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Virology 329 (20

Activation of HTLV-I gene transcription by protein tyrosine

phosphatase inhibitors

Melanie Langloisa,b, Brigitte Audeta,b, Eric Legaulta,b, Marie-Eve Parea,b, Michel Ouelleta,b,

Jocelyn Roya,b, Nancy Dumaisc, Jean-Michel Mesnardd, David M. Rothsteine,

Susan J. Marriottf, Michel J. Tremblaya,b, Benoit Barbeaua,b,*

aCentre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Quebec, Pavillon CHUL, Ste-Foy (Quebec), Canada G1V 4G2bDepartement de Biologie Medicale, Faculte de Medecine, Universite Laval, Ste-Foy (Quebec), Canada G1V 4G2

cDepartement de Biologie, Faculte des Sciences, Universite de Sherbrooke, Sherbrooke (Quebec), CanadadLaboratoires Infections Retrovirales et Signalisation Cellulaire, CRBM-CNRS UPR 1086, Montpellier 34060, France

eDepartment of Medicine, Yale Medical School, New Haven, CT 06510, USAfDepartment of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA

Received 30 March 2004; returned to author for revision 10 May 2004; accepted 8 September 2004

Available online 1 October 2004

Abstract

Human T-cell leukemia virus type I (HTLV-I) transcription generally depends on the ability of the viral Tax protein to bind the CREB

transcription factor and form an active complex by recruiting CBP/p300 coactivators to the long terminal repeat (LTR). Studies have

demonstrated that T-cell activating agents that stimulate CREB are potent inducers of HTLV-I transcription. Herein, we demonstrate that

bpV[pic], a protein tyrosine phosphatase (PTP) inhibitor activates the HTLV-I LTR in the presence and absence of Tax expression. Optimal

activation occurred at 8 h and was synergistic with forskolin or PGE2. Infected cell lines and cells transfected with HTLV-I proviral DNAwere

equally responsive to the synergistic effect of bpV and forskolin on HTLV-I gene expression. Activation of the LTR by bpV[pic] was T-cell

receptor-independent, but required ZAP70, calcineurin activity and functional calcium entry. Inhibition of the SHP-1 PTP was suggested to be

important. Transfection experiments with a CREB dominant-negative mutant and with isolated TRE1- or CREB-responsive reporter constructs

and treatment with the MDL-12,330A adenylate cyclase inhibitor all supported the involvement of a CREB/ATF family member in this bpV-

dependent activation of the HTLV-I LTR, although CREB itself did not seem to be involved. Analysis of HTLV-I reporter constructs containing

mutated CREB-binding sites also implied the involvement of another element in this activation. These results demonstrate for the first time a

powerful effect of PTP inhibitors on HTLV-I LTR activity and suggest participation of both CREB-dependent and -independent pathways in

this activation.

D 2004 Elsevier Inc. All rights reserved.

Keywords: HTLV-I; LTR; CREB; Protein tyrosine phosphatase

Introduction

Human T-cell leukemia virus type I (HTLV-I) is the

etiological agent of adult T-cell leukemia/lymphoma

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

doi:10.1016/j.virol.2004.09.003

* Corresponding author. Centre de Recherche en Infectiologie, RC709,

Centre Hospitalier Universitaire de Quebec, Pavillon CHUL 2705

Boulevard Laurier, Ste-Foy (Quebec), Canada G1V 4G2. Fax: +1 418

654 2715.

E-mail address: benoit.barbeau@crchul.ulaval.ca (B. Barbeau).

(ATLL) (Hinuma et al., 1981; Poiesz et al., 1980; Yoshida

et al., 1982) and the neurological disease HTLV-I-associated

myelopathy (also termed tropical spastic paraparesis)

(HAM/TSP) (Gessain et al., 1985; Osame et al., 1986;

Rodgers-Johnson et al., 1985). A number of other diseases

have been tentatively linked to HTLV-I infection in humans

although their association remains to be more clearly

ascertained (Uchiyama, 1997). HTLV-I is endemic in

specific regions of the world including Japan and the

Caribbean Islands. Between 10 and 20 million individuals

04) 395–411

M. Langlois et al. / Virology 329 (2004) 395–411396

are presently infected with HTLV-I; however, only 5% of

these infected individuals will eventually develop HTLV-I-

related diseases, while the rest will remain asymptomatic for

life (Uchiyama, 1997).

Infected cells are generally considered to be poor

producers of viral particles and only a very low proportion

of virions are infectious (Sommerfelt, 1999). It is thus

believed that HTLV-I replication in infected patients occurs

mainly through mitotic cell division, resulting in an

oligoclonal expansion of infected cells. The viral Tax

protein is an important T-cell proliferation factor thereby

contributing to the expansion of infected cells (for review,

see Smith and Greene, 1991; Yoshida et al., 1995).

However, when uninfected individuals become infected, a

transient burst of viral gene expression from the infected

cells promotes cell-to-cell transmission of HTLV-I, and

might be part of the two-step infection process previously

described in the squirrel monkey model (Kazanji et al.,

2000). Following the hypothesized cell contact-mediated

increase in viral gene expression, HTLV-I transcription is

believed to be mostly silent in the infected individual, which

might be important for allowing efficient immune escape.

Like all retroviruses, HTLV-I is bordered by sequences

known as long terminal repeats (LTRs). The 5VLTR contains

the major viral promoter and thus controls the expression of

HTLV-I genes. Several regions of the promoter have been

identified as important regulators of HTLV-I gene expres-

sion. Perhaps, the best-characterized transcriptional control

elements within the HTLV-I promoter are the Tax-responsive

element 1 (TRE1) sequences, which mediate the powerful

transactivation potential of Tax (Fujisawa et al., 1986;

Paskalis et al., 1986; Shimotohno et al., 1986). The three

imperfect TRE1 repeats span the U3 region of the LTR and

contain suboptimal cyclic AMP response element binding

protein (CREB) binding sites. Tax is thought to stabilize the

association of both CREB and CREB-binding protein (CBP)/

p300 with the LTR, thereby mediating its strong activation of

HTLV-I gene expression (Giebler et al., 1997; Harrod et al.,

1998; Kashanchi et al., 1998; Kwok et al., 1996; Yan et al.,

1998). Additional studies have also identified a second Tax-

responsive element within the LTR, TRE2 (Brady et al.,

1987), which contains sequences specific for the binding of

transcription factors such as NF-nB and Ets-1 (Bosselut et al.,

1990; Gitlin et al., 1991; Numata et al., 1991).

Little is known about regulation of the HTLV-I LTR in the

absence of Tax. This regulation is important in vivo since

Tax expression is generally low in HTLV-I-positive cells

isolated from infected individuals (Brauweiler et al., 1995).

A previous study has suggested that some of the TRE1

repeats are important for basal transcription (Barnhart et al.,

1997). These same sequences are also known to be important

determinants for HTLV-I LTR activation by cAMP-modulat-

ing agents such as forskolin (Nakamura et al., 1989; Poteat et

al., 1989). In contrast, agents that induce T-cell receptor

(TCR) aggregation have been shown to be poor activators of

the HTLV-I promoter (Copeland et al., 1994, 1995). A

phorbol 12-myristate 13-acetate (PMA)-responsive element

has been identified within the HTLV-I promoter and its

modulatory activity is mediated through Sp1 and p53

(Torgeman et al., 1999, 2001b). However, activation of the

LTR in response to PMA was shown to depend on the

apoptotic-inducing effects of PMA as well as other

apoptosis-inducing agents rather than transcriptional effects

(Torgeman et al., 2001a). A study by Lin et al. (1998)

demonstrated that phytohemagglutinin (PHA)/PMA in com-

bination with Tax resulted in high levels of HTLV-I

replication and that this induction was sensitive to inhibitors

of tyrosine phosphorylation.

