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Virology 330 (20

Feline immunodeficiency virus envelope glycoprotein mediates apoptosis

in activated PBMC by a mechanism dependent on gp41 function

Himanshu Garg1, Anjali Joshi, Wayne A. Tompkins*

Immunology Program, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA

Received 27 August 2004; returned to author for revision 17 September 2004; accepted 5 October 2004

Abstract

Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced

immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that

requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing

membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this

process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model. Our studies

demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a

mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner.

Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be

important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion

inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated

PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is

dependent on gp41 function.

D 2004 Elsevier Inc. All rights reserved.

Keywords: FIV; Envelope; Apoptosis; Fusion; Syncytia; CD134; CXCR4

Introduction

Feline Immunodeficiency Virus (FIV) infection, similar

to HIV-1, is characterized by a progressive and irrever-

sible depletion of CD4+ T lymphocytes leading to

immunodeficiency in cats. Various hypotheses have been

proposed for HIV-mediated T cell loss, including direct

cytopathic effect due to infection (Lenardo et al., 2002),

syncytia formation (Ferri et al., 2000), and autoimmune

killing of T cells (Golding et al., 1989). Several inves-

0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.virol.2004.10.007

* Corresponding author. Immunology Program, College of Veterinary

Medicine, North Carolina State University, 4700 Hillsborough Street,

Raleigh, NC 27606. Fax: +1 919 515 4237.

E-mail address: Wayne_Tompkins@ncsu.edu (W.A. Tompkins).1 Current address: National Cancer Institute, Frederick, MD 21702,

United States.

tigators have suggested that the loss of T cells in HIV

infection is partly due to apoptosis of uninfected bystander

cells (Alimonti et al., 2003; Famularo et al., 1997;

Gougeon and Montagnier, 1993; Meyaar et al., 1992;

Muro-Cacho et al., 1995). In this regard, Finkel et al.

(1995) showed that apoptosis of lymph node T cells in

HIV-infected individuals primarily occurs in uninfected

bystander cells that lie in close proximity to productively

infected cells. The mechanism(s) behind FIV-mediated T

cell depletion are not clear, although observations mostly

parallel findings in HIV-1.

Several viral proteins including HIV Env (Laurent-

Crawford et al., 1995), Vpr (Desai et al., 2002), and Tat

(Purvis et al., 1995) have been shown to induce apoptosis.

However, determinants of HIV pathogenicity most often

map to the Env glycoprotein (Etemad-Moghadam et al.,

2001), suggesting that it may be a key regulator of CD4+ T

04) 424–436

Fig. 1. Coculture of activated PBMC with Env-expressing cells results in

loss of viability. PBMC activated with 2 Ag/ml ConAwere cocultured with

either CrFK cells (Control) or Env-expressing CrFKenv/rev cells (Env) for

24 or 48 h. The non-adherent cells were collected after careful washing and

viability determined by MTT assay. Data represent mean F standard

deviation (SD) of quadruplicate observations. Statistical significance was

assessed by Student’s t test (*P b 0.01, **P b 0.001).

H. Garg et al. / Virology 330 (2004) 424–436 425

cell death. HIV Env is synthesized as a gp160 precursor that

is later proteolytically cleaved into the gp120 surface unit

and the gp41 transmembrane protein (Wyatt and Sodroski,

1998). While gp120 binds to CD4 and a chemokine co-

receptor (CXCR4/CCR5), the gp41 mediates fusion of viral

and cellular membranes leading to viral entry. Interestingly,

FIV shares CXCR4 as a co-receptor (Willett et al., 1997) for

Env binding, while the primary receptor has recently been

identified as CD134 (Shimojima et al., 2004). Membrane-

expressed Env glycoprotein also mediates fusion between

HIV/FIV-infected cells and uninfected bystander cells

expressing the necessary receptor/co-receptor, resulting in

syncytia formation. HIV Env-mediated syncytia, frequently

seen in in vitro infected T cell culture systems, undergo

apoptosis and have been suggested as a mechanism of T cell

loss by HIV (Ferri et al., 2000; Scheller and Jassoy, 2001).

Membrane expressed Env glycoprotein has been shown to

interact with bystander cells and cause apoptosis (Laurent-

Crawford et al., 1995), cytolysis (Nardelli et al., 1995), or

reduced proliferation and G2 phase arrest followed by

apoptosis (Kolesnitchenko et al., 1995; Schwartz et al.,

1994).

Studies with FIV have shown that T cells from

infected cats exhibit reduced survival in vitro and die of

apoptosis in a manner similar to that seen in HIV-1

infection (Guiot et al., 1997; Johnson et al., 1996; Momoi

et al., 1996; Tompkins et al., 2003). The earliest

distinctive pathological feature of FIV infections is a

decline in the CD4+ T cell population (Hoffmann-Fezer et

al., 1992) and an inversion of CD4/CD8 ratio (Novotney

et al., 1990). The mechanism(s) of immune dysfunction in

FIV infection are not clear, although Env glycoprotein of

FIV shares remarkably similarities to HIV Env in terms

of co-receptor usage and fusion mechanism. In this

context, the Env glycoprotein of FIV may be an

important target for further investigation. Although the

role of FIV Env in T cell apoptosis has not been studied,

it has been implicated in FIV-induced neuropathogenesis

(Johnston et al., 2002). Further, FIV Env is a potent

inducer of syncytia formation and similar to the X4 tropic

strains of HIV, utilizes CXCR4 as co-receptor for

mediating fusion and viral entry (Garg et al., 2004;

Willett et al., 1997).

