In primary effusion lymphoma cells, MYB transcriptionalrepression is associated with v-FLIP expression during latentKSHV infection while both v-FLIP and v-GPCR become involvedduring the lytic cycle

Kaposi sarcoma (KS)-associated herpesvirus (KSHV) is a

c2-herpesvirus originally identified in KS lesions obtained

from human immunodeficiency virus (HIV)-infected individ-

uals (Chang et al, 1994). Infection by KSHV is associated with

the development of all clinico-epidemiological forms of KS

(Moore et al, 1996; Ensoli et al, 2001), but is also associated

with other conditions, such as primary effusion lymphoma

(PEL) (Mesri et al, 1996; Nador et al, 1996; Said et al, 1996a,b;

Gessain et al, 1997; ), some aggressive forms of multicentric

Castleman disease (MCD) (Soulier et al, 1995; Weiss et al,

1998; Schulz, 2001; Cesarman & Mesri, 2007) and with some

rare other solid lymphomas (Chadburn et al, 2004; Carbone

et al, 2005; Deloose et al, 2005). Although studies on individ-

ual KSHV genes have provided more insights about KSHV

pathogenesis [for a review see (Rezaee et al, 2006)], the

mechanism by which the virus causes cancer is not completely

understood [for a recent review see (Cesarman & Mesri,

2007)].

In PEL derived cell-lines, KSHV encodes genes that latently

express three proteins from a multicistronic mRNA: v-FLIP

(viral FLICE inhibitory protein, ORF 71), v-Cyclin (ORF 72)

and LANA (ORF 73) (Dittmer et al, 1998; Sarid et al, 1999;

Vincent Lacoste,1* Christophe Nicot,2

Antoine Gessain,1 Francoise Valensi,3

Jean Gabarre,4 Hittu Matta,5 Preet M.

Chaudhary5 and Renaud Mahieux1

1Unite d’Epidemiologie et Physiopathologie des

Virus Oncogenes, Institut Pasteur, Paris, France,2Department of Microbiology, Molecular genetics

and Immunology, University of Kansas Medical

Center, Kansas City, KS, USA, 3Laboratoire

d’Hematologie, Hopital Necker, 4Service

d’Hematologie Clinique, Hopital de la Pitie

Salpetriere, Paris, France, and 5Division of

Hematology-Oncology and the Hillman Cancer

Center, Department of Medicine, University of

Pittsburgh Medical Center, Pittsburgh, PA, USA

Received 23 March 2007; accepted for

publication 17 May 2007

Correspondence: Renaud Mahieux, Unite

d’Epidemiologie et Physiopathologie des Virus

Oncogenes, Institut Pasteur, 28 rue du Docteur

Roux, 75724 Paris Cedex 15, France. E-mail:

rmahieux@pasteur.fr

*Present address: V. Lacoste, Laboratoire des

Interactions Virus-Hotes, Institut Pasteur de la

Guyane, Cayenne, French Guiana.

Summary

Primary effusion lymphoma (PEL) is a rare, distinct subtype of non-Hodgkin

lymphoma, which is associated with Kaposi sarcoma-associated herpesvirus

(KSHV) infection. Although MYB levels are high in most neoplastic B cells,

we found that, unexpectedly, both PEL cells and uncultured PEL patients’

samples contained very low levels of MYB mRNA when compared to B-cell

leukaemia samples obtained from KSHV) patients. These results were further

confirmed at the protein level. Both latent viral FLICE inhibitory protein

(v-FLIP) and early lytic viral G protein coupled receptor (v-GPCR) KSHV

proteins were found to activate nuclear factor (NF)-jB and transrepress

a MYB promoter reporter construct. In contrast, a dominant negative

inhibitor of NF-jB (IjB-a) mutant prevented v-FLIP and v-GPCR from

inhibiting MYB functions while a v-GPCR mutant that was impaired for NF-

jB activation could not repress the MYB construct. Transduction of a v-FLIP

expressing vector or stable transfection of v-GPCR both resulted in a marked

downregulation of the endogenous MYB protein expression. However, MYB

expression transactivated the lytic switch Replication and Transcription

Activator (RTA) promoter in transient transfection assays. Taken together,

our results demonstrate that, contrary to a number of other haematological

malignancies, MYB expression is not required for PEL cell proliferation.