Tyrosine phosphorylation is a very important event in

intracellular signaling cascades. In fact, early events

following T cell activation involve a sustained increase in

intracellular phosphotyrosine levels. A critical balance

between the activity of protein tyrosine kinases (PTK) and

protein tyrosine phosphatases (PTP) maintains a crucial

intracellular balance of tyrosine phosphorylation (Yakura,

1998). Some of the most important PTKs for T-cell

activation are p56lck, p59fyn, and ZAP70, while important

PTPs in T-cell activation include SHP-1, a downregulator of

T cell activation, and CD45, an abundant molecule having

both negative and positive effects on T-cell receptor-

mediated signaling (Yakura, 1998). Specific PTP inhibitors

have been used to examine the role of PTPs in T cell

activation. Evidence clearly suggests that these inhibitors

are potent T-cell activators, and strongly induce IL-2

secretion and the activity of transcription factors such as

AP-1 and NF-nB (Barbeau et al., 1997; Imbert et al., 1994,

1996a, 1996b; Ouellet et al., 1999, 2003a, 2003b; Secrist et

al., 1993). Recently, we demonstrated that PTP inhibitors

are also powerful activators of NFAT (Fortin et al., 2001).

Based on the results of Lin et al. (1998), we wanted to

determine whether PTP could modulate transcriptional

activity of the HTLV-I LTR. To directly test this hypothesis,

we used powerful PTP inhibitors known as bis-peroxovana-

dium (bpV) compounds (Posner et al., 1994). Incubation of

T cells with these compounds strongly stimulated activity of

the HTLV-I LTR, which could be further induced upon the

addition of forskolin. This activation was partly CREB-

dependent and required most components of the TCR

pathway. In addition, we identified the PTP, SHP-1, as an

upstream regulator of this induction. We conclude that PTPs

play an important role in the control of HTLV-I gene

expression and that inhibition of this activity leads to higher

levels of HTLV-I LTR activity.

Results

BpV[pic] is a powerful activator of HTLV-I LTR

transcription

We have previously used strong PTP inhibitors to study

the impact of tyrosine phosphorylation on gene regulation in

M. Langlois et al. / Virology 329 (2004) 395–411 397

the context of T cells (Barbeau et al., 1997; Fortin et al.,

2001; Ouellet et al., 1999). These compounds are termed

bis-peroxovanadium (bpV) (Posner et al., 1994) and were

demonstrated in our previous studies to strongly inhibit PTP

activity in Jurkat T cells. On the basis of a previous study

establishing that tyrosine phosphorylation might be required

for Tax-dependent HTLV-I LTR regulation (Lin et al.,

1998), we tested our bpV compounds in Jurkat cells to gain

a better understanding of the role played by tyrosine

phosphorylation in HTLV-I LTR gene expression.

Jurkat cells were transfected with an HTLV-I LTR

reporter vector (pHTLV-LUC) and a wild-type Tax expres-

sion vector or the empty vector. Different combinations of

activators were subsequently added to the transfected cells.

As depicted in Fig. 1A, stimulation of T cells by the bpV[pic]

PTP inhibitor led to a significant increase in Tax-dependent

LTR activation to levels higher than those measured in

untreated cells (36.7-fold). The addition of PMA with or

without ionomycin led to a significant but weaker induction

of Tax-dependent LTR activation. However, unexpectedly,

we found that bpV[pic] also induced a potent increase in

LTR gene expression in the absence of Tax expression. In

fact, LTR-dependent gene expression was induced 20-fold

following PTP inhibition, which was significantly greater

than induction measured in cells treated with PMA alone or

in combination with ionomycin (2.4 and 4.2-fold, respec-

tively). This increase in HTLV-I LTR activity could not be

generalized to all promoters as no similar induction in

reporter gene expression was noted when Jurkat cells

transfected with a h-actin promoter-driven h-gal vector

was treated with our PTP inhibitors (data not shown).

We next conducted a time course analysis to determine

the optimal time for HTLV-I LTR activation by bpV[pic].

Fig. 1B demonstrated that optimal HTLV-I LTR induction

occurred at 8 h following bpV[pic] treatment, reached a

plateau at 12 h, and then returned to basal level after 24 h of

stimulation. Such a pattern of induction has also been

observed in previous studies testing several activating

agents in T cells (including PTP inhibitors) (Fortin et al.,

Fig. 1. Activation of the HTLV-I LTR by bpV[pic] occurs in the presence and absen

10 Ag of the wild-type Tax expression vector or pHhPr.1-neo. In panel B, cells w

were either left untreated or stimulated with the following agents: PMA (20 ng/m

lysed and luciferase activity was measured after 8 h and at other time points p

triplicates F SD. Fold inductions in panel B were calculated as the ratio of the

results are representative of three independent experiments.

2001; Ouellet et al., 1999). In comparison, activation by

either PMA or the PMA/ionomycin combination continued

to increase after 24 h of incubation, a result which is

consistent with a previous study (Mor-Vaknin et al., 1997).

Activation of the HTLV-I LTR by bpV[pic] was also

detected in other T-cell lines such as SupT1 and HPB.ALL

(data not shown). These results demonstrate that PTP

inhibitors can independently induce activation of the

HTLV-I LTR in different T-cell lines, and that this LTR

activation is greater than other previously described

stimulation.

Activation of the HTLV-I LTR by bpV[pic] is synergistic

with forskolin

We next investigated possible synergistic activation of

the LTR by bpV[pic] and other known activators of HTLV-I

transcription. A series of different activators was tested

individually or in combination. These included PHA, PMA,

ionomycin, anti-CD3 (OKT3) and anti-CD28 (9.3) anti-

bodies, bpV[pic] and forskolin, a compound that upregu-

lates HTLV-I gene expression by activating the CREB

transcription factor (Nakamura et al., 1989; Poteat et al.,

1989). Jurkat cells were transfected with the pHTLV-LUC

vector and subsequently stimulated as indicated (Fig. 2A).

As demonstrated earlier, bpV[pic] potently induced HTLV-I

LTR-regulated gene expression while other activators

resulted in poor activation. When the PTP inhibitor was

combined with other activators, bpV-dependent induction

was either unaltered (PMA, ionomycin, anti-CD3/anti-

CD28), reduced (PMA/ionomycin) or was weakly enhanced

(PHA, PHA/PMA). The greatest synergistic activation of

the LTR was observed in the presence of forskolin and

bpV[pic]. A similar synergistic activation of the LTR by

bpV[pic] and forskolin was also observed in isolated

peripheral blood mononuclear cells (PBMCs) transfected

with the pHTLV-LUC vector (data not shown).

Synergistic activation of the LTR by forskolin and

bpV[pic] or PMA was compared at different concentrations

ce of Tax. (A) Jurkat cells were co-transfected with pHTLV-LUC (5 Ag) andere transfected with 15 Ag of pHTLV-LUC. At 24 h post-transfection, cells

l), PMA (20 ng/ml)/ionomycin (1 AM) and bpV[pic] (10 AM). Cells were

ost-induction for panel B. Luciferase values are reported as the mean of

mean value of treated samples over the mean of untreated samples. These

Fig. 2. Synergistic activation of the HTLV-I LTR by bpV[pic] and CREB-activating agent. Jurkat cells were transfected with 15 Ag of pHTLV-LUC. At 24 h

post-transfection, cells were either left untreated or stimulated with the following agents: (A) PHA (3 Ag/ml), PMA (20 ng/ml), ionomycin (1 AM), PHA (3

Ag/ml)/PMA (20 ng/ml), PMA (20 ng/ml)/ionomycin (1 AM), OKT3 (anti-CD3) (3 Ag/ml)/9.3 (anti-CD28) (1 Ag/ml), forskolin (100 AM) alone or in

combination with bpV[pic] (10 AM), (B) PMA (20 ng/ml) and bpV[pic] (10 AM) added in combination with increasing concentrations of forskolin (0.1, 1, 10,

100 AM), (C) combinations of increasing concentrations of forskolin (1, 10, 100 AM), bpV[pic] (0.1, 1, 10 AM) and Na3VO4 (0.25, 2.5, 25 mM), (D) bpV[pic]

(10 AM), PGE2 (100 nM), bpV[pic] (10 AM)/PGE2 (100 nM). After 8 h of stimulation (6h for panel A), cells were lysed and luciferase activity was measured.