Previously (Garg et al., 2004), we have shown that

coculture of FIV Env expressing (CrFKenv/rev) cells with

feline CD4+ T cell line (FCD4E) resulted in syncytia

formation, which could be blocked by CXCR4 antagonist

AMD3100 and gp41-specific fusion inhibitory peptide

T1971. In this report, we show that membrane-expressed

FIV Env is also capable of inducing apoptosis in

activated PBMC and that this phenomenon correlates

with Env-mediated fusion. This is the first report

indicating the potential of FIV envelope glycoprotein in

mediating T cell loss via a process that correlates

withgp41 function rather than gp120 binding to recep-

tor/co-receptor.

Results

FIV Env mediates cell death in activated PBMC via

apoptosis

A number of studies have suggested that the progressive

loss of CD4+ T cells in HIV and FIV infections is the result

of bystander cell death (Alimonti et al., 2003; Corbeil and

Richman, 1995; Laurent-Crawford et al., 1995; Tompkins et

al., 2003). Although numerous studies have been conducted

to elucidate the mechanism(s) of bystander cell death in HIV

infection, there have been no studies pertaining to FIV

infection in this regard. To address the hypothesis that FIV

Env is capable of inducing cell death in bystander T cells, a

coculture system was developed comprising of Env

expressing CrFKenv/rev cells as effector cells and Con-

canvalin A (ConA)-activated PBMC as target cells. The

model consisted of adherent effector cells and suspension

target cells, which allows easy separation of the two

populations for analysis. Previously, we have characterized

the CrFKenv/rev cell line expressing Env glycoprotein from

the NCSU1 isolate of FIV and studied the mechanisms of

FIV Env-mediated syncytia formation in IL-2-dependent

FCD4E cells (Garg et al., 2004). Activated PBMC were

cocultured with Env expressing CrFKenv/rev cells and the

viability of target cells was measured 24 and 48 h later by

MTT assay. As shown in Fig. 1, coculture of activated

PBMC with CrFKenv/rev cells resulted in loss of viability at

24 h as compared to cells cocultured with control CrFK cells

(P b 0.01), and this difference was more marked at 48 h post

coculture (P b 0.001).

To further characterize the loss of viability in target cells,

flow cytometry was used to assess cell morphology based

on forward (FSC) versus side scatter (SSC). Twenty-four

hours post coculture, the non-adherent cells were collected

H. Garg et al. / Virology 330 (2004) 424–436426

and analyzed by flow cytometry. The cells were gated into

R1 (high FCS, low SSC) and R2 (low FSC, high SSC) gates

representing live and dead cells, respectively. As depicted in

Fig. 2A, there was a significant decrease in percent cells in

R1 (live) gate when activated PBMC were cocultured with

CrFKenv/rev cells compared to control CrFK cells. Pooled

data from three independent experiments shows an approx-

imate 40% decrease in viability in PBMC cocultured with

Env-expressing cells compared to control.

To determine if FIV Env-mediated loss in cellular

viability was a result of apoptosis, the FSC/SSC gate was

applied to morphologically live cells (R1 gate) as depicted

in Fig. 2A and percent apoptosis determined by Annexin

V staining. Cells positive for Annexin V but negative for

propidium iodide (PI) were considered apoptotic. As

shown in Fig. 2B, a marked increase in apoptotic cells

was seen when target cells were cocultured with CrFKenv/

rev cells (12.6%) compared to control CrFK cells (6.2%).

Data from three separate experiments in Fig. 2B show a

twofold increase in percent apoptotic cells when cocul-

tured with Env-expressing cells compared to control cells

(P b 0.001).

Mitogenic stimulation is required for induction of apoptosis

by FIV Env and correlates with up-regulation of surface

CXCR4 but not CD134

To determine whether T cell activation was necessary

for induction of apoptosis, experiments were conducted

Fig. 2. Coculture of activated PBMC with Env-expressing cells results in morpho

with either CrFK cells (Control) or Env-expressing CrFKenv/rev cells (Env) for 24

by flow cytometry for morphological changes based on forward scatter (FSC

independent experiments is shown in the right panel. Data represent mean of triplic

post coculture, the non-adherent cells were stained with Annexin V-FITC and PI an

population (R1 gate) as in part A. Graphical representation of data from three ind

triplicate observations. Statistical significance was assessed by Student’s t test (*

with either resting cells or cells stimulated with ConA for

4 days. As shown in Fig. 3A, resting unstimulated PBMC

did not show any significant cell death or apoptosis when

cocultured with Env-expressing cells compared to activated

cells (P b 0.01), suggesting that cellular activation is

required for this process. The potential of FIV Env to

mediate cell death only in activated but not resting PBMC

suggests that there may be differences between the

receptor (CD134) and co-receptor (CXCR4) expression

levels between the two cell populations. Hence, we

determined the expression of these receptors on activated

versus resting cells as a determinant of differences seen in

the previous experiments. Flow cytometric analysis of

surface expression of CXCR4 and CD134 showed that

ConA mediated activation of feline PBMC results in up-

regulation of CXCR4 but not CD134 (Fig. 3B). This

suggests that Env-mediated apoptosis in ConA-activated

PBMC correlates with up-regulation of surface CXCR4

expression and leads to the possibility that the majority of

dying cells are more likely to be CXCR4 positive than

CD134 positive.