Repressing MYB expression also helps in maintaining the virus in latency.

Keywords: Kaposi sarcoma, nuclear factor-jB, herpes virus, non-Hodgkin

lymphoma, B cells, molecular studies.

research paper

ª 2007 The AuthorsJournal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 487–501 doi:10.1111/j.1365-2141.2007.06697.x

Talbot et al, 1999). Their expression is only very slightly

enhanced following sodium butyrate or phorbol ester tetra-

decanoyl phorbol acetate (TPA) exposure (Jeong et al, 2001;

Paulose-Murphy et al, 2001; Fakhari & Dittmer, 2002). ORF 71

(v-FLIP) transcription is complex. Mono- and bi-cistronic

v-FLIP mRNAs can be detected by reverse transcription

polymerase chain reaction (RT-PCR) in growing cells

(Grundhoff & Ganem, 2001). The function of the low abundant

monocistronic v-FLIP form, which is TPA-inducible, is unclear

(Grundhoff & Ganem, 2001). v-FLIP proteins have also been

characterised in other c2-herpesviruses (Bertin et al, 1997;

Wang et al, 1997). These proteins contain two death effector

domains (Thome et al, 1997) and were originally believed to

protect cells against death receptor-induced apoptosis (Djerbi

et al, 1999; Sturzl et al, 1999; Belanger et al, 2001). However,

recent studies suggest that KSHV v-FLIP does not act as an

inhibitor of caspase 8 and is primarily involved in nuclear factor

(NF)-jB activation (Chugh et al, 2005). As v-FLIP is expressed

in KSHV-latently infected KS spindle cells, it might therefore

favour tumour formation (Sturzl et al, 1999).

The early lytic KSHV viral G protein coupled receptor

(v-GPCR) protein (Nador et al, 2001) is transcribed and

translated from ORF 74 and shares high homology with

human chemokine receptor CXCR2 (Cesarman et al, 1996).

v-GPCR mRNA was detected primarily during lytic replication,

either by northern-blot or by RT-PCR, in a small fraction of

KS lesions and also in the PEL cell lines that have been tested

(Cesarman et al, 1996; Kirshner et al, 1999; Chiou et al, 2002).

v-GPCR stimulates signalling pathways linked to cell prolifer-

ation in a constitutive (agonist-independent) way (Bais et al,

1998). v-GPCR can also directly transform rodent cells in vitro

and induces KS-like lesions in transgenic animals (Arvanitakis

et al, 1997; Bais et al, 1998; Yang et al, 2000).

Several reports have demonstrated that NF-jB factors are

constitutively present in the nuclei of both KSHV latently-

infected PEL cell-lines and in uncultured PEL samples, when

compared to other non-KSHV B-cell lines (Keller et al, 2000;

Liu et al, 2002). KSHV v-FLIP has been shown to play

a significant role in this process (Cesarman & Mesri, 2007).

v-FLIP activates NF-jB by recruitment to a high molecular

weight inhibitor jB kinase (IKK) complex, consisting of IKK-

a, IKK-b and IKK-c/NEMO (Liu et al, 2002; Field et al, 2003)

and then interacting with tumour necrosis factor receptor

associated factors (TRAFs) (Guasparri et al, 2006). Interest-

ingly, this feature is not shared by other v-FLIP proteins, such

as E8 and MC159L (Chaudhary et al, 1999). Consistent with

these observations, inhibiting v-FLIP expression with specific

small interfering (si)RNA results in a significant inhibition of

NF-jB activity in PEL cells (Guasparri et al, 2004). It was also

shown that ectopic expression of v-FLIP in human umbilical

vein endothelial cells induced NF-jB activation and is

associated with the induction of spindle cell morphology

(Matta et al, 2007). At the early lytic stage, v-GPCR can also

activate NF-jB (Couty et al, 2001; Montaner et al, 2001;

Schwarz & Murphy, 2001; Cannon et al, 2003).

