetherand how primary HIV Env variants induce apoptosis inbystander cells, whether there is variability in bystander apo-ptosis induction between different HIV subtypes, and whetherspecific genetic signatures in the Env glycoprotein are associ-ated with increased bystander apoptosis phenotype.

To achieve these objectives, we developed a high throughputassay that can determine the relative bystander apoptosis-in-ducing activity of primary HIV-1 Envs. Our caspase-based assaywas highly reproducible and correlated well with other meth-ods of apoptosis determination. More importantly, the assay

could distinguish small changes in bystander apoptosis-induc-ing activity between various HIV Env variants tested. With thehelp of this assay, an important characteristic of HIV Env can beadded to the profile of patient viruses.

We found that the bystander apoptosis-inducing activity ofprimary Envs of R5 tropism varies considerably. Although wedid not find subtype-specific differences in our panel, it shouldbe noted that the assay was done using a reference panel of Envsthat represents a highly diverse set of Envs. Whether certainsubtypes contain high apoptosis-inducing Envs that are over-represented in the population can only be determined by study-ing larger populations of Envs directly from patients. We alsofound that virion infectivity correlated with bystander apopto-sis-inducing activity in our panel. This does not come as a sur-prise because both virion infectivity and bystander apoptosisare dependent on the function of the Env glycoprotein.Although as a group, the Envs showed these differences, thebystander apoptosis and virion infectivity of individual Envs didnot necessarily correlate in every case. We did find some Envsthat showed high infectivity but low bystander apoptosis andvice versa. These findings underscore both the genotypic andphenotypic variability of Envs seen in patients. This also sug-gests that in some cases, the viruses may be capable of infection/replication in the absence of inducing bystander apoptosis.Such non-pathogenic viruses probably contain changes in theEnv glycoprotein that alter their apoptosis phenotype whilemaintaining virion infectivity. This is consistent with our pre-vious findings, whereby a single amino acid change in the Envglycoprotein altered bystander apoptosis and CD4 loss in ahumanized mouse model of HIV infection (11) while maintain-ing virus infectivity. However, from a viral evolution point ofview, the acquisition of bystander apoptosis-inducing activityby a virus must come with some selective advantage, and in thiscase, it might be increased fitness/infectivity. This increasedfitness may help in virus dissemination via infection with cell-free virus in different anatomical sites.

Putative N-glycosylation sites have been shown to vary con-siderably in Env glycoproteins from HIV-infected patientsbased on the stage of the disease (18, 33). During the chronicphase, HIV Env has been shown to contain more PNGS com-pared with the acute phase of infection (33). This same charac-teristic is reflected in viruses isolated from late stages of thedisease, whereby loss of PNGS is observed during late versuschronic viruses (32). Furthermore, the presence of higher gly-cosylation or PNGS has also been associated with reduced viralfitness (32). The question that arises is what mediates theincrease in PNGS during the chronic phase of HIV infections.The leading hypothesis is that the neutralizing antibody pres-sure is the primary driving force behind PNGS evolution inHIV. This is supported by the fact that the presence of PNGS on

FIGURE 8. Phylogenetic analysis of primary Envs to identify sequences that share genetic signatures but vary in apoptosis induction. A, phylogeny ofprimary Envs by a maximum likelihood method based on the HKY model conducted in MEGA5 (51). �-Distribution (five categories) with shape parameter(0.4327) was used to model evolutionary rate differences among the sites. Pairs of high-low bystander apoptosis are colored with the same color. Shown aresequence signatures of high and low apoptosis inducers at amino acid positions 425 (B) and 476 (C). The high apoptosis-inducing Envs predominantly bear anAsn at position 425 and an Arg at position 476, whereas low apoptosis-inducing Envs bear an Arg at position 425 and a Lys at position 476. D, sequenceharmony score coloring of GP120 in complex with CD4. The red colored residue is sequence harmony’s best estimate of an important residue distinguishinghigh/low apoptosis, but based on its structural position away from CD4, such an effect is not intuitively clear. The further ranking can be seen by color transitionfrom red to white to blue.

FIGURE 9. Structure (Protein Data Bank entry 2B4C) of GP120 (gray rib-bon) binding to CD4 (yellow ribbon) rendered using Yasara (55). Arg-476(red residue) from gp120 is in close proximity with Gln-25 and Ser-23 (pinkresidues) from CD4. Asn-425 (blue residue) from GP120 is also within 5 Å ofPhe-43 and Arg-59 (light blue residues) from CD4.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

HIV Env is responsible for masking the epitopes and increasingneutralization resistance (33–35). However, this phenomenonmay also come at the price of reduced viral fitness (77), andhence, when the opportunity arises, as in the case of late stageswhere the immunocompromised patients are no longer able tomount an effective neutralizing antibody response, the virusreverts to low PNGS, increased fitness, and possibly increasedbystander apoptosis phenotype.