CD134 can act as a receptor for fusion mediated by

FIV-NCSU1 Env glycoprotein

In the previous experiment, apoptosis in ConA-stimu-

lated cultures did not correlate with surface expression of

CD134. Hence, we attempted to investigate whether the

NCSU1 isolate of FIV similar to other primary isolates

logical changes and apoptosis. (A) ConA-activated PBMC were cocultured

h. The non-adherent cells were collected after careful washing and analyzed

) versus side scatter (SSC). Graphical representation of data from three

ate observations from three independent experiments. (B) Twenty-four hours

d analyzed by flow cytometry for apoptosis. The gate was applied to the live

ependent experiments is shown in the right panel. Data are mean F SD of

P b 0.01).

Fig. 3. Mitogenic stimulation of PBMC is required for induction of apoptosis via FIV Env. (A) Feline PBMC were either cultured in media alone (Resting) or in

media supplemented with 2 Ag/ml ConA (Activated) for 4 days followed by coculture with either Env-expressing or control CrFK cells for 24 h. Apoptosis was

subsequently determined by Annexin V and PI staining. (B) Flow cytometric analysis of surface expression of CXCR4 and CD134 in PBMC either with

(Activated) or without (Resting) stimulation with ConA for 4 days. Thin line: secondary antibody alone, bold line: specific staining for CXCR4 or CD134.

Numbers represent the percentage of cells expressing CXCR4 or CD134.

H. Garg et al. / Virology 330 (2004) 424–436 427

utilizes CD134 as the primary receptor. For this purpose, a

syncytia forming assay was conducted using CD134 trans-

fected HeLa cells and CrFKenv/rev cells to determine

whether FIV-NCSU1 binds/utilizes CD134. Syncytia form-

ing assay in Fig. 4 shows that expression of feline CD134

in HeLa cells renders them permissive to FIV-NCSU1 Env-

mediated fusion, which otherwise does not mediate fusion

in parental HeLa cells (Fig. 4) as shown previously (Garg

et al., 2004). For control purposes, fusion with CrFKpet

cells is shown, which fuse with HeLa cells irrespective of

CD134 expression (Fig. 4). Interestingly, though CXCR4

antagonist AMD3100 blocked fusion under different con-

ditions, anti-human CD134 antibody previously shown to

cross-react with feline CD134 (Shimojima et al., 2004)

failed to block the fusion process (Table 1).

CXCR4 binding and not CD134 is required for FIV

Env-mediated apoptosis

Studies with HIV-1 have implicated CD4 and CXCR4

binding/signaling as one of the mechanisms for HIV Env-

mediated apoptosis in bystander T cells (Arthos et al., 2002;

Roggero et al., 2001). Hence, we wished to study whether

FIV Env binding to its surface receptor (CD134) and co-

receptor (CXCR4) was required for induction of apoptosis.

Our data in Fig. 4 indicates that FIV-NCSU1 similar to other

primary isolates of FIV binds CD134, although a blocking

antibody or reagent specific to feline CD134 is not available.

To study the role of CD134 binding in FIV Env-mediated

apoptosis, we utilized the cross-reactive monoclonal anti-

body to deplete activated PBMC of CD134-positive cells to

Fig. 4. FIV-NCSU1 can utilize feline CD134 to mediate fusion. Untransfected HeLa cells or those transfected with feline CD134 expressing plasmid (HeLa

CD134) were cocultured either with cells expressing FIV-NCSU1 Env (NCSU1) or with CrFKpet cells chronically infected with FIV-Petaluma isolate

(Petaluma). The cells were fixed and stained with Giemsa stain. Representative photomicrographs are shown. Magnification 200�.

H. Garg et al. / Virology 330 (2004) 424–436428

high purity (Fig. 5A) using Fluorescence Activated Cell

Sorting (FACS) and then determine percent apoptosis either

in total PBMC or CD134-depleted cells. As shown in

Fig. 5B, there was no difference in apoptosis induced by

FIV-NCSU1 Env in either total or CD134-depleted PBMC,

suggesting that CD134 binding may not be an absolute

requirement for this process. Further, infection studies with

CD134-depleted PBMC (data not shown) indicated that they

are equally infectable with FIV-NCSU1, suggesting that

although NCSU1 can utilize CD134 it may not be restricted

to using this receptor in primary cells.

Our next objective was to determine whether CXCR4

binding was required for FIV Env-mediated apoptosis.