Luciferase values were reported as the mean of triplicates F SD. Fold inductions were calculated as the ratio of the mean value of treated samples over the

mean value of untreated samples. These results are representative of three independent experiments.

M. Langlois et al. / Virology 329 (2004) 395–411398

in Jurkat cells transfected with pHTLV-LUC (Fig. 2B).

Although a significant increase was apparent for all tested

activators at higher concentrations of forskolin, the greatest

synergistic induction was observed in the presence of the

bpV[pic]/forskolin combination. Also, maximal synergistic

activation with bpV[pic] occurred at a lower concentration

of forskolin (1 AM) than needed to induce synergistic

activation with PMA.

Sodium orthovanadate is another PTP inhibitor, which

has been suggested to be less potent than bpV[pic] (Posner

et al., 1994). LTR activity in response to different

concentrations of bpV[pic] or sodium orthovanadate in the

presence or absence of forskolin was analyzed in Jurkat cells

(Fig. 2C). Sodium orthovanadate did not induce HTLV-I

LTR-mediated gene expression either alone or in the

presence of forskolin, indicating that bpV[pic] was likely

more inhibitory for PTPs and consequently a better activator

of HTLV-I LTR transcription. These results also indicated

that the action of bpV[pic] occurred over a very narrow

concentration range and was not measurable at 1 AM either

with or without forskolin.

Since the synergy of bpV[pic] and forskolin might be

driven by the ability of forskolin to activate CREB, we

tested PGE2, another CREB activator, which we have

recently shown to activate the HTLV-I LTR upon concom-

itant TCR activation (Dumais et al., 2003). Jurkat cells

transfected with pHTLV-LUC were treated with bpV[pic]

alone or in combination with PGE2. As demonstrated in Fig.

2D, the combination of PGE2 and bpV[pic] resulted in

synergistic activation of the HTLV-I LTR supporting a role

for CREB in bpV[pic]-mediated activation of the LTR. As

we have previously demonstrated (Dumais et al., 2003),

PGE2 alone was a poor activator of the HTLV-I LTR.

To determine whether this bpV-dependent activation of

the HTLV-I LTR was reporter-dependent, the GFP reporter

gene was positioned downstream of the HTLV-I LTR.

Typically, less than 1% of Jurkat cells transfected with the

pHTLV-GFP vector showed a positive GFP signal. In Jurkat

M. Langlois et al. / Virology 329 (2004) 395–411 399

cells co-transfected with pHTLV-GFP and the Tax expres-

sion vector, GFP signals could be detected in up to 6% of

the analyzed cells (data not shown). Therefore, Jurkat cells

were transfected with pHTLV-GFP and subsequently

stimulated with different activating agents for 8 h. A small

increase in the number of cells positive for GFP expression

was observed following PMA/ionomycin treatment (1.93%)

(Table 1). Interestingly, a greater increase in positive cells

was measured in bpV[pic]-treated cells (3.12%). The

highest level of GFP-positive cells was observed when

bpV[pic] was used in combination with forskolin (6.62%).

These results demonstrate potent activation of the HTLV-I

LTR in response to a PTP inhibitor as compared to other

known T cell activators. Our results further indicate the

synergistic action of bpV[pic] with CREB-activating agents.

Activation of HTLV-I gene expression from full-length

proviral DNA and integrated proviral DNA by bpV[pic] in

combination with forskolin

To evaluate bpV[pic] activation of HTLV-I LTR tran-

scription in the context of full-length HTLV-I proviral DNA,

a luciferase reporter gene was inserted in frame with the

envelope ATG initiation codon from the K30 proviral DNA

clone without altering the splice donor site for multiply

spliced mRNA. This proviral DNA was directly transfected

into Jurkat cells and the transfected cells were subsequently

treated with activators. As depicted in Fig. 3A, activation by

bpV[pic] and all other tested activators led to a weak

induction, which was less than 2-fold. However, the

combination of bpV[pic] and forskolin induced luciferase

expression by 9.1-fold 8 h after stimulation, and this activity

was greatly enhanced at 24 h post-stimulation (129.7-fold)

and sustained up to at least 72 h post-treatment.

Previous experiments have indicated differences in the

regulation of HTLV-I gene transcription when integrated

into cellular DNAversus an episomal form (Lemasson et al.,

2002; Okada and Jeang, 2002). To confirm our results with

integrated proviral DNA, we performed experiments with

HTLV-I-infected MJ cells, which are CD3- and CD4-

positive T-cells. At different time points after stimulation,

supernatants from the MJ cells were quantitated for the

presence of the HTLV-I p19 antigen. As illustrated in Fig.

3B, at 24 h post-stimulation, only the bpV[pic]/forskolin

combination led to a significant increase in supernatant-

Table 1

Induction of HTLV-I-driven GFP gene expression by bpV[pic]

Treatment Percentage of positive

cells (%)a

NT 1.23

PMA/ionomycin 1.93

bpV[pic] 3.12

bpV[pic]/forskolin 6.62

a Percentage of positive cells for the GFP signal was determined at 8 h after

stimulation by FACS analysis.

associated p19 antigen. While bpV[pic] alone had little

effect on the production of HTLV-I p19 in the supernatant of

MJ cells throughout the time course, bpV[pic]/forskolin led

to a sustained increase in p19 production, to higher levels

than observed following treatment with the combination of

PMA/ionomycin. Forskolin-like PMA had no detectable

effect on the level of extracellular p19 antigen (data not

shown). These results support the notion that bpV com-

pounds can activate the HTLV-I LTR in the context of

episomal DNA as well as the integrated proviral DNA form,

although forskolin was required to observe this induction in

MJ cells.

Effect of early TCR-like cell signaling mediated by bpV

compounds on HTLV-I LTR activation

Our results confirmed that TCR-mediated T cell activa-

tion results in weak activation of the HTLV-I LTR. Since

PTP inhibitors can activate multiple transcription factors

through TCR-like events (Ouellet et al., 2003a,b), we next

examined the effect of bpV[pic] on the activation of HTLV-I

LTR transcription in response to TCR signaling pathways.

To determine whether the T-cell receptor is required for

activation of HTLV-I LTR transcription by bpV[pic], both

Jurkat and J.RT3 (a TCR-negative derivative) cells were

transfected with the pHTLV-LUC reporter construct and

transfected cells were then exposed to various agents.

bpV[pic]-mediated LTR activation (with or without added

forskolin) was decreased in J.RT3 cells as compared to

Jurkat cells, although a significant induction of the HTLV-I

promoter was still observed in J.RT3 cells (Fig. 4A). Other

activating agents showed similar abilities to activate the

LTR in both Jurkat cell lines. These results suggest that the

TCR is not absolutely necessary for bpV[pic] induction of

HTLV-I LTR-mediated gene expression and agree with our

observation of HTLV-I LTR activation in the TCR-negative

SupT1 cell line (data not shown).

ZAP70 is another important player in T cell signaling.

P116 cells are Jurkat cell derivatives that are negative for

ZAP70 (Williams et al., 1998). A stable transfectant (P116

clone 39) has been generated from this clone and

expresses normal levels of ZAP70. P116, P116 clone 39,

and Jurkat cells were transfected with pHTLV-LUC and

then stimulated with the different activators (Fig. 4B).

Comparison of the different cell lines showed that P116

cells had the weakest HTLV-I LTR response to bpV[pic].

P116 clone 39 showed an increased HTLV-I LTR response

to bpV stimulation and reached levels of activation close

to that observed in similarly stimulated and transfected

Jurkat cells. ZAP70 expression was also required for

activation of the HTLV-I LTR by the bpV[pic]/forskolin

combination. In general, activation by PMA, PMA/

ionomycin or forskolin was not statistically different in

the presence or absence of ZAP70. This was expected due

to the fact that these agents activate the signaling pathway

downstream of ZAP70.