Our study demonstrates that many of the characteristics ofEnv glycoprotein, including PNGS, neutralizing antibody sen-

sitivity, and viral fitness, that have previously been shown tocorrelate with CD4 loss and disease progression are also inci-dentally associated with bystander apoptosis induction. Thepreliminary hypothesis that emerges from our study is that dur-ing the chronic phase of the disease, immune pressure selectsfor Envs with higher PNGS. This potentially results in a loss/reduction in bystander apoptosis, which may be reflected in theslower depletion of CD4� T cells seen during the chronic phase.Subsequently, the gradual loss of CD4 cells results in an immu-nocompromised state that paves the way for the viruses with

FIGURE 10. Mutation of residues Arg-476 and Asn-425 to R476K and N425R, respectively, leads to a reduction in Env-mediated bystander apoptosis-inducing activity. HeLa cells transfected with WT Env clones or their corresponding N425R (A) or R476K (B) counterparts were assayed for bystander apoptosisinduction as described in Fig. 1. C, gain of function analysis for the 11034 and 11035 Env pairs. HeLa cells transfected with the indicated WT DNAs or their gainof function counterpart mutants were analyzed for bystander apoptosis induction. Data represent mean � S.D. (error bars) from triplicate observations. Shownis pairwise analysis of the nearest neighbors 11034 and 11035 for various Env characteristics like apoptosis (D), virus infectivity (E), antibody neutralization (F),and PNGs (G). This pair represents the perfect case scenario for all of the parameters studied. H, detection of Env expression for various WT and mutant Env pairsused in the study by HIV Ig-mediated immunoprecipitation.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

lower PNGS, increased fitness, and possibly higher bystanderapoptosis-inducing phenotype to emerge and precipitate arapid CD4 loss. Although our study only provides circumstan-tial evidence of this correlation, it does open the door to furtherstudies focused on this aspect.

Interestingly, most of the PNGS differences as well as the 12most relevant sites found to be associated with the apoptosisphenotype were in the gp120 region, with many mappingaround the CD4 binding site. The fusion process mediated byEnv is a concerted effort between both the gp120 and the gp41subunits of HIV Env. Because the gp120 subunit is exposed tohost defense mechanisms, it is not surprising that the largestvariability is seen in this subunit, possibly via antibody pressure.It is also not surprising that most of the changes associated withbystander apoptosis induction also map to gp120. Supportingthese findings, Holm et al. (78) demonstrated that changes inthe gp120 co-receptor binding domain can affect Env functionand bystander apoptosis.

As mentioned above, one of our major objectives was also todetermine whether certain HIV phenotypic and genotypic sig-natures correlate with bystander apoptosis and, more specifi-cally, whether sequence analysis alone may be able to predictthe bystander apoptosis-inducing phenotype of HIV Envs. Tothis end, we adopted a novel pairwise analysis approach thatfocused on phylogenetically closely related Env pairs with dif-fering apoptosis activity in an attempt to identify candidateamino acid signatures related to this unique phenotype. TheEnv fusogenic activity has been associated with a specific stageof disease and specific residues like Asn-362 (66). Recently,Wade et al. (27) demonstrated that Env glycoproteins derivedfrom late stage patients are more fusogenic and induceenhanced CD4� T cell apoptosis compared with early stageEnvs. In our study, we identified a total of 12 amino acid posi-tions that were potentially associated with high bystander apo-ptosis-inducing Envs, including the previously mapped Asn-362 linked to increased fusogenicity. Among these, R476K andN425R were identified as novel sites through our pairwise anal-ysis and molecular modeling. We also confirmed the potential

role of these amino acid signatures experimentally using site-directed mutagenesis.

The primary function of HIV Env is to mediate fusion of theviral and cellular membranes to facilitate entry of the virus coreinto cells. However, the same phenomenon can also mediatefusion between an infected and uninfected bystander cell. Thisfusion process and the intermediate of this phenomenonreferred to as hemifusion (kiss of death) have been shown to bethe primary cause of bystander apoptosis in in vitro culturesystems (40, 41, 59, 79). Most of the data supporting the role ofEnv-mediated fusion/hemifusion in bystander apoptosis comesfrom laboratory-adapted viruses. Hence, in this study, we alsoasked whether bystander apoptosis mediated by primary HIV-1Envs is dependent on the Env fusogenic activity or gp41 func-tion. We indeed demonstrate that bystander apoptosis induc-tion by primary Envs is dependent on gp41 function (Envfusogenic activity). Interestingly, fusogenic activity of Env gly-coprotein has been indirectly associated with HIV pathogenesisin clinical studies where the presence of a highly fusogenic syn-cytia-inducing phenotype has been associated with poor prog-nosis in patients (80, 81), increased pathogenesis (23), andCXCR4 (82) tropism of viruses. The syncytia-inducing pheno-type has also been associated with increased pathogenesis in theSCID-hu mouse model. In SCID-hu mice, the lack of CD4 T cellloss by certain CCR5 tropic laboratory strains underscores thisdifference (83). However, it has also been demonstrated thatCCR5 tropic viruses from late stages of disease are more fuso-genic, which correlates with pathogenicity (26). This is furthersupported by recent studies by Wade et al. (27), demonstratingthe apoptotic potential of late stage R5 viruses. Probably thestrongest evidence for a role of Env membrane-fusing activityand HIV pathogenesis and CD4 decline comes from SHIV stud-ies in rhesus macaques, where the pathogenicity of the SHIV-KB9 variant can be linked directly to the membrane fusionactivity of the virus (84 – 88). The fact that primary Env-medi-ated bystander apoptosis in our study could be inhibited byinhibiting Env-mediated fusion using a gp41 inhibitor adds fur-ther support for the role of membrane fusion in this phenome-non. Recently, Hunt et al. (89) have shown the clinical benefitsof maraviroc and ENF therapy that target Env, supporting therole of Env in HIV pathogenesis.

Our findings suggest that bystander apoptosis-inducingactivity of HIV Env may be an important phenotypic character-istic that warrants studies in a larger panel of Envs for tworeasons. 1) It will help strengthen the hypothesis that bystanderapoptosis is an important phenomenon for CD4 loss in HIVinfection. 2) With a larger panel of Envs, we will be able toobtain enough preliminary data to determine whether certaingenetic signatures are associated with bystander apoptosis,much in line with the current methods for determining co-re-ceptor tropism using sequence information alone.