Previously, we have shown that CXCR4 antagonist

AMD3100 can block fusion mediated by FIV-NCSU1 Env

in FCD4E cells in a dose dependent manner (Garg et al.,

2004). Based on these findings, we expected to block FIV

Env-mediated apoptosis in activated feline PBMC using

AMD3100. As shown in Fig. 5C, AMD3100 blocked Env-

Table 1

AMD3100 but not anti-CD134 monoclonal antibody blocks syncytia

formation under different coculture conditions

Medium AMD Anti-CD134

CRFKrev/env + HeLa � � �CrFKrev/env + HeLa CD134 + � +

CrFKpet + HeLa + � +

CrFKpet + HeLa CD134 + � +

Cultures were set up with different cell types as described in Fig. 4. In

some wells, AMD3100 or anti-CD134 antibody was added to block

fusion. Twenty-four hours post-coculture, cells were fixed, stained with

Giemsa stain, and subsequently photomicrographed. Presence of syncytia

under different culture conditions was recorded as (+) and absence of

syncytia as (�).

mediated apoptosis in ConA-stimulated feline PBMC in a

dose-dependent manner. The above results confirm that

apoptosis seen in our model was due to membrane-

expressed FIV Env and that CXCR4 binding was required

for this process.

FIV Env-mediated apoptosis is dependent on gp41 function

FIV Env-mediated fusion can be inhibited by gp41-

specific peptide T1971 that corresponds to the C terminal

heptad repeat region of gp41. T1971, an FIV-specific

peptide, binds gp41 at a post-CXCR4 binding stage

inhibiting the six-helix hairpin structure formation thereby

blocking fusion (Garg et al., 2004; Medinas et al., 2002).

Interestingly, in our coculture system, T1971 was able to

block FIV Env-mediated apoptosis in a dose-dependent

manner (Fig. 6A). This suggests that CXCR4 binding alone

is not sufficient for Env-induced apoptosis and that steps

subsequent to CXCR4 binding involving gp41 function are

important in mediating the apoptotic process.

To further establish the above findings, we utilized a

gp41 mutant defective in mediating fusion in our study. A

conserved tryptophan-rich region in HIV gp41 comprising

the membrane proximal ectodomain, also referred to as the

pre-transmembrane (pre-TM) region (Suarez et al., 2000),

has been identified as being critical for fusion (Salzwedel et

al., 1999). Previously, we have identified a similar

conserved region in FIV gp41 (Garg et al., 2004) and

established a fusion defective mutant, W(1–3)A, by replac-

ing the tryptophans (W) at position 766, 769, and 772 with

alanines (A) as represented in Fig. 6B. We assayed the

apoptosis inducing potential of W(1–3)A mutant and

compared it to the wild-type (WT) clone. As shown in

Fig. 5. FIV Env-mediated apoptosis correlates with CXCR4 binding but not with CD134 binding. (A) ConA-activated PBMC were depleted of CD134+ cells

using the MoFlo fluorescence activated cells sorter. The purity of CD134-depleted cells is shown along with total PBMC. Numbers represent the percentage of

cells within that quadrant. (B) Either total PBMC or PBMC depleted of CD134+ cells were cocultured with Env-expressing or control cells. Apoptosis was

determined 24 h later by Annexin V and PI staining. (C) Total PBMC activated with 2 Ag/ml ConA for 4 days were cocultured with either CrFK cells or Env-

expressing CrFKenv/rev cells for 24 h. CXCR4 antagonist AMD3100 was added at the indicated concentrations at the time of coculture. Apoptosis was

determined by Annexin V staining followed by flow cytometry 24 h post coculture. Data are mean F SE from triplicate observations from two independent

experiments.

H. Garg et al. / Virology 330 (2004) 424–436 429

Fig. 6C, the fusion defective mutant W(1–3)Awas unable to

induce apoptosis in activated PBMC compared to the WT

Env (P b 0.01) under similar expression levels (Fig. 6C

inset). These findings further suggest that a fusion com-

petent FIV Env glycoprotein is required for induction of

apoptosis.

Syncytia formation per se is not sufficient for induction of

apoptosis

Our previous experiments suggest a correlation bet-

ween fusion and apoptosis even though syncytia formation

is not evident in cocultures using PBMC. Previously, we

have utilized the feline CD4+ T cell line, FCD4E cells that

form extensive syncytia when cocultured with CrFKenv/

rev cells (Garg et al., 2004). To determine whether

syncytia formation was sufficient to induce apoptosis in

our system, we cocultured FCD4E cells with Env-

expressing cells and compared them to PBMC for

apoptosis induction. As shown in Fig. 7A, FCD4E cells

did not undergo significant apoptosis when cocultured

with Env-expressing cells compared to activated PBMC.

However, under similar conditions, FCD4E cells formed

extensive syncytia while in cocultures using activated

Fig. 6. FIV Env-mediated apoptosis is dependent on gp41 function. (A)

ConA-activated PBMC were cocultured either with CrFK cells or Env-

expressing CrFKenv/rev cells for 24 h. Gp41 C-terminal-specific inhibitory

peptide T1971 was added at the indicated concentrations at the time of

coculture. Apoptosis was determined by Annexin V staining 24 h post

coculture followed by flow cytometry. Data are mean F SE of triplicate

observations from two independent experiments. (B) Schematic diagram of

FIV gp41 and fusion-defective mutant W(1–3)A. The position of heptad

repeats (HR1 and HR2), transmembrane region (TM), and pre-trans-

membrane region (pre-TM) is shown. (C) CrFK cells were transiently

transfected with either wild type (pWT) or mutant Env (pW(1–3)A), or

vector control (pControl) plasmids. Inset shows protein expression by

Western blot. Forty-eight hours post-transfection, cells were cocultured with

ConA-activated PBMC. Twenty-four hours post-coculture, suspension cells

were collected after careful washing, stained with Annexin V and PI and

analyzed by flow cytometry for apoptosis. Data represent mean F SD of

triplicate observations. Statistical significance was assessed by Student’s

t test (*P b 0.01).