MYB was identified more than 20 years ago as the cellular

homolog of the transforming gene of two avian leukaemia

viruses (AMV and E26) (Baluda & Reddy, 1994). The MYB gene

family consists of three members: MYBL1, MYBL2 and MYB

(Klempnauer & Bishop, 1984; Oh & Reddy, 1999). The MYB

transcript encodes a 75 kDa protein, MYB, that functions both

as a transcription factor and as a repressor (Introna & Golay,

1999; Ness, 1999; Bender et al, 2004). Expression of MYB is

mainly seen in haematopoietic tissue (Oh & Reddy, 1999). It is

generally considered that the expression of MYB is high in

immature thymocyte progenitor cells and decreases during

terminal differentiation to mature blood cells (Gonda & Metcalf,

1984). However, this paradigm is not always true, since

B-chronic lymphocytic leukaemia (B-CLL) cells do not express

MYB, whilst immunoblastic and more mature cells, such as

plasmablastic B cells, do express MYB (Bading et al, 1988).

A number of neoplastic B-cells, i.e. pre-B acute lymphocytic

leukaemia (ALL), Epstein–Barr virus lymphoblastoid B cells

(EBV-LCL), non-Hodgkin lymphomas, Burkitt lymphoma cell

lines, null and common ALL, express high levels of MYB

mRNA (Golay et al, 1996). MYB is critical for proliferation of

these leukaemic cells, since preventing MYB expression results

in cell death (Weston, 1999). Several reports have also shown

that MYB is overexpressed in diverse types of cancers, such as

neuroblastomas, colon carcinomas, breast carcinomas and

certain types of leukaemia (Alitalo et al, 1984; Griffin & Baylin,

1985; Guerin et al, 1990; Davies et al, 1999; Lipsick & Wang,

1999; Weston, 1999). The expression levels of MYB in KSHV-

infected PEL cells are unknown.

We have previously shown that human T-cell leukaemia

virus type 1 (HTLV-1) infected cell lines express very low levels

of MYB (Nicot et al, 2000a). These mature, activated T cells

(Hermine et al, 1998) display a constitutive NF-jB activity

that is due to the expression of the viral transactivator protein

Tax (Sun & Yamaoka, 2005). We have also established that

MYB functions are repressed by NF-jB factors in Tax-

expressing cells (Nicot et al, 2001).

The present study found that, contrary to most other

B-lymphomas tested to date (Golay et al, 1996), both MYB

transcript and MYB protein levels were low in latently-infected

PEL cell lines and in uncultured PEL samples. We then

determined which of the latent KSHV proteins was responsible

for this effect. Susequent to this, having observed that MYB

mRNA was further downregulated when PEL cells were treated

with TPA and having considered that v-GPCR could also

activate NF-jB, we then assessed whether v-GPCR expression

was sufficient to repress MYB function. We confirmed that

both v-FLIP and v-GPCR induced NF-jB and showed that the

ectopic expression of v-FLIP or of v-GPCR was sufficient for

repressing endogenous MYB in transfection or transduction

assays. While it has been shown that Rta alone is sufficient to

disrupt latency and to activate the expression of lytic genes

(Deng et al, 2000), our results demonstrate that MYB also

activates the Rta promoter. These data suggest that v-FLIP and

v-GPCR play a dual role in disturbing the cell cycle by

V. Lacoste et al

ª 2007 The Authors488 Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 487–501

repressing c-Myb functions and also in preventing the switch

to the lytic phase of viral replication.

Materials and methods

Cell lines and patient specimens

BCBL1 and BC3 cells are KSHV+/EBV) while BBG1, BC1 and

BC2 cells are KSHV+/EBV+ (Cesarman et al, 1995; Arvanitakis

et al, 1996; Renne et al, 1996; Morand et al, 1999). Nalm-6,

697, DHL-9 and DHL-4 B-cell lines are not infected with

KSHV (Golay et al, 1996; Heckman et al, 2000). Jurkat

(HTLV-1)) and C8166 (HTLV+) are T cells. Cells were grown

in RPMI 1640 medium (Life Technologies, Cergy Pontoise,

France) supplemented with 10% heat-inactivated fetal bovine

serum (FBS) (HyClone� Europe, Erembodegem, Belgium),

1% l-glutamine (Life Technologies) and 1% penicillin/strep-

tomycin (Life Technologies) at 37�C in 5% CO2. When

needed, cells were split at a concentration of 250 · 103 cells/ml

24 h before TPA treatment. TPA (Sigma, St Quentin Fallavier,

France) was used at 20 ng/ml for 48 h.