Although the Env glycoprotein has previously been charac-terized for numerous phenotypic properties like infectivity, co-receptor use, neutralizing antibody sensitivity, and PNGS,bystander apoptosis induction is a unique phenotype of HIVthat has previously not been studied, probably due to a lack ofan appropriate high throughput assay. Because a number ofthese characteristics of the Env glycoprotein are associated with

FIGURE 11. Bystander apoptosis induction by primary Envs requires CCR5binding as well as gp41 function. HeLa cells were transfected with the indi-cated Envs. The next day, SupT-R5-H6 cells were added to each well in thepresence of medium alone, enfuvirtide (2 �M), or maraviroc (1 �M). After anovernight incubation, the cells were analyzed for caspase-3 activity using theCaspase-Glo 3/7 assay as described in the legend to Fig. 1. Data representmean � S.D. (error bars) of triplicate observations from three independentexperiments.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

disease progression and CD4 loss, our findings provide furthersupport for the phenomenon of bystander apoptosis in CD4loss and the role of Env glycoprotein in this process. The actualapoptotic potential of Env variants may best be calculated usingour assay and pairwise computational analysis. We believe thatusing this strategy, we will be able to generate a database thatcan be used for developing a methodology for predictingbystander apoptosis. Recently, a machine-learning method hasbeen developed for identifying genetic signatures in HIV Envpredictive of HIV-associated dementia (90) based on a databaseof HIV Env sequences from the brain (91). Using similar strat-egies, our study should lead to development of an HIV Envsequence database characterizing the bystander apoptosispotential of Envs. Our study establishes a first set of preliminaryrules like PNGS and specific amino acid signatures that can befurther developed into an algorithm. Ultimately, this shouldlead to determination of bystander apoptosis phenotypes ofEnvs by sequence analysis alone. Whether these findings wouldhave a diagnostic value remains to be seen.

Acknowledgments—We are grateful to the National Institutes ofHealth AIDS Research and Reference Reagent Program for supplyingvaluable reagents used in this study.

REFERENCES1. Gandhi, R. T., Chen, B. K., Straus, S. E., Dale, J. K., Lenardo, M. J., and

Baltimore, D. (1998) HIV-1 directly kills CD4� T cells by a Fas-independ-ent mechanism. J. Exp. Med. 187, 1113–1122

2. Garg, H., Mohl, J., and Joshi, A. (2012) HIV-1 induced bystander apopto-sis. Viruses 4, 3020 –3043

3. Douek, D. (2007) HIV disease progression. Immune activation, microbes,and a leaky gut. Top. HIV Med. 15, 114 –117

4. Meythaler, M., Martinot, A., Wang, Z., Pryputniewicz, S., Kasheta, M.,Ling, B., Marx, P. A., O’Neil, S., and Kaur, A. (2009) Differential CD4�

T-lymphocyte apoptosis and bystander T-cell activation in rhesus ma-caques and sooty mangabeys during acute simian immunodeficiency virusinfection. J. Virol. 83, 572–583

5. van Gils, M. J., Edo-Matas, D., Bowles, E. J., Burger, J. A., Stewart-Jones,G. B., and Schuitemaker, H. (2011) Evolution of human immunodefi-ciency virus type 1 in a patient with cross-reactive neutralizing activity inserum. J. Virol. 85, 8443– 8448

6. Gougeon, M. L., and Montagnier, L. (1993) Apoptosis in AIDS. Science260, 1269 –1270

7. Oyaizu, N., McCloskey, T. W., Than, S., Hu, R., and Pahwa, S. (1995)Mechanism of apoptosis in peripheral blood mononuclear cells of HIV-infected patients. Adv. Exp. Med. Biol. 374, 101–114

8. Oyaizu, N., McCloskey, T. W., Coronesi, M., Chirmule, N., Kalyanaraman,V. S., and Pahwa, S. (1993) Accelerated apoptosis in peripheral bloodmononuclear cells (PBMCs) from human immunodeficiency virus type-1infected patients and in CD4 cross-linked PBMCs from normal individu-als. Blood 82, 3392–3400

9. Finkel, T. H., Tudor-Williams, G., Banda, N. K., Cotton, M. F., Curiel, T.,Monks, C., Baba, T. W., Ruprecht, R. M., and Kupfer, A. (1995) Apoptosisoccurs predominantly in bystander cells and not in productively infectedcells of HIV- and SIV-infected lymph nodes. Nat. Med. 1, 129 –134

10. Meythaler, M., Pryputniewicz, S., and Kaur, A. (2008) Kinetics of T lym-phocyte apoptosis and the cellular immune response in SIVmac239-in-fected rhesus macaques. J. Med. Primatol. 37, 33– 45

11. Garg, H., Joshi, A., Ye, C., Shankar, P., and Manjunath, N. (2011) Singleamino acid change in gp41 region of HIV-1 alters bystander apoptosis andCD4 decline in humanized mice. Virol. J. 8, 34

12. Perfettini, J. L., Castedo, M., Roumier, T., Andreau, K., Nardacci, R., Pia-centini, M., and Kroemer, G. (2005) Mechanisms of apoptosis induction

by the HIV-1 envelope. Cell Death Differ. 12, 916 –92313. Ahr, B., Robert-Hebmann, V., Devaux, C., and Biard-Piechaczyk, M.