Fig. 7. Fusion per se is not sufficient for induction of apoptosis by FIV Env.

(A) Either ConA activated PBMC or highly fusogenic FCD4E cells were

cocultured with Env-expressing or control cells for 24 h. Apoptosis was

determined by Annexin V staining followed by flow cytometry. Data are

mean F SD of triplicate observations. (B) Photomicrographs of the

different cocultures as described in A 24 h post-coculture and prior to

analysis of apoptosis. Magnification 200�.

H. Garg et al. / Virology 330 (2004) 424–436430

PBMC syncytia formation was not evident (Fig 7B). This

suggests that syncytia formation per se may not be

sufficient for FIV Env-mediated apoptosis, although the

fusogenic potential or membrane disruption capacity of

gp41 is necessary.

FIV Env-mediated apoptosis correlates with Env-mediated

fusion/gp41 function

Our coculture experiments with PBMC did not show

significant syncytia formation (Fig. 7B) even though we

believe that small syncytia comprising of a few cells maybe

formed. Fluorescent dye transfer assay may identify small

syncytia or cells that have undergone cytoplasmic content

mixing due to fusion pore formation. Hence, we utilized a dye

transfer assay to determine the population of cells undergoing

apoptosis, and whether there was a correlation between FIV

Env-mediated apoptosis and fusion. For this purpose, FIV

Env-expressing cells were labeled with CMTMR (Cell

Tracker Orange) dye prior to coculture with CMFDA (Cell

Tracker Green) labeled PBMC for studying fusion. For

apoptotic assays, only the Env-expressing cells were labeled

with CMTMR prior to coculture with unlabeled PBMC and

followed by staining with Annexin V-FITC post coculture.

Fig. 8. FIV Env-mediated apoptosis correlates with Env-mediated fusion. CrFKenv/rev or control CrFK cells were pre-labeled with CMTMR (red) and

cocultured with CMFDA (green) labeled PBMC (A,B) or unlabeled PBMC (C) for 24 h. (A) CMFDA-labeled PBMC were cocultured with CMTMR-labeled

CrFK control cells or Env-expressing CrFKenv/rev cells for 24 h. Following coculture, the non-adherent cells were collected, cytospun onto slides, and

observed by fluorescent microscopy. Images acquired for red and green fluorescence were overlaid using Adobe Photoshop software. Arrows indicate

multicellular syncytia appearing as yellow in the overlay. Arrowheads indicate single cells that are double positive. (B) Fusion was estimated 24 h post-

coculture by flow cytometry as CMTMR- and CMFDA-positive cells. (C) Apoptosis was detected by Annexin V staining followed by flow cytometry 24 h

post-coculture. For B and C, numbers represent the percentage of cells within each quadrant or within each specified gate. (D) Morphology of different cell

populations (gates R2 through R5) in B and C based on forward scatter (FSC) and side scatter (SSC). (E) Graphical representation of data for (B) and (C). Data

are mean F SD of triplicate observations.

H. Garg et al. / Virology 330 (2004) 424–436 431

H. Garg et al. / Virology 330 (2004) 424–436432

Twenty-four hours post coculture, fusion was quantified by

flow cytometry as CMTMR+ CMFDA+ double-positive cells

and apoptosis was determined by staining with FITC labeled

Annexin V (CMTMR+/�Annexin+/�). As seen in Fig. 8A,

fused cells (appearing as yellow in overlay) were distinctly

seen by fluorescent microscopy when activated PBMC

were cocultured with Env-expressing CrFKenv/rev cells

compared to control cells. Fusion here did not necessarily

represent syncytia (arrows) as single cells that were double

positive were also seen (arrowheads) most likely due to

formation of fusion pore without progressing to complete

fusion. A quantification of fusion/dye transfer by flow

cytometry revealed that approximately 10% of the cells

were double positive in Env cocultures (11.88%) when

compared to background control cocultures (1.62%). We

then determined percent apoptosis in cocultured target cells

by Annexin V staining under identical coculture conditions

(except that the target PBMC were unlabeled). Interest-

ingly, a similar increase of 10% (5.94–17.01%) was seen

in CMTMR+Annexin V+ cells in Env cocultures compared

to control cocultures. Further, there was no significant

increase in apoptosis of CMTMR� cells (9.17–10.54%,

Fig. 8C). Since the previously unlabelled target cells

(activated PBMC) had taken up the CMTMR dye, this

population most likely represents cells that have formed

fusion pores with Env-expressing cells. To further validate

the above findings, we determined whether the fused and

apoptotic populations were morphologically similar based

on the FSC/SSC characteristics. As seen in Fig. 8D, the

fused CMTMR+CMFDA+ (R2 gate) cells were almost

identical to the apoptotic CMTMR+Annexin V+ (R4 gate)

cells based on forward and side scatter characteristics.