Fig. 3. Activation of HTLV-I proviral gene expression by bpV[pic]. (A) Jurkat cells were transfected with 30 Ag of 5VK30-LUC/3VK30-ligated DNA mixture.

At 24 h post-transfection, cells were either left untreated or stimulated with the following conditions: PMA (20 ng/ml), PMA (20 ng/ml)/ionomycin (1 AM),

forskolin (100 AM), bpV[pic] (10 AM) and bpV[pic] (10 AM)/forskolin (100 AM). After 8, 24, 48, and 72 h of stimulation, cells were lysed and luciferase

activity was measured. (B) MJ cells were left untreated or stimulated for 24, 48, and 72 h with the following conditions: PMA (20 ng/ml)/ionomycin (1 AM),

bpV[pic] (10 AM) and bpV[pic] (10 AM)/forskolin (100 AM). Supernatants were then harvested and p19 antigen was quantitated. Luciferase values and p19

antigen levels were reported as the mean of triplicates F SD. In panel A, fold inductions were calculated as the ratio of the mean value of treated samples over

the mean value of untreated samples. These results are representative of three independent experiments.

M. Langlois et al. / Virology 329 (2004) 395–411400

Since the calcineurin serine/threonine phosphatase plays

an important role in activating several transcription factors

in T cells, we assessed the effect of this serine/threonine

phosphatase on bpV[pic]-mediated HTLV-I LTR activation.

Jurkat cells transfected with the pHTLV-LUC vector were

treated with FK506 or CsA, two specific calcineurin

inhibitors, prior to stimulation (Fig. 4C). Both of these

inhibitors reduced the capacity of bpV[pic] and bpV[pic]/

forskolin activators to induce HTLV-I LTR activity. Since

calcium is a crucial signaling mediator in the activity of

calcineurin, we assessed the role of intracellular calcium

mobilization in bpV[pic]-dependent activation of the HTLV-

I LTR. PMA/ionomycin activation of the HTLV-I LTR was

reduced in the transfected CJ1.1 cell line, deficient for

capacitative calcium entry, as compared to the wild type CJ

parental clone (Fig. 4D). Importantly, a similarly reduced

LTR response was observed in this calcium-deficient cell

line when cells were stimulated with the PTP inhibitor (with

or without forskolin), suggesting that calcium plays an

important role in activation of the HTLV-I LTR by PTP

inhibition. These results demonstrate that certain cellular

components involved in TCR-mediated signaling play a role

in the bpV[pic]-mediated signaling cascade that leads to

HTLV-I LTR activation. However, the TCR was not

specifically required in this pathway.

BpV[pic] activation of HTLV-I LTR activity is counteracted

by SHP-1 expression

Activation of the HTLV-I LTR by the PTP inhibitor

suggested the involvement of one or more PTPs in this

activation pathway. We looked at the possible role of two

important PTPs, CD45 and SHP-1, initially focusing on

CD45 as a target of bpV[pic]. JKF cells and the CD45-

deficient K6D cell line derived from JKF cells were

transfected with pHTLV-LUC and subsequently treated

with the activating agents. No significant differences in

LTR response to any of the tested activators including

bpV[pic] were detected (data not shown). The Jurkat-

derived CD45-negative J45.01 cell line was also compared

to Jurkat cells, and again no significant difference in

response was observed when cells transfected with

pHTLV-LUC were treated with our PTP inhibitor (data

not shown). We then turned our attention to SHP-1. This

PTP is required to terminate the TCR-initiated cascade and

has been observed to dephosphorylate several crucial

players in this cascade. To determine whether SHP-1 was

targeted by bpV[pic], a vector expressing SHP-1 or the

empty vector were co-transfected with pHTLV-LUC into

Jurkat cells and cells were then stimulated with the different

activators (Fig. 5). We predicted that overexpression of a

PTP targeted by bpV[pic] should block activation of the

HTLV-I LTR. In fact, although SHP-1 expression did not

affect the response of the HTLV-I LTR following PMA or

PMA/ionomycin induction, its expression significantly

reduced bpV[pic]- and bpV[pic]/forskolin-dependent

HTLV-I LTR activation. These results thus suggest that

SHP-1 inhibition might be responsible for activation of the

HTLV-I LTR following bpV[pic] treatment.

Role of the LTR region containing TRE1 sequences in

bpV[pic]-mediated activation of the HTLV-I transcription

TRE1 sequences in the HTLV-I LTR contain CREB-

binding sites that are important for positive regulation of

HTLV-I transcription. To determine the potential role of

these sequences in bpV-mediated LTR activation, LTR

deletion mutants were generated with or without the TRE1

Fig. 4. TCR-like signaling is involved in the activation of the HTLV-I LTR by bpV[pic]. pHTLV-LUC (15 Ag) was transfected into Jurkat (panels A, B and C),

J.RT3 (panel A), P116 and P116 clone 39 (panel B), CJ parental and CJ1.1 (panel D). In panel C, transfected cells were pretreated for 15 min with FK506 (10

ng/ml) or CsA (100 ng/ml) before activation. At 24 h post-transfection, cells were either left untreated or stimulated with the following agents: PMA (20 ng/

ml), PMA (20 ng/ml)/ionomycin (1 AM), forskolin (100 AM), bpV[pic] (10 AM) and bpV[pic] (10 AM)/forskolin (100 AM). After 8 h of stimulation, cells were

lysed and luciferase activity was measured. Luciferase values were reported as the mean of triplicates F SD. Fold inductions were calculated as the ratio of the

mean value of treated samples over the mean value of untreated samples. These results are representative of three independent experiments.

M. Langlois et al. / Virology 329 (2004) 395–411 401

sequences. These constructs were transfected into Jurkat

cells and, after cell activation, extracts were prepared and

luciferase activity was quantitated (Fig. 6A). Removal of the

5VLTR sequence up to position �325 (pHTLV-LUCDSmaI)

did not significantly alter LTR activation by any of the

tested activators including bpV[pic]. However, deletion up

to position �57, which removed all three TRE1 sequences,

completely abolished activation mediated by bpV either

alone or in the presence of forskolin. To confirm the

importance of the sequence contained between position

�325 and �57, the SmaI/NdeI fragment (harboring all 3

TRE1s) was cloned upstream of a minimal promoter (TATA

box) and the luciferase reporter gene (pGL2tata

HTLV(�325/�57)). BpV[pic] treatment of Jurkat cells

transfected with this construct resulted in a significant

induction of luciferase activity that was absent in cells

transfected with the pGL2tata control vector (Fig. 6B and

data not shown). Interestingly, the activation was more

pronounced than the one measured with the pHTLV-LUC

construct. In fact, in Jurkat cells, a significant response of

the pGL2tata HTLV(�325/�57) construct was observed

following treatment with forskolin, PMA, or PMA/ionomy-

cin. Similar transfection results in Jurkat cells were obtained

when the HTLV-I TRE1-containing cassette was positioned

upstream of the SV40 promoter and the luciferase reporter

gene (pGL2-Promoter HTLV(�325/�57)) (data not shown).

These results suggest that the TRE1 sequences play an

important role in activation of the HTLV-I LTR mediated by

bpV[pic].

Role of a CREB/ATF family member in HTLV-I LTR

activation induced by bpV[pic]

Since CREB is an important positive modulator of the

HTLV-I LTR TRE1 sequences, we tested its potential role in

modulation of HTLV-I LTR activity by bpV[pic]. To

Fig. 6. An LTR region containing TRE1 sequences mediates bpV[pic] activatio

pHTLV-LUC (panels A and B), pHTLV-LUCDSmaI (panel A), pHTLV-LUCDNd

transfection, cells were either left untreated or stimulated with the following agents

bpV[pic] (10 AM) and bpV[pic] (10 AM)/forskolin (100 AM). After 8 h of stimula

values were measured as the mean of triplicatesF SD. Fold inductions were calcul

untreated samples. Tested constructs are depicted above each panel in which the

three independent experiments.