(2004) Apoptosis of uninfected cells induced by HIV envelope glycopro-teins. Retrovirology 1, 12

14. Shankarappa, R., Margolick, J. B., Gange, S. J., Rodrigo, A. G., Upchurch,D., Farzadegan, H., Gupta, P., Rinaldo, C. R., Learn, G. H., He, X., Huang,X. L., and Mullins, J. I. (1999) Consistent viral evolutionary changes asso-ciated with the progression of human immunodeficiency virus type 1 in-fection. J. Virol. 73, 10489 –10502

15. Edo-Matas, D., Rachinger, A., Setiawan, L. C., Boeser-Nunnink, B. D., van’t Wout, A. B., Lemey, P., and Schuitemaker, H. (2012) The evolution ofhuman immunodeficiency virus type-1 (HIV-1) envelope molecular prop-erties and coreceptor use at all stages of infection in an HIV-1 donor-recipient pair. Virology 422, 70 – 80

16. Navis, M., Matas, D. E., Rachinger, A., Koning, F. A., van Swieten, P.,Kootstra, N. A., and Schuitemaker, H. (2008) Molecular evolution of hu-man immunodeficiency virus type 1 upon transmission between humanleukocyte antigen disparate donor-recipient pairs. PLoS One 3, e2422

17. Hahn, B. H., Gonda, M. A., Shaw, G. M., Popovic, M., Hoxie, J. A., Gallo,R. C., and Wong-Staal, F. (1985) Genomic diversity of the acquired im-mune deficiency syndrome virus HTLV-III. Different viruses exhibitgreatest divergence in their envelope genes. Proc. Natl. Acad. Sci. U.S.A.82, 4813– 4817

18. Curlin, M. E., Zioni, R., Hawes, S. E., Liu, Y., Deng, W., Gottlieb, G. S., Zhu,T., and Mullins, J. I. (2010) HIV-1 envelope subregion length variationduring disease progression. PLoS Pathog. 6, e1001228

19. Shankarappa, R., Gupta, P., Learn, G. H., Jr., Rodrigo, A. G., Rinaldo, C. R.,Jr., Gorry, M. C., Mullins, J. I., Nara, P. L., and Ehrlich, G. D. (1998) Evo-lution of human immunodeficiency virus type 1 envelope sequences ininfected individuals with differing disease progression profiles. Virology241, 251–259

20. Gray, L., Roche, M., Churchill, M. J., Sterjovski, J., Ellett, A., Poumbourios,P., Sherieff, S., Wang, B., Saksena, N., Purcell, D. F., Wesselingh, S., Cun-ningham, A. L., Brew, B. J., Gabuzda, D., and Gorry, P. R. (2009) Tissue-specific sequence alterations in the human immunodeficiency virus type 1envelope favoring CCR5 usage contribute to persistence of dual-tropicvirus in the brain. J. Virol. 83, 5430 –5441

21. Schuitemaker, H., van ’t Wout, A. B., and Lusso, P. (2011) Clinical signif-icance of HIV-1 coreceptor usage. J. Transl. Med. 9, S5

22. Edo-Matas, D., van Dort, K. A., Setiawan, L. C., Schuitemaker, H., andKootstra, N. A. (2011) Comparison of in vivo and in vitro evolution ofCCR5 to CXCR4 coreceptor use of primary human immunodeficiencyvirus type 1 variants. Virology 412, 269 –277

23. Schuitemaker, H., Koot, M., Kootstra, N. A., Dercksen, M. W., de Goede,R. E., van Steenwijk, R. P., Lange, J. M., Schattenkerk, J. K., Miedema, F.,and Tersmette, M. (1992) Biological phenotype of human immunodefi-ciency virus type 1 clones at different stages of infection. Progression ofdisease is associated with a shift from monocytotropic to T-cell-tropicvirus population. J. Virol. 66, 1354 –1360

24. Jansson, M., Backstrom, E., Bjorndal, A., Holmberg, V., Rossi, P., Fenyo,E. M., Popovic, M., Albert, J., and Wigzell, H. (1999) Coreceptor usage andRANTES sensitivity of non-syncytium-inducing HIV-1 isolates obtainedfrom patients with AIDS. J. Hum. Virol. 2, 325–338

25. Esbjornsson, J., Månsson, F., Martınez-Arias, W., Vincic, E., Biague, A. J.,da Silva, Z. J., Fenyo, E. M., Norrgren, H., and Medstrand, P. (2010) Fre-quent CXCR4 tropism of HIV-1 subtype A and CRF02_AG during late-stage disease. Indication of an evolving epidemic in West Africa. Retrovi-rology 7, 23

26. Olivieri, K., Scoggins, R. M., Bor, Y. C., Matthews, A., Mark, D., Taylor,J. R., Jr., Chernauskas, D., Hammarskjold, M. L., Rekosh, D., and Camerini,D. (2007) The envelope gene is a cytopathic determinant of CCR5 tropicHIV-1. Virology 358, 23–38

27. Wade, J., Sterjovski, J., Gray, L., Roche, M., Chiavaroli, L., Ellett, A., Jako-bsen, M. R., Cowley, D., Pereira Cda, F., Saksena, N., Wang, B., Purcell,D. F., Karlsson, I., Fenyo, E. M., Churchill, M., and Gorry, P. R. (2010)Enhanced CD4� cellular apoptosis by CCR5-restricted HIV-1 envelopeglycoprotein variants from patients with progressive HIV-1 infection. Vi-rology 396, 246 –255




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

28. Bjorndal, A., Sonnerborg, A., Tscherning, C., Albert, J., and Fenyo, E. M.(1999) Phenotypic characteristics of human immunodeficiency virus type1 subtype C isolates of Ethiopian AIDS patients. AIDS Res. Hum. Retrovi-ruses 15, 647– 653