Moreover, they were morphologically distinct from

CMTMR negative (R3 gate) and non-apoptotic (R5 gate)

cells. These results suggest that apoptosis in feline PBMC

was primarily occurring post fusion pore formation as a

result of coculture with Env-expressing cells, indicating a

strong correlation between FIV Env-mediated fusion and

apoptosis.

Discussion

Feline immunodeficiency virus infection is a relevant and

well-characterized animal model for human AIDS (English

et al., 1993; Mizuno et al., 2001). Based on the similarities

between FIV and HIV pathogenesis, it is speculated that

both viruses share common mechanism(s) for inducing

immunodeficiency. Even though it has been known for

sometime that FIV causes T cell depletion in a manner

similar to HIV-1 (Guiot et al., 1997; Johnson et al., 1996;

Tompkins et al., 2003), the molecular mechanisms involved

in FIV-induced T cell loss have not been established. The

Env glycoprotein of HIV has been implicated in apoptotic

killing of bystander T cells leading to immunodeficiency

(Etemad-Moghadam et al., 2001). Conflicting reports

suggest the role of gp120 binding to CD4 and chemokine

receptors versus fusion mediated by Env glycoprotein in

initiating apoptotic signaling. The objective of the present

study was to determine (1) whether FIV Env glycoprotein

was capable of inducing apoptosis in bystander cells and (2)

if this process was dependent on receptor (CD134), co-

receptor (CXCR4) binding, or Env-mediated fusion.

The fact that FIV Env utilizes CD134 and not CD4 as its

primary receptor (Hosie et al., 1993; Shimojima et al., 2004)

but can still initiate apoptosis in bystander cells indicates

that CD4 signaling may not be necessary for lentiviral

Env-mediated apoptosis. We show here that membrane-

expressed FIV Env can induce apoptosis in activated PBMC

but not resting cells, and this correlates with increased

surface expression of the CXCR4 but not CD134. While

CXCR4 is a well-known activation marker (Bleul et al.,

1997), our observations with regard to lack of CD134 up-

regulation following ConA stimulation are consistent with

findings in other species where ConA-mediated activation

failed to up-regulate surface CD134 expression (Barten et

al., 2001). Interestingly, similar to HIV-1, FIV Env binding

to CXCR4 is required for apoptosis, suggesting that it is a

common requirement for both HIV and FIV Env-induced T

cell death. However, inhibition of FIV Env-mediated

apoptosis at steps post CXCR4 binding using either gp41-

specific inhibitory peptides or a mutation in gp41 pre-

transmembrane region suggests that CXCR4 itself may not

be sufficient for activating the apoptotic signaling pathway.

Although our studies regarding the primary receptor CD134

were not conclusive in the absence of suitable blocking

antibodies, inhibition of the fusion process at steps post

receptor and co-receptor binding abrogates apoptosis,

suggesting that the binding of CD134 and/or CXCR4 is

not sufficient for induction of FIV-NCSU1 Env-mediated

apoptosis. The observation that fusion-inhibiting agents/

mutations inhibit T cell apoptosis indicates a strong

correlation between the two processes. Our findings are

contrary to the hypothesis that CXCR4 binding and signal-

ing is sufficient for lentivirus Env-mediated apoptosis

(Arthos et al., 2002; Penn et al., 1999), but are in agreement

with recent reports showing that HIV Env-mediated

apoptosis can be blocked by T20, an HIV gp41-specific

fusion inhibitor (Barretina et al., 2003; Blanco et al., 2003;

Scheller and Jassoy, 2001).

The correlation between HIV Env-mediated fusion and

apoptosis remains controversial. While some reports show

the potential of soluble Env glycoprotein to induce

apoptosis in T cells (Corbeil and Richman, 1995), others

have demonstrated the necessity of membrane-bound Env

glycoprotein for induction of apoptosis (Laurent-Crawford

et al., 1995). Though it has been established that HIV Env

can signal via chemokine receptors (CXCR4/CCR5)

(Davis et al., 1997), and some authors suggest that HIV

Env mediates apoptotic signaling via engaging CD4 and/or

CXCR4 (Arthos et al., 2002; Vlahakis et al., 2001), others

have shown that signaling via either of these receptors is

H. Garg et al. / Virology 330 (2004) 424–436 433

not required for HIV Env-mediated apoptosis (Biard-

Piechaczyk et al., 2000; Blanco et al., 1999). While

pathogenicity of HIV is often related to the fusogenic

potential of the Env glycoprotein (Alimonti et al., 2003;

Gougeon and Montagnier, 1993), the requirement of fusion

between infected and uninfected bystander cells for

apoptosis remains controversial.