Fig. 5. SHP-1 expression blocks bpV[pic] activation of the HTLV-I LTR.

Jurkat cells were co-transfected with 5 or 10 Ag of the SHP-1 WT

expression vector (or the control pSFFVneo vector) along with 10 Ag of

pHTLV-LUC. At 24 h post-transfection, cells were either left untreated or

stimulated with the following agents: PMA (20 ng/ml), PMA (20 ng/ml)/

ionomycin (1 AM), forskolin (100 AM), bpV[pic] (10 AM) and bpV[pic] (10

AM)/forskolin (100 AM). After 8 h of stimulation, cells were lysed and

luciferase activity was measured. Luciferase values were reported as the

mean of triplicatesF SD. Fold inductions were calculated as the ratio of the

mean value of treated samples over the mean value of untreated samples.

These results are representative of three independent experiments.

M. Langlois et al. / Virology 329 (2004) 395–411402

directly address the role of a CREB/ATF family member in

bpV[pic]-mediated LTR activation, a dominant negative

CREB mutant, KCREB, was transfected into Jurkat cells

with the pHTLV-LUC vector (Fig. 7A). Compared to

transfections with the empty vector, KCREB-expressing

Jurkat cells showed less induction of the HTLV-I LTR by

bpV[pic] and bpV[pic]/forskolin. No statistically significant

differences in LTR activity were observed following

stimulation with PMA, PMA/ionomycin, or forskolin in

cells transfected with KCREB versus the empty vector.

These results suggest that a member of the CREB tran-

scription factor family is required for bpV[pic] activation of

the HTLV-I LTR. To confirm the involvement of a CREB/

ATF member, cells were treated with increasing subcyto-

toxic concentrations of MDL-12,330A, an adenylate cyclase

inhibitor, which thus diminishes the level of intracellular

cAMP. Jurkat cells transfected with pHTLV-LUC were less

responsive to bpV[pic] or bpV[pic]/forskolin stimulation

upon MDL-12,330A treatment, dropping to 40% to 45%

from fold activation levels measured in untreated cells,

while limited effect was noted on PMA/Ionomycin-acti-

vated cells. Forskolin was also sensitive to this inhibitor

while PMA-stimulated cells were not affected (data not

shown).

n of HTLV-I transcription. Jurkat cells were co-transfected with 15 Ag of

eI/SmaI (panel A) or pGL2tata HTLV(�325/�57) (panel B). At 24 h post-

: PMA (20 ng/ml), PMA (20 ng/ml)/ionomycin (1 AM), forskolin (100 AM),

tion, cells were lysed and luciferase activity was measured. Mean luciferase

ated as the ratio of the mean value of treated samples over the mean value of

filled boxes represent the TRE1 repeats. These results are representative of

Fig. 7. Implication of a CREB/ATF factor in bpV[pic]-dependent activation of HTLV-I LTR. Jurkat cells were co-transfected with 5 or 10 Ag of expression

vectors of KCREB (or the pRC/RSV control vector) with 10 Ag of pHTLV-LUC (panel A). In panel B, Jurkat cells were transfected with 15 Ag of pHTLV-LUCand pretreated or not with MDL-12,330A (100 AM and 250 AM) for 1 h prior to activation. In panel C, Jurkat cells were transfected with 15 Ag of pCRE-LUC.

In panels D and E, Jurkat cells were co-transfected with 10 Ag of expression vectors for CREB WT or CREB F/Yor the pcDNA.CF empty vector and 10 Ag of

pGL2tata HTLV(�325/�57) (panel D) or pNF-nB-LUC (panel E). At 24 h post-transfection, cells were either left untreated or stimulated with the following

agents: PMA (20 ng/ml) (except for panels B, D and E), PMA (20 ng/ml)/ionomycin (1 AM), forskolin (100 AM) (except for panels B, D and E), bpV[pic] (10

AM), and bpV[pic] (10 AM)/forskolin (100 AM). After 8 h of stimulation (in panel C, after 2, 4, 6, 8, and 24 h), cells were lysed and luciferase activity was

measured. Luciferase values were reported as the mean value of triplicates F SD. Fold inductions were calculated as the ratio of the mean value of treated

samples over the mean value of untreated samples. In panel B, values are presented as percentage of fold activation with 100% representing folds obtained in

cells not treated with the inhibitor. These results are representative of three independent experiments.

M. Langlois et al. / Virology 329 (2004) 395–411 403

M. Langlois et al. / Virology 329 (2004) 395–411404

To further demonstrate that bpV[pic] activated a member

of the CREB/ATF family, Jurkat cells were transfected with

pCRE-LUC, which contains 4 tandem repeats of the CRE

element (Fig. 7C). Upon activation of the transfected Jurkat

cells, reporter gene expression was responsive to several

stimulating agents. While forskolin and bpV[pic] both

activated this reporter weakly, forskolin alone was a more

potent activator of CREB-dependent transcription than

bpV[pic] alone. However, the combination of bpV[pic]/

forskolin again led to a significant increase in CREB-

dependent luciferase expression. As expected, PMA and

PMA/ionomycin were poor inducers of luciferase gene

expression in these transfected cells.

We directly tested the possible role of CREB family

members in activation of the HTLV-I LTR by bpV[pic] by

overexpressing the ATF/CREB family member in Jurkat

cells. Expression vectors for wild type CREB or a

constitutively active version of CREB were transfected into

Jurkat cells along with pGL2tata HTLV(�325/�57) and

then stimulated with different activators (Fig. 7D). To our

surprise, expression of wild type or constitutively active

CREB resulted in a significant reduction in the activation of

pGL2tata HTLV(�325/�57) by bpV[pic] or bpV[pic]/

forskolin, while no comparable effects were observed in

PMA/ionomycin-stimulated cells. As a control, Jurkat cells

were similarly co-transfected with the CREB expression

vectors and pNF-nB-LUC (Fig. 7E). In contrast to the

results presented above, CREB expression had no effect on

NF-nB-dependent luciferase expression regardless of the

tested activator. This result is likely due to competitive

binding of the overexpressed CREB protein to the LTR,

thereby displacing a functional CREB family member. It

appears unlikely that the reduced HTLV-I LTR activity

observed upon CREB expression and bpV[pic] treatment is

simply an effect of squelching the CBP/p300 co-activators

since a similar effect was not observed on a NF-nBdependent promoter. Together, these results suggest that a

CREB family member, but not CREB itself, is involved in

bpV[pic]-mediated activation of the HTLV-I LTR.

A second factor is required for bpV-induced HTLV-I LTR

activity

To more directly address the role of TRE1 CREB-

binding sites in activation of the HTLV-I LTR following

PTP inhibition, different HTLV-I LTR constructs containing

mutations in 1, 2, or all 3 TRE1 repeats were used (Fig. 8A).

Reduced bpV[pic] activation was reproducibly observed

when these constructs were transfected into Jurkat cells and

was most evident when all three TRE1 sequences were

mutated (Fig. 8B). However, even in this triple TRE1-

mutated construct (pGL3-21PMD/11T), a significant level

of bpV-dependent activation was still apparent and

accounted for more than 50% of the activity observed with

the wild-type LTR. A similar trend was detected with the

bpV[pic]/forskolin combination. PMA and PMA/ionomycin

induced luciferase gene expression to similar levels for all 8

tested LTR constructs. In contrast to these results, co-

transfection of a Tax expression vector with pGL3-21PMD/

11T showed a greater than 90% reduction in luciferase

expression from that observed on pGL3-U3R at both 24 and

48 h post-transfection (Fig. 8C). These data are consistent

with the idea that activation of the HTLV-I LTR by bpV[pic]

is not exclusively dependent on the CREB-binding elements

located in the TRE1 repeats.

Discussion

In this study, we were interested in assessing the

involvement of PTP in the transcriptional regulation of

HTLV-I. A previous study had suggested that tyrosine

phosphorylation plays an important role in HTLV-I tran-

scription by the Tax protein combined with a PHA

stimulation (Lin et al., 1998). Indeed, in this study, we

demonstrated that the use of a PTP inhibitor termed

bpV[pic], which is known to increase intracellular phos-

photyrosine levels, resulted in a significant increase in

HTLV-I LTR activity.