29. McDermott, A. B., and Koup, R. A. (2012) CD8� T cells in preventing HIVinfection and disease. AIDS 26, 1281–1292

30. Streeck, H., and Nixon, D. F. (2010) T cell immunity in acute HIV-1 infec-tion. J. Infect. Dis. 202, S302–S308

31. Mosier, D. E. (2005) HIV-1 envelope evolution and vaccine efficacy. Curr.Drug Targets Infect. Disord. 5, 171–177

32. Borggren, M., Repits, J., Sterjovski, J., Uchtenhagen, H., Churchill, M. J.,Karlsson, A., Albert, J., Achour, A., Gorry, P. R., Fenyo, E. M., and Jansson,M. (2011) Increased sensitivity to broadly neutralizing antibodies of end-stage disease R5 HIV-1 correlates with evolution in Env glycosylation andcharge. PLoS One 6, e20135

33. Sagar, M., Wu, X., Lee, S., and Overbaugh, J. (2006) Human immunodefi-ciency virus type 1 V1-V2 envelope loop sequences expand and add gly-cosylation sites over the course of infection, and these modifications affectantibody neutralization sensitivity. J. Virol. 80, 9586 –9598

34. van Gils, M. J., Bunnik, E. M., Boeser-Nunnink, B. D., Burger, J. A., Ter-louw-Klein, M., Verwer, N., and Schuitemaker, H. (2011) Longer V1V2region with increased number of potential N-linked glycosylation sites inthe HIV-1 envelope glycoprotein protects against HIV-specific neutraliz-ing antibodies. J. Virol. 85, 6986 – 6995

35. Wei, X., Decker, J. M., Wang, S., Hui, H., Kappes, J. C., Wu, X., Salazar-Gonzalez, J. F., Salazar, M. G., Kilby, J. M., Saag, M. S., Komarova, N. L.,Nowak, M. A., Hahn, B. H., Kwong, P. D., and Shaw, G. M. (2003) Anti-body neutralization and escape by HIV-1. Nature 422, 307–312

36. Li, S., Juarez, J., Alali, M., Dwyer, D., Collman, R., Cunningham, A., andNaif, H. M. (1999) Persistent CCR5 utilization and enhanced macrophagetropism by primary blood human immunodeficiency virus type 1 isolatesfrom advanced stages of disease and comparison to tissue-derived isolates.J. Virol. 73, 9741–9755

37. Gray, L., Sterjovski, J., Churchill, M., Ellery, P., Nasr, N., Lewin, S. R.,Crowe, S. M., Wesselingh, S. L., Cunningham, A. L., and Gorry, P. R.(2005) Uncoupling coreceptor usage of human immunodeficiency virustype 1 (HIV-1) from macrophage tropism reveals biological properties ofCCR5-restricted HIV-1 isolates from patients with acquired immunode-ficiency syndrome. Virology 337, 384 –398

38. Thomas, E. R., Dunfee, R. L., Stanton, J., Bogdan, D., Taylor, J., Kunstman,K., Bell, J. E., Wolinsky, S. M., and Gabuzda, D. (2007) Macrophage entrymediated by HIV Envs from brain and lymphoid tissues is determined bythe capacity to use low CD4 levels and overall efficiency of fusion. Virology360, 105–119

39. Sterjovski, J., Churchill, M. J., Roche, M., Ellett, A., Farrugia, W., Wessel-ingh, S. L., Cunningham, A. L., Ramsland, P. A., and Gorry, P. R. (2011)CD4-binding site alterations in CCR5-using HIV-1 envelopes influencinggp120-CD4 interactions and fusogenicity. Virology 410, 418 – 428

40. Garg, H., Joshi, A., Freed, E. O., and Blumenthal, R. (2007) Site-specificmutations in HIV-1 gp41 reveal a correlation between HIV-1-mediatedbystander apoptosis and fusion/hemifusion. J. Biol. Chem. 282, 16899–16906

41. Garg, H., and Blumenthal, R. (2006) HIV gp41-induced apoptosis is me-diated by caspase-3-dependent mitochondrial depolarization, which is in-hibited by HIV protease inhibitor nelfinavir. J. Leukoc. Biol. 79, 351–362

42. Meissner, E. G., Coffield, V. M., and Su, L. (2005) Thymic pathogenicity ofan HIV-1 envelope is associated with increased CXCR4 binding efficiencyand V5-gp41-dependent activity, but not V1/V2-associated CD4 bindingefficiency and viral entry. Virology 336, 184 –197

43. Meissner, E. G., Duus, K. M., Gao, F., Yu, X. F., and Su, L. (2004) Charac-terization of a thymus-tropic HIV-1 isolate from a rapid progressor. Roleof the envelope. Virology 328, 74 – 88

44. Joshi, A., Nyakeriga, A. M., Ravi, R., and Garg, H. (2011) HIV ENV glyco-protein-mediated bystander apoptosis depends on expression of theCCR5 co-receptor at the cell surface and ENV fusogenic activity. J. Biol.Chem. 286, 36404 –36413

45. Platt, E. J., Wehrly, K., Kuhmann, S. E., Chesebro, B., and Kabat, D. (1998)Effects of CCR5 and CD4 cell surface concentrations on infections by

macrophagetropic isolates of human immunodeficiency virus type 1. J. Vi-rol. 72, 2855–2864

46. Kulkarni, S. S., Lapedes, A., Tang, H., Gnanakaran, S., Daniels, M. G.,Zhang, M., Bhattacharya, T., Li, M., Polonis, V. R., McCutchan, F. E.,Morris, L., Ellenberger, D., Butera, S. T., Bollinger, R. C., Korber, B. T.,Paranjape, R. S., and Montefiori, D. C. (2009) Highly complex neutraliza-tion determinants on a monophyletic lineage of newly transmitted sub-type C HIV-1 Env clones from India. Virology 385, 505–520