Tumoral lymphoid cells were obtained from the pleural or

ascitic liquids of five PEL patients (four KSHV+/HIV+ and one

KSHV+/HIV)) by centrifugation and were frozen in liquid

nitrogen. These patients have been described elsewhere (Boul-

anger et al, 2005). Six B-leukaemia (KSHV)) samples were also

used as controls. Among them, three were classified as BI null

(patients B, Z, N), two as BII common (patients K and D) and

one as BIII pre-B (patient A) according to the European Group

for the Immunological Characterization of Acute Leukaemias

(EGIL) classification.

Immunoblot analyses

Cells were washed in phosphate-buffered saline and lysed

(Tris–HCl 50 mmol/l pH 7Æ4, 120 mmol/l NaCl, 5 mmol/l

EDTA, 0Æ5% NP-40, 50 mmol/l NaF, 0Æ2 mmol/l Na3VO4,

1 mmol/l DTT) for 20 min on ice in the presence of protease

inhibitors (Complete, Boehringer, Mannheim, Germany).

The lysate was then centrifuged for 20 min at 4�C and the

supernatant frozen at )80�C. Protein concentration was

determined by Bradford assays (Biorad laboratories, Hercu-

les, CA, USA). 10%, 12% and 16% Tris-glycine-gels (Invi-

trogen, Groningen, the Netherlands) were used. Each sample

(except in the case of v-GPCR containing extracts) was

heated at 95�C for 5 min in Laemli buffer and run. After

transfer to an Immobilon polyvinylidene difluoride (PVDF)

membrane (Biorad Laboratories), Western-blots were per-

formed as previously described (Mahieux et al, 2000). Signals

were detected either with the Supersignal� West Pico

chemiluminescent substrate kit or with the Supersignal�West Dura Extended duration substrate kit (Pierce, Perbio

Sciences, Brebieres, France). When necessary, the membranes

were stripped using the Re-Western-blot recycling kit

(Chemicon, Temecula, CA, USA) and re-probed.

Antibodies

b-Tubulin (sc-9104), actin (sc-1616) and MYB (sc-517) antibod-

ies were purchased from Santa Cruz (Santa Cruz biotechnology,

Santa Cruz, CA, USA). Polyclonal anti-GFP rabbit antiserum was

purchased from Invitrogen (R-970-01). Anti v-GPCR antibody

(v-GPCR-N) was obtained from Dr G Hayward (Chiou et al,

2002) and used at 1:2700 dilution. Anti-rabbit or anti-mouse

horseradish peroxidase-conjugated secondary antibodies

(Amersham Biosciences, GE Healthcare, Saclay, France) were

used at 1:45 000 and 1:30 000 dilutions respectively.