Our first experiments were conducted to assess the role

of bpV[pic] in Tax-mediated LTR activation. These results

indicated that bpV[pic] could significantly induce HTLV-I

LTR activity in both Tax-expressing and non-Tax-express-

ing cells. Time course experiments permitted us to conclude

that this induction was likely mediated by direct activation

of a signaling pathway since the optimal effect was at 8 h,

consistent with several studies from our group using

different activators that act directly on specific transcription

factors such as NFAT and NF-nB (Barbeau et al., 1997;

Fortin et al., 2001; Ouellet et al., 1999). In addition, the

kinetic response of the HTLV-I LTR induced by bpV[pic]

was drastically different (at least in Jurkat cells) from the

activation pattern of PMA, recently linked to apoptosis

(Torgeman et al., 2001a). These results with bpV[pic]

demonstrate the potential of TCR-like signaling cascades

to positively modulate HTLV-I gene expression.

A series of experiments also indicated that the combina-

tion of forskolin and bpV[pic] resulted in increased

activation of the HTLV-I LTR, while other agents in

combination with bpV[pic] generally did not affect the

bpV[pic] response. In contrast, sodium orthovanadate had

no such effect, which is consistent with the fact that bpV

compounds are more efficient PTP inhibitors than sodium

orthovanadate. It is also possible that bpV compounds might

target different PTPs than those inhibited by sodium

orthovanadate. In the presence of bpV[pic], forskolin

efficiently induced the HTLV-I LTR to greater levels than

PMA. Similar to bpV[pic] (discussed below), PHA and

PMA are likely to be weak activators of CREB and might

also activate other HTLV-I-regulatory factors that synergize

with CREB. This is consistent with our finding that

bpV[pic] strongly synergizes with PGE2, another CREB

Fig. 8. CREB-dependent and -independent mechanisms regulate bpV-mediated activation of the HTLV-I LTR. (A) Vectors containing wild type or mutated

CREB-binding sites in the TRE1 sequence located proximal (P), middle (M) or distal (D) from the HTLV-I promoter CAP site. (B) Jurkat cells were transfected

with 15 Ag of pGL3-U3R, pGL3-21P/11T, pGL3-21M/11T, pGL3-21D/11T, pGL3-21PM/11T, pGL3-21PD/11T, pGL3-21MD/11T or pGL3-21PMD/11T. At

24 h post-transfection, cells were either left untreated or stimulated with the following agents: PMA (20 ng/ml), PMA (20 ng/ml)/ionomycin (1 AM), forskolin

(100 AM), bpV[pic] (10 AM), and bpV[pic] (10 AM)/forskolin (100 AM). After 8 h of stimulation, cells were lysed and luciferase activity was measured.

Luciferase values were reported as the mean value of triplicates F SD. Fold inductions were calculated as the ratio of the mean value of treated samples over

the mean value of untreated samples. (C) Jurkat cells were co-transfected with 10 Ag of the Tax expression vector (or the empty pHhPr.1-neo vector) and 10 Agof either pGL3-U3R or pGL3-21PMD/11T. Cells were lysed 24 or 48 h after transfection and luciferase activity was assessed. Luciferase values were reported

as the mean of triplicates F SD. Fold inductions were calculated as the ratio of the mean value of Tax-transfected cells over the mean value of empty vector-

transfected cells. These results are representative of three independent experiments.

M. Langlois et al. / Virology 329 (2004) 395–411 405

activator, to regulate the HTLV-I LTR. We have previously

described a synergism between PGE2 and antigen-like

stimulation, which appears to use a similar mechanism to

activate the HTLV-I LTR (Dumais et al., 2003).

In light of previous publications addressing the tran-

scriptional regulation of integrated HTLV-I DNA (Lemas-

son et al., 2002; Okada and Jeang, 2002), we also

investigated the modulation of HTLV-I replication in

HTLV-I-infected cells following stimulation. We found a

significant increase in viral particle production as deter-

mined by p19 antigen levels following bpV[pic] treatment

in the presence of forskolin. Furthermore, transient trans-

fection of a proviral DNA construct containing the

luciferase reporter gene confirmed that activation of the

HTLV-I LTR in the context of proviral DNA was also

observed in cells treated with forskolin and bpV[pic]. This

activation occurred at later time points than similarly

treated pHTLV-LUC-transfected cells. The multiple splic-

ing events that are required for expression of viral gene

products likely contributed to the delay in detecting peak

viral replication responses to PTP inhibition. BpV[pic], as

well as other activators added alone, resulted in poor

activation of the HTLV-I LTR in the context of proviral

DNA. Although this result requires further investigation, it

is possible that the negative regulatory functions of the

viral HBZ and p30II proteins could contribute to the lack

of response to these activators (Gaudray et al., 2002; Nicot

et al., 2004). Despite this caveat, the above results do

suggest that PTPs may influence HTLV-I gene expression

in vivo.

BpV compounds have been previously shown to utilize

signaling pathways similar to those induced by the TCR

M. Langlois et al. / Virology 329 (2004) 395–411406

(Fortin et al., 2001; Ouellet et al., 1999, 2003a,b).

Activation of the HTLV-I LTR by these inhibitors was not

surprisingly found to depend on ZAP70, calcineurin activity

and calcium mobilization, but did not require the expression

of cell surface TCR. These signaling molecules are also

involved in bpV-mediated NF-nB and NFAT activation

(Fortin et al., 2001; Ouellet et al., 1999). These results

support the idea that TCR signaling can activate the HTLV-I

LTR. This is especially relevant since our data suggest that

the PTP targeted by bpV[pic], permitting activation of the

HTLV-I LTR, is likely to be SHP-1, a crucial downregulator

of TCR signaling.

Our results strongly suggest that the TRE1 repeats are

important for PTP-dependent activation of the HTLV-I LTR.

Indeed, isolated TRE1 sequences were sufficient to confer

bpV[pic] responsiveness to a minimal promoter. We also

suggested that CREB activation and binding to the CREB-

binding site within the TRE1 repeats is important for

activation of HTLV-I transcription. Furthermore, point

mutation of the CREB-binding site in all three TRE1

repeats reduced the ability of the PTP inhibitor to induce

transcription. Parallel experiments conducted with the

pCRE-LUC plasmid also showed that it was activated by

bpV[pic] in synergy with forskolin. The ability of our PTP

inhibitors to activate CREB/ATF family members is not

surprising in light of a previous study indicating that PTP

activity inhibits CREB activation (Champion-Arnaud et al.,

1991). It is interesting to note the difference between

activation of the CREB consensus sequence (pCRE-LUC)

and the CREB-binding site in the TRE1 sequence by the

combination of bpV[pic] and forskolin. These dissimilar

responses might reflect a minimal involvement of CREB in

activation of the HTLV-I LTR following PTP inhibition (see

below). We are presently making efforts to identify the

CREB/ATF family member involved in HTLV-I LTR

activation by bpV[pic], and our results suggest that CREB

itself is not a likely candidate. Indeed, Tax-mediated

modulation of HTLV-I gene expression was affected by

CREB expression in a manner similar to that observed with

bpV[pic]-dependent stimulation of the HTLV-I LTR

(Gachon et al., 1998). It is tempting to speculate that

CREB2 might be the relevant factor, although experiments

conducted with a CREB2 expression vector indicated no

increasing effects by this family member on our observed

bpV-mediated induction of HTLV-I LTR activity in Jurkat

cells (data not shown). Other family members are known to

bind to the TRE1 regions in vivo (Lemasson et al., 2002)

and might thus be involved.