47. Li, M., Gao, F., Mascola, J. R., Stamatatos, L., Polonis, V. R., Koutsoukos,M., Voss, G., Goepfert, P., Gilbert, P., Greene, K. M., Bilska, M., Kothe,D. L., Salazar-Gonzalez, J. F., Wei, X., Decker, J. M., Hahn, B. H., andMontefiori, D. C. (2005) Human immunodeficiency virus type 1 envclones from acute and early subtype B infections for standardized assess-ments of vaccine-elicited neutralizing antibodies. J. Virol. 79, 10108–10125

48. Wei, X., Decker, J. M., Liu, H., Zhang, Z., Arani, R. B., Kilby, J. M., Saag,M. S., Wu, X., Shaw, G. M., and Kappes, J. C. (2002) Emergence of resistanthuman immunodeficiency virus type 1 in patients receiving fusion inhib-itor (T-20) monotherapy. Antimicrob. Agents Chemother. 46, 1896 –1905

49. Derdeyn, C. A., Decker, J. M., Bibollet-Ruche, F., Mokili, J. L., Muldoon,M., Denham, S. A., Heil, M. L., Kasolo, F., Musonda, R., Hahn, B. H., Shaw,G. M., Korber, B. T., Allen, S., and Hunter, E. (2004) Envelope-constrainedneutralization-sensitive HIV-1 after heterosexual transmission. Science303, 2019 –2022

50. Williamson, C., Morris, L., Maughan, M. F., Ping, L. H., Dryga, S. A.,Thomas, R., Reap, E. A., Cilliers, T., van Harmelen, J., Pascual, A., Ramjee,G., Gray, G., Johnston, R., Karim, S. A., and Swanstrom, R. (2003) Char-acterization and selection of HIV-1 subtype C isolates for use in vaccinedevelopment. AIDS Res. Hum. Retroviruses 19, 133–144

51. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S.(2011) MEGA5. Molecular evolutionary genetics analysis using maximumlikelihood, evolutionary distance, and maximum parsimony methods.Mol. Biol. Evol. 28, 2731–2739

52. Huang, C. C., Lam, S. N., Acharya, P., Tang, M., Xiang, S. H., Hussan, S. S.,Stanfield, R. L., Robinson, J., Sodroski, J., Wilson, I. A., Wyatt, R., Bewley,C. A., and Kwong, P. D. (2007) Structures of the CCR5 N terminus and ofa tyrosine-sulfated antibody with HIV-1 gp120 and CD4. Science 317,1930 –1934

53. Huang, C. C., Tang, M., Zhang, M. Y., Majeed, S., Montabana, E., Stanfield,R. L., Dimitrov, D. S., Korber, B., Sodroski, J., Wilson, I. A., Wyatt, R., andKwong, P. D. (2005) Structure of a V3-containing HIV-1 gp120 core. Sci-ence 310, 1025–1028

54. McLellan, J. S., Pancera, M., Carrico, C., Gorman, J., Julien, J. P., Khayat, R.,Louder, R., Pejchal, R., Sastry, M., Dai, K., O’Dell, S., Patel, N., Shahzad-ul-Hussan, S., Yang, Y., Zhang, B., Zhou, T., Zhu, J., Boyington, J. C.,Chuang, G. Y., Diwanji, D., Georgiev, I., Kwon, Y. D., Lee, D., Louder,M. K., Moquin, S., Schmidt, S. D., Yang, Z. Y., Bonsignori, M., Crump, J. A.,Kapiga, S. H., Sam, N. E., Haynes, B. F., Burton, D. R., Koff, W. C., Walker,L. M., Phogat, S., Wyatt, R., Orwenyo, J., Wang, L. X., Arthos, J., Bewley,C. A., Mascola, J. R., Nabel, G. J., Schief, W. R., Ward, A. B., Wilson, I. A.,and Kwong, P. D. (2011) Structure of HIV-1 gp120 V1/V2 domain withbroadly neutralizing antibody PG9. Nature 480, 336 –343

55. Krieger, E., Koraimann, G., and Vriend, G. (2002) Increasing the precisionof comparative models with YASARA NOVA. A self-parameterizing forcefield. Proteins 47, 393– 402

56. Eswar, N., Eramian, D., Webb, B., Shen, M. Y., and Sali, A. (2008) Proteinstructure modeling with MODELLER. Methods Mol. Biol. 426, 145–159

57. Li, H., Robertson, A. D., and Jensen, J. H. (2005) Very fast empirical pre-diction and rationalization of protein pKa values. Proteins 61, 704 –721

58. Katoh, K., Kuma, K., Toh, H., and Miyata, T. (2005) MAFFT version 5.Improvement in accuracy of multiple sequence alignment. Nucleic AcidsRes. 33, 511–518

59. Blanco, J., Barretina, J., Ferri, K. F., Jacotot, E., Gutierrez, A., Armand-Ugon, M., Cabrera, C., Kroemer, G., Clotet, B., and Este, J. A. (2003)Cell-surface-expressed HIV-1 envelope induces the death of CD4 T cellsduring GP41-mediated hemifusion-like events. Virology 305, 318 –329

60. Biard-Piechaczyk, M., Robert-Hebmann, V., Richard, V., Roland, J., Hip-skind, R. A., and Devaux, C. (2000) Caspase-dependent apoptosis of cells




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

expressing the chemokine receptor CXCR4 is induced by cell membrane-associated human immunodeficiency virus type 1 envelope glycoprotein(gp120). Virology 268, 329 –344

61. Garg, H., Joshi, A., and Blumenthal, R. (2009) Altered bystander apoptosisinduction and pathogenesis of enfuvirtide-resistant HIV type 1 Env mu-tants. AIDS Res. Hum. Retroviruses 25, 811– 817