Northern-blot

Total RNA was extracted using the RNeasy Mini kit (QIAGEN,

Dusseldorf, Germany), precipitated overnight at )20�C in

isopropanol containing 10% diethylpyrocarbonate-treated

3 mol/l sodium acetate, washed in ethanol 70% and resuspended

in formazol (Euromedex, Souffelweyersheim, France). RNAs

(8 lg) were size-fractionated by 2Æ2 mol/l formaldehyde-agarose

gel electrophoresis, transferred to MagnaGraph nylon mem-

branes (MSI, Westboro, MA, USA), and ultraviolet cross-linked

as previously described (Hans et al, 2001). The blots were

hybridised overnight at 42�C in ULTRAhybTM buffer (Ambion,

Inc., Austin, TX, USA) containing 25 ng of a a32P dCTP DNA

probe (Prime-it RmT; Stratagene, La Jolla, CA, USA). After high

stringency washes [0Æ2 · standard saline phosphate/EDTA

(SSPE), 0Æ2% sodium dodecyl sulphate (SDS) at 42�C, twice],

the blots were exposed to a PhosphorImager screen. The blots

were stripped (2 mmol/l EDTA, 5 mmol/l Tris, pH 7Æ5, 0Æ1%

SDS) before being re-hybridised. The MYB and GAPDH probes

were generated from RT-PCR products (Table I). The v-GPCR

probe (1241 bp) was amplified by PCR using the following

primers: ORF74F2 5¢-AGC ACT AGG TTA GGT TGA AAG

CCG-3¢, ORF74B5 5¢-GGG ATC AGA CCC CTC ATT TAA TCG-

3¢. Actin probe was a generous gift from Dr J Pidoux, Pasteur

Institute. The RT-PCR and PCR products were cloned by TA

cloning (Invitrogen-Novex) in the pCR2.1 vector. Signals were

quantified using the ImageQuant program (Molecular Dynamics,

GE Healthcare, Saclay, France) and corrected for GAPDH.

Reverse-transcription PCR

Reverse transcription polymerase chain reaction was carried

out using the QIAGEN OneStep RT-PCR kit (QIAGEN) as

recommended by the manufacturer. The experimental PCR

conditions as well as the c-myb (Walker et al, 1998), GAPDH

and v-FLIP (Grundhoff & Ganem, 2001) primers and probes

sequences are described in Table I. PCR cycling conditions

were designed to avoid the plateau stage.

Transient transfection and luciferase assays

Jurkat cells were transfected using either the Superfect

procedure (QIAGEN) as previously described (Mahieux et al,

Inhibition of MYB functions by KSHV v-FLIP and v-GPCR

ª 2007 The AuthorsJournal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 487–501 489

2000) or Lipofectamine 2000 (Invitrogen) using the manufac-

turer’s protocol. 293T cells were transfected with the Polyfect

reagent (QIAGEN). The amount of DNA transfected was

equalised by addition of a pCDNA3.1 control empty vector. All

the transfections were carried out in the presence of a

pRSV-RL vector (Promega France, Charbonnieres, France) to

normalise the results for the transfection efficiencies. Reporter

activities were assayed 24 h post-transfection using the Dual–

Luciferase reporter assay system (Promega France). Luciferase

assays were performed with a Berthold LB9500C luminometer

as described elsewhere (Mahieux et al, 2000). The MYB, ORF-

K13 (v-FLIP), pcKSHV-GPCRwt, pKSHV-GPCRwt-EGFP,

pKSHV-GPCRD5-EGFP, IjB-aS32/36A, pRP-luc, MRE-luc and

NF-jB-luc constructs have been described elsewhere (Sun

et al, 1993; Guerra et al, 1995; McCann et al, 1995; Chaudhary

et al, 1999; Deng et al, 2000; Nicot et al, 2000b, 2001; Schwarz

& Murphy, 2001).

v-FLIP-recombinant lentiviral vector

A three plasmid expression system derived from HR’CMVLacZ

(Naldini et al, 1996) was used to generate vector particles by

transient transfection of 293T cells using the calcium phosphate

co-precipitation technique as described (Zennou et al, 2000).

The pTRIP v-FLIP plasmid was constructed by replacing the

BamHI/XhoI fragment of pTRIP GFP by a v-FLIP cDNA

fragment amplified by PCR adding BamHI and XhoI restriction

sites in 5¢ and 3¢ respectively. PCR primers were as follows:

v-FLIPBams 5¢-CGGGATCCGCCACCATGGCCACTTAC-

GAGGTTCTCTG-3¢ and v-FLIPXhoas 5¢-GTCCGCTCGAGC-

GGCTATGGTGTATGGCGATAGTG-3¢. The encapsidation

plasmid (p8.2) provides vector structural, enzymatic and

accessory HIV proteins (Zufferey et al, 1997) and the VSV-G

envelope expression plasmid (pHCMV-G) encodes envelope for

viral particle production. The vector stocks were treated with

DNaseI prior to ultracentrifugation. Quantification of vector

stocks was realised by estimation of the amount of p24 capsid

protein by enzyme-linked immunosorbent assay (NEN kit) as

described by the manufacturer. Vector stocks contained 70 ng

p24/ll, verified by triplicate estimation. Jurkat cells (250 · 103)

were transduced with 1Æ5 lg of pTRIP v-FLIP vector and the

cells were collected and proteins extracted after 24 h.