Our results strongly argue for the involvement of a

transcription factor that binds outside of the CRE-like

consensus sequence. Based on experiments using the TRE1-

mutated HTLV-I LTRs, this putative factor appears to

contribute more than 50% of the induction mediated by

bpV[pic]. In the presence of forskolin, bpV[pic] induction

was more sensitive to the mutated TRE1 repeats, which

might be expected given the known activation of CREB by

forskolin. In fact, the synergy between bpV[pic] and

forskolin is consistent with the weak CREB activating

potential of bpV[pic] alone and strong enhancement of the

CREB activating potential by forskolin. There are several

possible candidates for the identity of this other factor. First,

the TRE2 sequence harbors a binding site for the GLI2

protein, which was initially suggested to be involved in Tax-

mediated activation of the HTLV-I LTR (Dan et al., 1999;

Tanimura et al., 1998). When the LTR was mutated to knock

out the GLI2-binding site in TRE2, there was no apparent

difference in response of the mutant and wild-type LTRs to

bpV[pic] stimulation (B.A. and B.B., unpublished results).

This suggests that GLI2 is not involved in the activation of

the LTR by bpV[pic]. Previous studies have shown that

PMA is a good activator of HTLV-I LTR transcription and

that this activation depended on a region containing Sp1-

and p53-binding sites (Torgeman et al., 2001b). PTP

inhibitors might activate these factors; however, the effect

of PMA is believed to increase with time and to be

enhanced by PKC inhibition. This type of kinetic activation

was not observed for our PTP inhibitor, although these

results do not completely rule out the possible involvement

of these factors in bpV[pic]-mediated activation of HTLV-I

transcription. Since other transcription factor-binding sites

(including the recently described serum response element

(Wycuff et al., 2004)) have been identified within the

HTLV-I LTR, further studies will be needed to identify the

transcription factor that mediates bpV[pic] activation of the

HTLV-I LTR.

The data presented in this study argues that constitutive

expression of various cellular PTPs provide an important

mechanism to suppress HTLV-I transcription in infected

cells thereby contributing to their immune escape. It remains

possible that an increase in oxidative state might also be

induced through the peroxo group of the bpV[pic] com-

pound as described previously (Baier-Bitterlich et al., 1996;

Sasada et al., 2002), and could partially contribute to

activation of the HTLV-I LTR. However, our analysis of the

signaling pathway activated by bpV[pic] suggests that the

main mechanism behind the modulation of HTLV-I gene

expression by bpV[pic] involves the inhibitory potential of

PTP activity. PTP inhibition (possibly by SHP-1) provides a

possible mechanism for activation of latent HTLV-I proviral

DNA. In fact, it has been shown that HTLV-I-infected cells

are frequently negative for SHP-1 expression (Cheng et al.,

2004; Migone et al., 1998), although the absence of SHP-1

expression is also observed in other leukemic cell lines and

is thus not exclusive to HTLV-I-infected transformed cells.

Since it has been suggested that activation of HTLV-I gene

expression might be sudden and dependent on cell-to-cell

contact, PTP inhibition could potentially turn on a signaling

cascade, resulting in rapid turn on of viral gene expression,

viral particle production and transmission to uninfected

cells.

Our results indicate a strong transcriptional upregulation

of the HTLV-I LTR by the bpV[pic] PTP inhibitor in T cells.

M. Langlois et al. / Virology 329 (2004) 395–411 407

The implication of PTP inhibition in positive regulation of

the HTLV-I gene transcription needs to be further addressed

with respect to key events in HTLV-I replication such as

HTLV-I transmission to uninfected cells. We are presently

addressing these issues to better identify the transcription

factors involved in bpV-dependent modulation of HTLV-I

transcription.

Materials and methods

Cell lines and PBMCs

Jurkat (clone E6-1) (Weiss et al., 1984), and Jurkat-

derived J45.01 (Koretsky et al., 1991), J.RT3 (Morley et al.,

1988) and P116 (Williams et al., 1998) cell lines were

included in this study. J45.01 (generously provided by Dr.

Arthur Weiss, Howard Hughes Medical Center San Fran-

cisco, CA), J.RT3 and P116 (kindly given by Dr. Robert T.

Abraham, The Mayo Clinic Rochester, MN) are deficient in

CD45, TCR and ZAP70/Syk expression, respectively. P116

clone 39 was derived from P116 and has been recomple-

mented for ZAP70 expression. Jurkat derivatives deficient

in a mode of calcium entry dependent on extracellular

calcium known as capacitative calcium entry (CJ.1) and the

parental CJ clone (Fanger et al., 1995) were kindly provided

by Dr. Richard S. Lewis (Stanford University School of

Medicine, Stanford, CA). The CD45-negative K6D cell line

has been previously described and is a clone derived from

the JKF cell line stably expressing antisense CD45 mRNA

(McKenney et al., 1995). The HPB.ALL cell line (Koretsky

et al., 1990) is a TCR-positive T cell line with a more

immature phenotype than Jurkat cells and was generously

provided by Dr. Arthur Weiss. The SupT1 T-cell line (Smith

et al., 1984) is negative for the TCR and was obtained from

the NIH AIDS Repository Reagent Program (Rockville,

MD). The HTLV-I-infected MJ cell line (Popovic et al.,

1983) produces infectious particles and was purchased from

the American Type Culture Collection (Manassas, VA). Cell

lines were maintained in RPMI-1640 medium supplemented

with 10% FBS (Hyclone Laboratories, Logan, UT),

glutamine (2 mM), penicillin G (100 U/ml), and streptomy-

cin (100 Ag/ml). G418 (Invitrogen Life Technologies,

Burlington, Canada) and Hygromycin B (Calbiochem, San

Diego, CA) were added to the appropriate concentrations as

indicated. PBMCs from healthy donors were isolated by

Ficoll-Hypaque density gradient centrifugation as previ-

ously described (Fortin et al., 1999).

Plasmid constructs

The pHTLV-LUC vector (Geleziunas et al., 1998) was

kindly provided by Dr. Warner C. Greene (The J. Gladstone

Institutes, San Francisco, CA) and contains the HTLV-I LTR

cloned upstream of the luciferase reporter gene in the pGL2-

Basic vector (Promega, Madison, WI). The pHTLV-GFP

construct was generated by excising the luciferase gene/

poly(A) segment from pHTLV-LUC and inserting a green

fluorescent protein (GFP)/SV40 poly(A) fragment isolated

from the HindIII/BamHI-digested phGFP-S65T vector (BD

Biosciences Clontech, Palo Alto, CA). Deletion mutants of

pHTLV-LUC were generated by self-ligation after digestion

with SmaI (pHTLV-LUCDSmaI) or SmaI and NdeI

(pHTLV-LUCDNdeI/SmaI). The pGL2tata-LUC vector

was constructed by subcloning a synthetic double-stranded

oligonucleotide containing a TATA box and extremities

compatible with NheI/HindIII (produced from hybridized 5V-CTAGCGGGTATATAATGGATCCA-3V and 5V-AGCTTG-GATCCATTATATACCCG-3V oligonucleotides) into the

digested pGL2-Basic vector (Promega). The pGL2tata

HTLV(�325/�57) construct was then generated by cloning

the SmaI/NdeI blunted fragment from the HTLV-I LTR into

the SmaI site of the pGL2tata-LUC vector. A similar cloning

procedure was used to insert this HTLV-I LTR fragment into

the SmaI-digested pGL2-Promoter vector (Promega) gen-

erating pGL2-Promoter HTLV(�325/�57). Constructs were

also created with previously described HTLV-I LTRs

(Barnhart et al., 1997) being either wild type or mutated

for one, two, or all three CREB consensus sequences located

in the TRE1 regions. These LTRs were excised from their

vector by XhoI/HindIII and cloned into the pGL3-Basic

vector (Promega) using the same restriction site. These

vectors are termed pGL3-U3R, pGL3-21P/11T, pGL3-21M/

11T, pGL3-21D/11T, pGL3-21PM/11T, pGL3-21PD/11T,

pGL3-21MD/112T and pGL3-21PMD/11T, and are wild

type or mutated in the proximal (P), middle (M) and/or distal

(D) TRE1 sequences. Expression vectors for the wild type

Tax protein and the empty vector (pHhPr.1-neo) (Matsu-

moto et al., 1997) were kindly provided by Dr. Masataka

Nakamura (Tokyo Medical and Dental University, Tokyo,

Japan). The pNF-nB-LUC and pCRE-LUC vectors were

obtained from Stratagene (La Jolla, CA) and contain

multiple NF-nB- and CREB-binding sites, respectively,

positioned upstream of a minimal promoter and the

luciferase reporter gene. Vectors encoding wild-type SHP-

1 as well as the empty vector pSFFVneo (Plas et al., 1996)

were provided by Dr. Matthew L. Thomas (Washington

University School of Medicine, Saint Louis, MI). The

CREB dominant negative mutant KCREB and the control

empty vector (Walton et al., 1992) were provided by Dr.