62. Gnanakaran, S., Daniels, M. G., Bhattacharya, T., Lapedes, A. S., Sethi, A.,Li, M., Tang, H., Greene, K., Gao, H., Haynes, B. F., Cohen, M. S., Shaw,G. M., Seaman, M. S., Kumar, A., Gao, F., Montefiori, D. C., and Korber, B.(2010) Genetic signatures in the envelope glycoproteins of HIV-1 thatassociate with broadly neutralizing antibodies. PLoS Comput. Biol. 6,e1000955

63. Rong, R., Li, B., Lynch, R. M., Haaland, R. E., Murphy, M. K., Mulenga, J.,Allen, S. A., Pinter, A., Shaw, G. M., Hunter, E., Robinson, J. E., Gnana-karan, S., and Derdeyn, C. A. (2009) Escape from autologous neutralizingantibodies in acute/early subtype C HIV-1 infection requires multiplepathways. PLoS Pathog. 5, e1000594

64. de Mendoza, C., Van Baelen, K., Poveda, E., Rondelez, E., Zahonero, N.,Stuyver, L., Garrido, C., Villacian, J., and Soriano, V. (2008) Performanceof a population-based HIV-1 tropism phenotypic assay and correlationwith V3 genotypic prediction tools in recent HIV-1 seroconverters. J. Ac-quir. Immune Defic. Syndr. 48, 241–244

65. Xu, S., Huang, X., Xu, H., and Zhang, C. (2007) Improved prediction ofcoreceptor usage and phenotype of HIV-1 based on combined features ofV3 loop sequence using random forest. J. Microbiol. 45, 441– 446

66. Sterjovski, J., Churchill, M. J., Ellett, A., Gray, L. R., Roche, M. J., Dunfee,R. L., Purcell, D. F., Saksena, N., Wang, B., Sonza, S., Wesselingh, S. L.,Karlsson, I., Fenyo, E. M., Gabuzda, D., Cunningham, A. L., and Gorry,P. R. (2007) Asn 362 in gp120 contributes to enhanced fusogenicity byCCR5-restricted HIV-1 envelope glycoprotein variants from patients withAIDS. Retrovirology 4, 89

67. Pirovano, W., Feenstra, K. A., and Heringa, J. (2006) Sequence comparisonby sequence harmony identifies subtype-specific functional sites. NucleicAcids Res. 34, 6540 – 6548

68. Garg, H., and Blumenthal, R. (2008) Role of HIV Gp41 mediated fusion/hemifusion in bystander apoptosis. Cell Mol. Life Sci. 65, 3134 –3144

69. Denizot, M., Varbanov, M., Espert, L., Robert-Hebmann, V., Sagnier, S.,Garcia, E., Curriu, M., Mamoun, R., Blanco, J., and Biard-Piechaczyk, M.(2008) HIV-1 gp41 fusogenic function triggers autophagy in uninfectedcells. Autophagy 4, 998 –1008

70. Finkel, T. H., and Banda, N. K. (1994) Indirect mechanisms of HIV patho-genesis. How does HIV kill T cells? Curr. Opin. Immunol. 6, 605– 615

71. Castedo, M., Perfettini, J. L., Andreau, K., Roumier, T., Piacentini, M., andKroemer, G. (2003) Mitochondrial apoptosis induced by the HIV-1 enve-lope. Ann. N.Y. Acad. Sci. 1010, 19 –28

72. Varbanov, M., Espert, L., and Biard-Piechaczyk, M. (2006) Mechanisms ofCD4 T-cell depletion triggered by HIV-1 viral proteins. AIDS Rev. 8,221–236

73. Espert, L., Denizot, M., Grimaldi, M., Robert-Hebmann, V., Gay, B.,Varbanov, M., Codogno, P., and Biard-Piechaczyk, M. (2007) Autophagyand CD4� T lymphocyte destruction by HIV-1. Autophagy 3, 32–34

74. Gordon, S. N., Dunham, R. M., Engram, J. C., Estes, J., Wang, Z., Klatt,N. R., Paiardini, M., Pandrea, I. V., Apetrei, C., Sodora, D. L., Lee, H. Y., Haase,A. T., Miller, M. D., Kaur, A., Staprans, S. I., Perelson, A. S., Feinberg, M. B.,and Silvestri, G. (2008) Short-lived infected cells support virus replication insooty mangabeys naturally infected with simian immunodeficiency virus. Im-plications for AIDS pathogenesis. J. Virol. 82, 3725–3735

75. Gougeon, M. L., Colizzi, V., Dalgleish, A., and Montagnier, L. (1993) Newconcepts in AIDS pathogenesis. AIDS Res. Hum. Retroviruses 9, 287–289

76. Laurent-Crawford, A. G., Coccia, E., Krust, B., and Hovanessian, A. G.(1995) Membrane-expressed HIV envelope glycoprotein heterodimer is apowerful inducer of cell death in uninfected CD4� target cells. Res. Virol.146, 5–17

77. Bunnik, E. M., Lobbrecht, M. S., van Nuenen, A. C., and Schuitemaker, H.(2010) Escape from autologous humoral immunity of HIV-1 is not asso-ciated with a decrease in replicative capacity. Virology 397, 224 –230

78. Holm, G. H., Zhang, C., Gorry, P. R., Peden, K., Schols, D., De Clercq, E.,and Gabuzda, D. (2004) Apoptosis of bystander T cells induced by humanimmunodeficiency virus type 1 with increased envelope/receptor affinityand coreceptor binding site exposure. J. Virol. 78, 4541– 4551

79. Meissner, E. G., Zhang, L., Jiang, S., and Su, L. (2006) Fusion-inducedapoptosis contributes to thymocyte depletion by a pathogenic human im-munodeficiency virus type 1 envelope in the human thymus. J. Virol. 80,11019 –11030