v-GPCRwt and GPCRD5 stable cell lines

pKSHV-GPCRwt-EGFP, pKSHV-GPCRD5-EGFP plasmids

(Schwarz & Murphy, 2001) were transfected in Jurkat cells

(Qiagen, Superfect). After 48 h, the cell culture medium was

replaced and the transfected cells were grown in the presence of

Geneticin (400 lg/ml). The level of MYB and of GFP protein

expression was evaluated by Western-blot after 3 weeks of

culture in the presence of the selective agent.

Results

Transcriptional repression of the endogenous MYBpromoter in PEL cell lines and in ex vivo PEL patientssamples

MYB is overexpressed in a number of B and T lymphomas

(Golay et al, 1996; Poenitz et al, 2005). Indeed, ablation of

MYB has even been tested as a potential therapy for human

Table I. Reverse transcription polymerase chain reaction primers, probes and amplification conditions.

Oligonucleotide name 5¢ fi 3¢ sequence Orientation Product size No. cycles

GAPDH

GAPDHs AAG GTG AAG GTC GGA GTC AAC GG +

GAPHas CAT GAG TCC TTC CAC GAT ACC AA ) 518 bp 23

GAPDHpr TCC TGC ACC ACC AAC TGC TTA GCA C + (60�C)*

MYB�c-Mybs AAA AGC CAG CCA GCC AGC AG +

c-Mybas CTG TGC CAC CCG GGG TAG CT ) 377 bp 23

c-Mybpr CAT TTG ATG GGT TTT GCT CAG GCT + (60�C)*

v-FLIP�v-FLIPs CGC TAA CAG GGG AAA CGT TAA CCT GC + 1200 bp ) TPA (bicistronic) 30

v-FLIPas GCG CCA CGA AGC AGT CAC GTC CC ) 470 bp + TPA (monocistronic) (61�C)*

v-FLIPpr GTT TCC GCG CCA CCT CAC AGA GAA CCT +

Trip-v-FLIP–

Trip-v-FLIPs GTT AGC GGA ATG TCT GTT TCG TG +

Trip-v-FLIPas CGA TAG TGT TGG GAG TGT GAT GG ) 390 bp 30

Trip-v-FLIPpr AGC GGG CAC AAT GAG TTA TTT CAG C +

+, sense; ), antisense.

*Annealing temperature.

�As described in Walker et al, 1998.

�As described in Grundhoff and Ganem (2001).

–Used in Fig 6.

V. Lacoste et al

ª 2007 The Authors490 Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 487–501

leukaemia (Ratajczak et al, 1992a,b; Gewirtz, 1999; Luger et al,

2002). To determine whether PEL cells also express high levels

of MYB, RNA was extracted from PEL cell lines as well as from

uncultured PEL patient samples and MYB mRNA levels were

quantified by Northern-blot (Fig 1A–C). Jurkat cells that

express MYB were also used as positive control. In all KSHV

cell lines tested, MYB levels were lower than in Jurkat cells

(Fig 1A and C, left panel).