R.H. Goodman (Vollum Institute for Advanced Biochemical

Research, Portland, OR). Vectors expressing wild type

CREB or the constitutively active CREB F/Y mutant (Du

et al., 2000) were obtained from Dr. Marc Montminy (The

Salk Institute for Biological Studies, La Jolla, CA). We

constructed the empty vector termed pcDNA.CF by self-

ligation after excision of the insert.

Generation of luciferase-containing HTLV-I proviral DNA

The 5VK30-Luc vector was derived from the pre-

viously described K30 plasmid harboring a full-length

M. Langlois et al. / Virology 329 (2004) 395–411408

and infectious HTLV-I proviral DNA clone (Zhao et al.,

1995) obtained from the NIH AIDS Repository Reagent

Program. A BamHI fragment was isolated from this

construct (position 5102 to 6107) and cloned into

pBlueScript SK (Stratagene). This fragment contained

the ATG initiation codon for the env, tax, and rex

genes. A two-step PCR mutagenesis protocol directed at

position 5550 to 5552 (48 nucleotides downstream of

the ATG codon) mutated the ATC sequence to GAG

thereby generating a SacI site. Briefly, the 5V-CAGTTCTGCCCCCTCGAGCTCGGTGATTACAGCCC-

CAG-3V/Reverse primer and 5V-CTGGGGCTGTAAT-

CACCGAGCTCGAGGGGGCAGAACTG-3V/Universal

primer combinations were used for amplification of 5V and 3Vends of the BamHI fragment. Both amplified products

were mixed and amplified as a complete fragment with

pBlueScript Reverse and Universal primers. The luciferase

gene was amplified from pGL3basic with oligonucleotides

(5V-TGGAGCTCGTTGGTAAAGCCACCATGGAA-

GACGCCAAAAAC-3V and 5V-ACGAGCTCATCAATG-TATCTTATCATGTCTGCTCGAAGC-3V) to generate SacI

sites at each end, and then cloned in frame with the HTLV-

I ATG codon into the SacI-generated site of the K30

BamHI fragment. Sequencing of each PCR-amplified

product was carried out to ascertain proper sequence.

The BamHI fragment was subsequently cloned into the

K30 vector, after BamHI digestion, to generate 5V K30-LUC. Since the 3V end of the K30 proviral DNA had been

deleted in this construct, complete full-length proviral

DNA was recreated by ligating this 5V K30 end (EcoRI/

SalI digestion) with the 3VK30 fragment (SalI/EcoRI). The

ligation mixture was then directly transfected into T cell

lines.

Antibodies and reagents

BpV[pic] was synthesized as previously described

(Posner et al., 1994) and supplied by Dr. R. Faure (CHUL

Research Center, Ste-Foy, Canada). Briefly, V2O5 was

dissolved in an aqueous KOH solution and then mixed

with 30% H2O2 and the picolinic acid ancillary ligand in

addition to ethanol for optimal precipitation. Character-

ization of the bpV molecule was carried out by infrared 1H

NMR and vanadium-51 (51V) NMR spectroscopy. Stock

solutions of bpV[pic] molecules (1 mM in phosphate-

buffered saline, pH 7.4) were kept at �20 8C until used.

Sodium orthovanadate (Sigma, St Louis. MO) was freshly

dissolved in 10 mM HEPES, pH 7.4. FK506 and cyclo-

sporin A (CsA) were purchased from Fujisawa (Osaka,

Japan) and Sigma, respectively. Antibodies from the anti-

CD3 OKT3 hybridoma were purified with mAbTrap protein

G affinity columns (Pharmacia LKB Biotechnology AB,

Uppsala, Sweden). Purified anti-CD28 antibodies (clone

9.3) were provided by Dr. Jeffrey A. Ledbetter (Bristol-

Myers Squibb Pharmaceutical Research Institute, Princeton,

NJ).

Transfections and reporter gene assays

Transient transfections of T-cell lines using the DEAE-

Dextran method were performed as previously described

(Barbeau et al., 1997). Electroporation of PBMCs was

conducted according to Hughes and Pober’s protocol

(Hughes and Pober, 1996). Transiently transfected cells

were seeded at a density of 105 cells/well in 96-well plates

and left unstimulated or treated for 8 h (unless specified)

with bpV[pic] (10 AM), PHA-P (3 Ag/ml), PMA (20 ng/ml)

(Sigma), ionomycin (1 AM) (Iono; Calbiochem), anti-CD3

antibody (clone OKT3) (3 Ag/ml), anti-CD28 antibody

(clone 9.3) (1 Ag/ml), forskolin (100 AM) (BioMol,

Plymouth Meeting, PA) and prostaglandin E2 (100 nM)

(PGE2; Sigma) in a final volume of 200 Al. For some

experiments, prior to activation, cells were treated with

FK506 (10 ng/ml) or CsA (100 ng/ml) for 15 min or with

MDL-12,330A (100–250 AM) (Calbiochem) for 1 h at 37

8C. Cells were then lysed in a 1� lysis buffer (25 mM Tris

phosphate, pH 7.8, 2 mM DTT, 1% Triton X-100, 10%

glycerol). Luciferase activity was determined as follows.

After a freeze/thaw cycle, 20 Al of cellular extract was

transferred to a 96-well luminometer plate, and luciferase

activity was quantitated on a Dynex MLX microplate

luminometer (MLX; Dynex Technologies, Chantilly, VA)

following the addition of 100 Al of luciferase buffer (20

mM tricine, 1.07 mM (MgCO3)4dMg(OH)2d 5 H2O, 2.67

mM MgSO4, 0.1 mM EDTA, 220 AM Coenzyme A, 4.7

AM d-Luciferin potassium salt, 530 AM ATP, 33.3 mM

DTT). Values from the luminometer are expressed as

Relative Light Units (RLU). Fold induction was calculated

as the ratio between the mean values F standard deviation

(SD) of the luciferase activity of treated over untreated

samples. Cells transfected with the pHTLV-GFP vector

were fixed in a solution of 1� phosphate-buffered saline

(PBS) containing methanol-free 1% paraformaldehyde and

subsequently analyzed by flow cytometry for GFP detection

(EPICS XL, Coulter Corp., Miami, FL).

Detection of supernatant HTLV-I p19 antigen

HTLV-I-producing MJ cells (1 � 106 cells/ml) were

treated with PMA (20 ng/ml)/ionomycin (1 AM), bpV[pic]

(10 AM) and bpV[pic] (10 AM)/forskolin (100 AM).

Supernatants were subsequently harvested at 24, 48, and

72 h post-activation. Quantification of HTLV-I p19 antigen

in the collected supernatant was performed using an ELISA

HTLV-I p19 antigen kit (Zeptometrix, Buffalo, NY) as

described by the manufacturer.

Acknowledgments

We thank Dr. Maurice Dufour for FACS analyses. This

work was supported by a grant from the Fonds de

Recherche en Sante du Quebec (FRSQ) (#003318). Dr.

M. Langlois et al. / Virology 329 (2004) 395–411 409

Michel J. Tremblay holds a Tier 1 Canada Research Chair in

Human Retrovirology. Dr. Benoit Barbeau is the recipient of

a Chercheur-Boursier Junior 2 award from the FRSQ.

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