80. Koot, M., van ’t Wout, A. B., Kootstra, N. A., de Goede, R. E., Tersmette,M., and Schuitemaker, H. (1996) Relation between changes in cellularload, evolution of viral phenotype, and the clonal composition of viruspopulations in the course of human immunodeficiency virus type 1 infec-tion. J. Infect. Dis. 173, 349 –354

81. Spijkerman, I., de Wolf, F., Langendam, M., Schuitemaker, H., andCoutinho, R. (1998) Emergence of syncytium-inducing human immuno-deficiency virus type 1 variants coincides with a transient increase in viralRNA level and is an independent predictor for progression to AIDS. J. In-fect. Dis. 178, 397– 403

82. van Rij RP, Blaak, H., Visser, J. A., Brouwer, M., Rientsma, R., Broersen, S.,de Roda Husman AM, and Schuitemaker, H. (2000) Differential corecep-tor expression allows for independent evolution of non-syncytium-induc-ing and syncytium-inducing HIV-1. J. Clin. Invest. 106, 1569

83. Kaneshima, H., Su, L., Bonyhadi, M. L., Connor, R. I., Ho, D. D., andMcCune, J. M. (1994) Rapid-high, syncytium-inducing isolates of humanimmunodeficiency virus type 1 induce cytopathicity in the human thymusof the SCID-hu mouse. J. Virol. 68, 8188 – 8192

84. Karlsson, G. B., Halloran, M., Schenten, D., Lee, J., Racz, P., Tenner-Racz,K., Manola, J., Gelman, R., Etemad-Moghadam, B., Desjardins, E., Wyatt,R., Gerard, N. P., Marcon, L., Margolin, D., Fanton, J., Axthelm, M. K.,Letvin, N. L., and Sodroski, J. (1998) The envelope glycoprotein ectodo-mains determine the efficiency of CD4� T lymphocyte depletion in simi-an-human immunodeficiency virus-infected macaques. J. Exp. Med. 188,1159 –1171

85. Cayabyab, M., Karlsson, G. B., Etemad-Moghadam, B. A., Hofmann, W.,Steenbeke, T., Halloran, M., Fanton, J. W., Axthelm, M. K., Letvin, N. L.,and Sodroski, J. G. (1999) Changes in human immunodeficiency virus type1 envelope glycoproteins responsible for the pathogenicity of a multiplypassaged simian-human immunodeficiency virus (SHIV-HXBc2). J. Virol.73, 976 –984

86. LaBonte, J. A., Patel, T., Hofmann, W., and Sodroski, J. (2000) Importanceof membrane fusion mediated by human immunodeficiency virus enve-lope glycoproteins for lysis of primary CD4-positive T cells. J. Virol. 74,10690 –10698

87. Etemad-Moghadam, B., Sun, Y., Nicholson, E. K., Fernandes, M., Liou, K.,Gomila, R., Lee, J., and Sodroski, J. (2000) Envelope glycoprotein determi-nants of increased fusogenicity in a pathogenic simian-human immuno-deficiency virus (SHIV-KB9) passaged in vivo. J. Virol. 74, 4433– 4440

88. Etemad-Moghadam, B., Rhone, D., Steenbeke, T., Sun, Y., Manola, J.,Gelman, R., Fanton, J. W., Racz, P., Tenner-Racz, K., Axthelm, M. K.,Letvin, N. L., and Sodroski, J. (2001) Membrane-fusing capacity of thehuman immunodeficiency virus envelope proteins determines the effi-ciency of CD� T-cell depletion in macaques infected by a simian-humanimmunodeficiency virus. J. Virol. 75, 5646 –5655

89. Hunt, P. W., Shulman, N. S., Hayes, T. L., Dahl, V., Somsouk, M., Funder-burg, N. T., McLaughlin, B., Landay, A. L., Adeyemi, O., Gilman, L. E.,Clagett, B., Rodriguez, B., Martin, J. N., Schacker, T. W., Shacklett, B. L.,Palmer, S., Lederman, M. M., and Deeks, S. G. (2013) The immunologiceffects of maraviroc intensification in treated HIV-infected individualswith incomplete CD4� T-cell recovery. A randomized trial. Blood 121,4635– 4646

90. Holman, A. G., and Gabuzda, D. (2012) A machine learning approach foridentifying amino acid signatures in the HIV env gene predictive of de-mentia. PLoS One 7, e49538

91. Holman, A. G., Mefford, M. E., O’Connor, N., and Gabuzda, D. (2010)HIVBrainSeqDB. A database of annotated HIV envelope sequences frombrain and other anatomical sites. AIDS Res. Ther. 7, 43




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Tek Ng, Sebastian Maurer-Stroh and Himanshu GargAnjali Joshi, Raphael T. C. Lee, Jonathan Mohl, Melina Sedano, Wei Xin Khong, Oon

Genetic Signatures of HIV-1 Envelope-mediated Bystander Apoptosis

doi: 10.1074/jbc.M113.514018 originally published online November 21, 20132014, 289:2497-2514.J. Biol. Chem.

10.1074/jbc.M113.514018Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2013/11/21/M113.514018.DC1

http://www.jbc.org/content/289/5/2497.full.html#ref-list-1

This article cites 91 references, 31 of which can be accessed free at


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M113.514018

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;289/5/2497&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/289/5/2497

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=289/5/2497&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/289/5/2497

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2013/11/21/M113.514018.DC1

http://www.jbc.org/content/289/5/2497.full.html#ref-list-1

http://www.jbc.org/

genetic signatures of hiv-1 envelope-mediated bystander apoptosis

Documents