HIV infections are characterized highly cytopathic

syncytia-inducing (SI) and relatively less pathogenic non-

syncytia-inducing (NSI) strains based on the in vitro

phenotype of the viruses. The likelihood of finding SI

phenotype in a patient is associated with poor immuno-

logical parameters, rapid CD4+ T cell loss and disease

progression (Koot et al., 1993; Richman and Bozzette,

1994). The mechanism by which SI strains tend to be more

pathogenic than NSI strains is not established. SI strains

utilize CXCR4 as co-receptor, which is expressed practi-

cally on all CD4+ T cells while NSI strains generally utilize

CCR5, which is expressed only on a limited number of

CD4+ T cells especially in the gut-associated lymphoid

tissue (Anton et al., 2000). Based on co-receptor expression

by T cells, X4 viruses tend to be more infectious, which may

be a cause of their increased pathogenicity (Fouchier et al.,

1996). On the other hand, cells infected with SI strains

would be more effective at causing apoptosis of CXCR4

expressing bystander T cells via binding by Env glycopro-

tein and mediating fusion. Even though NSI strains are

considered less cytopathic, they have been shown to be

capable of CD4+ T cell killing by a mechanism that is not

directly related to the rate of viral replication (Yu et al.,

1994). In a recent report, LaBonte et al. (2003) have shown

that CCR5 utilizing viruses can cause cytolysis of CCR5+

target cells by a process that is again dependent on

membrane fusion.

Our findings utilizing the highly fusogenic FCD4E cells

suggests that although syncytia formation itself may not be

the determining factor in inducing apoptosis, the fusogenic

potential and membrane disruption properties of FIV Env

are required for this process. Further analysis of this

phenomenon is necessary utilizing membrane specific

lipophilic dyes and nondiffusible cytoplasmic labeling. We

believe that the discrepancy between FCD4E cells and

PBMC might be due to differences in membrane structure

and organization as well as signaling factors. This correlates

with the observation that although pathogenicity of HIV

correlates with the fusogenic potential of Env glycoprotein,

formation of syncytia in vivo are rare.

Based on the above evidence, it is clear that interaction of

lentiviral Env glycoprotein with target cell membranes may

result in membrane destabilization subsequently leading to

apoptosis of target cells. Our findings are in agreement with

others (Roumier et al., 2003), further strengthening the

hypothesis that gp41-mediated membrane perturbation may

be important for FIV as well as HIV-induced apoptosis. This

is the first report showing the potential of membrane-

expressed FIV Env to induce apoptosis in bystander cells.

Also, by blocking Env-mediated apoptosis at the level of

gp41, we show a strong correlation between fusion and

apoptosis mediated by FIV Env. Our findings not only

strengthen the hypothesis that lentiviral Env glycoproteins

may be involved in T cell apoptosis but also indicate strong

similarities between FIV and HIV pathogenesis.

Materials and methods

Cells and reagents

Crandell Feline Kidney (CrFK), CrFK cells expressing

FIV-NCSU1 Env glycoprotein (CrFKenv/rev) (Garg et al.,

2004), and CrFKpet (CrFK cells chronically infected with

the Petaluma isolate of FIV) were maintained in Dulbecco’s

Modified Eagle’s Medium (DMEM) (Mediatech) supple-

mented with 10% fetal bovine serum (FBS), penicillin (100

U/ml), and streptomycin (100 Ag/ml). Medium for

CrFKenv/rev cells was supplemented with G-418 (Gibco-

BRL, Gaithesberg, MD) at 700 Ag/ml. HeLa cells were

maintained in DMEM medium supplemented with 10%

FBS, penicillin (100 U/ml) and streptomycin (100 Ag/ml).

FCD4E, a CD4+ T cell line highly permissive for FIV-

NCSU1 infection (English et al., 1993) was cultured in

RPMI medium supplemented as described below. Periph-

eral blood lymphocytes were obtained from FIV negative

cats by Percoll density gradient centrifugation as described

previously (English et al., 1993). The donor cats were

housed at the Laboratory Animal Resource facility of the

College of Veterinary Medicine, North Carolina State

University, as per the federal guidelines and institutional

policies. Purified PBMC were cultured in RPMI 1640

medium (Mediatech) supplemented with 10% FBS, strep-

tomycin (100 Ag/ml), penicillin (100 IU/ml), l-glutamine

(2 mM), HEPES (15 mM), sodium pyruvate (2 mM) and h-mercaptoethanol (2.5 � 10�5 M). PBMC were activated

with 2 Ag/ml ConA (Sigma) and cultured for 4 days prior

to use in the coculture experiments. Plasmid expressing

feline CD134 was a kind gift from Dr. Brian Willett

(University of Glasgow). CXCR4 antagonist AMD3100

was a kind gift from Dr. Edward Hoover (Colorado State

University). FIV gp41-specific fusion inhibitor T1971,

shown to inhibit FIV Env-mediated fusion, was a kind gift

from Robyn Medinas (Trimeris Inc. Durham, NC).

Flow cytometry and cell sorting

Cell surface CXCR4 expression was measured using the

cross-reacting anti-CXCR4 monoclonal antibody (44717,

BD Biosciences) followed by staining with Phycoerythrin

(PE) conjugated goat anti-mouse IgG (Jackson ImmunoR-

esearch Laboratories, PA). PE-conjugated anti CD134 anti-

body BerACT35 (Ancell Corporation, Bayport, MN)

previously shown to cross-react with the feline CD134

homolog (Shimojima et al., 2004) was used to detect surface

H. Garg et al. / Virology 330 (2004) 424–436434

CD134 expression. Cells were acquired using the FACSCa-

libur flow cytometer (BD Biosciences) and analyzed using

the CellQuest software. For blocking experiments, the

nonconjugated anti-CD134 antibody was used. PBMC were

sorted into purified CD134+ Gaithesberg and CD134�

populations using a high-speed MoFlo fluorescence acti-

vated cell sorter (DakoCytomation) at the flow cytometry

facility at North Carolina State University.

Coculture experiment

CrFK or CrFKenv/rev cells were seeded in 24-well plates

at 2 � 105 cells/well and allowed to adhere overnight. After

24 h, medium was aspirated from the wells and 4 � 105

PBMC activated with 2 Ag/ml ConA for 4 days were added

to the wells. Inhibitors AMD3100 or T1971 were added at

the time of coculture. The cells were cocultured for 24 or

48 h. Subsequently, the suspension cells were collected by

careful washing and used for viability or apoptotic assays.

Transfection of cells

HeLa cells were transfected using Effectene transfection

reagent (Qiagen, CA) as per the manufacturer’s instructions.

Twenty-four hours post transfection, the cells were seeded

in 96-well plates and used for syncytia forming assay.

Syncytia-forming assay

HeLa cells either untransfected or transfected with feline

CD134 were seeded in 96-well plates at 104 cells per well

and allowed to adhere overnight. Subsequently, CrFK cells

expressing FIV-NCSU1 Env (CrFKenv/rev) or chronically

infected with FIV-Petaluma (CrFKpet) were added at 1000

cells per well. In some wells, either AMD3100 or anti-

CD134 antibody was added for blocking. The cells were

cocultured for 24 h after which the plates were fixed and

stained with Giemsa stain as described previously (Garg

et al., 2004).

Measurement of viability

Viability of PBMC was measured by the MTT [3-(4,5-

dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide]

(Sigma) assay as described previously (Mosmann, 1983)

with slight modifications. Briefly, ConA-activated PBMC

were cocultured with either CrFK or CrFKenv/rev cells for

24 or 48 h following which the non-adherent cells were

collected and re-plated in 96-well plates at 100 Al/well.Thirty microlitres of MTT reagent (5 mg/ml in PBS) was

added to each well and incubated for 4 h. Subsequently, the

plates were centrifuged, supernatant discarded and 100 Al of0.1 N HCl in isopropanol added to the wells. The plates

were incubated for an additional 1 h and then 100 Al ofdistilled water added per well. Finally, the plates were read

at 530/650 nm in an ELISA plate reader (Tecan).

Detection of apoptosis

Morphological change in suspension cells was detected

24 h post coculture by measuring the forward scatter (FSC)

versus side scatter (SSC) by flow cytometry on a FACSCa-

libur flow cytometer (BD Biosciences). At least 15000

events were collected and analyzed using the CellQuest

software. Apoptosis was detected by staining suspension

cells with FITC-conjugated Annexin V, a 35–36 kDa protein

that binds to phosphatidylserine exposed on the surface of

apoptotic cells. Twenty-four hours post coculture, the

suspension cells were stained with Annexin V and PI

(Roche Diagnostics, IN) as per the manufacturer’s instruc-

tions and analyzed by flow cytometry. Cells positive for

Annexin V but negative for PI were considered apoptotic,

while PI-positive cells were excluded as dead cells.

Inhibitors AMD3100 or T1971 were added at the time of

coculture for some experiments.

Detection of fusion and apoptosis using fluorescent labeling

of cells

To determine FIV Env-mediated fusion in activated

PBMC, a two-color dye redistribution assay was developed.

Briefly, CrFK or CrFKenv/rev cells were labeled with

cytoplasmic dye 5- and 6-{[(4-choloromethyl) benzoyl]

amino} tetramethyl rhodamine (CMTMR/Cell Tracker

Orange, Molecular Probes) (5AM in PBS for 30 min),

washed and seeded in 24-well plates at 2 � 105 cells per

well. Next day, activated PBMC labeled with cytoplasmic

dye 5-chloromethylfluorescein diacetate (CMFDA/Cell

Tracker Green, Molecular Probes) (0.5 AM in PBS for 30

min) were added to the wells at 4 � 105 cells per well. The

cells were cocultured for 24 h, following which suspension

cells were analyzed either by fluorescent microscopy or by

flow cytometry. A similar assay with slight modification

was used to detect apoptosis in activated PBMC after

coculture with Env-expressing cells. CrFKenv/rev or non-

transfected CrFK cells labeled with CMTMR dye were

cocultured with unlabeled ConA-activated PBMC. Apopto-

sis was detected the subsequent day by staining suspensions

cells with Annexin V. Cells positive for Annexin V but

negative for CMTMR were single unfused PBMC that were

apoptotic. Cells positive for Annexin V as well as CMTMR

were Env-expressing cells undergoing apoptosis, most

likely post fusion.

Acknowledgments

The authors would like to thank Dr. Edward Hoover

(Colorado State University) for AMD3100, Robyn Medinas

(Trimeris Inc. Durham, NC) for T1971, and Dr. Brian

Willett (University of Glasgow) for the feline CD134

expression plasmid. The authors are also thankful to Dr.

Frederick Fuller (N.C. State University) for critical review

H. Garg et al. / Virology 330 (2004) 424–436 435

of the manuscript. This work was supported in part by

National Institute of Health grant AI43858.

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