In HTLV-1 infected cells, MYB downregulation is due to

persistent NF-jB activity (Nicot et al, 2001). KSHV encodes at

least two proteins that have the ability of activating the NF-jB

pathway: v-FLIP, which is expressed both at the latent and the

lytic KSHV stages (Liu et al, 2002), and v-GPCR (Schwarz &

Murphy, 2001), which is only expressed at the early lytic stage

(Nador et al, 1996; Paulose-Murphy et al, 2001). To determine

the correlation between v-FLIP or v-GPCR expression and MYB

levels, PEL cells were treated with TPA, an inducer of the lytic

cycle (Miller et al, 1997; Yu et al, 1999; Paulose-Murphy et al,

2001). Following TPA treatment, MYB levels significantly

decreased in all PEL cell lines tested (Fig 1A, see lanes 3, 5, 7,

9, 11 versus 2, 4, 6, 8, 10 and Fig 1C for quantification). Such

a decrease was not observed in DHL9 B-cells, which are not

infected with KSHV (data not shown). This suggested that the

reduction of MYB mRNA was most probably the result of

a change in the KSHV gene expression. As NF-jB is also

activated in uncultured PEL samples (Keller et al, 2000), we

performed the same experiments with uncultured PEL patients

cells (Fig 1A, right panel). MYB levels were variable but always

lower in the PEL samples than in Jurkat cells (Fig 1A right panel

see lanes 13–17 versus 12 and Fig 1C for quantification).

Moreover, the magnitude of MYB downregulation in the

uncultured PEL samples versus Jurkat cells was comparable to

that observed in the PEL cell lines before TPA treatment, i.e.

during the latent stage. As a control, GAPDH levels were assessed

(Fig 1B). Altogether, these results suggest that MYB downreg-

ulation in the uncultured PEL patient cells is probably linked to

the ability of the v-FLIP protein to induce the NF-jB pathway.

As controls, RNA was extracted from KSHV) B-cell lines

(n ¼ 4), as well as from KSHV) B-leukaemia patient samples

(n ¼ 5) (Fig 1D–F). The MYB level in these samples was then

compared to that of KSHV+ BC2 cells. All but one of the

KSHV-uninfected cell lines tested were found to contain

higher levels of MYB than BC2 cells (Nalm-6: 2Æ6-fold; 697:

5Æ2-fold; DHL-9: 1Æ5-fold) (Fig 1D–F). Similarly, five out of

five non-KSHV B-leukaemia patient samples contained higher

levels of MYB mRNA than in BC2 cells (patient B: twofold;

patient Z: 2Æ3-fold; patient K: 7Æ5-fold; patient D: 1Æ7-fold and

patient A: sevenfold) (Fig 1D–F). It is worth noting that

among the PEL cell-lines used in this work, BC2 displayed 1Æ5-

to 10-fold higher MYB mRNA levels than the other KSHV PEL

cell lines (see Fig 1C). This enabled us to estimate that BC1,

BC3, BBG1 and BCBL1 PEL cells contained 2Æ25- to 52-fold

less MYB mRNA than the KSHV) B-cell controls and 2Æ55- to

70-fold less MYB mRNA than the KSHV) B-leukaemia

controls. DHL-4 cells, which are not infected with KSHV,

were also used as controls because they are known not to

contain detectable MYB mRNA (Heckman et al, 2000). As

control, GAPDH mRNA levels were assessed (Fig 1E).

As previously published, nuclear protein extracts obtained

from four different PEL cell lines used in this study confirmed

the presence of activated NF-jB complexes (data not shown).

As v-FLIP is the latent protein that is responsible for such

activation, we wanted to determine whether MYB mRNA

downregulation was correlated with v-FLIP expression in cells

that express latent KSHV. To this end, we first measured

v-FLIP expression both in latently infected cells but also after

TPA treatment. KSHV-infected cells can employ two different

strategies for v-FLIP expression. The use of the internal

ribosome entry site within the bicistronic ORF 72/71 mRNA

represents the dominant strategy for v-FLIP expression in

latency. As the bicistronic ORF 72/71 RNA is at least 100- to

500-fold more abundant than the monocistronic ORF 71 RNA,

translation of the latter RNA is predicted to make only a small

contribution to the intracellular pool of v-FLIP in latency

(Grundhoff & Ganem, 2001). We first performed Northern-

blot analysis for v-FLIP using either untreated or TPA-treated

BC3 cells (data not shown). As previously reported (Talbot

et al, 1999), the v-FLIP containing 5Æ32 and 1Æ7 kb messages

were only marginally affected by TPA, while the appearance of

the v-FLIP monocistronic transcript could not be detected

(data not shown).

Reverse transcription polymerase chain reaction analysis was

therefore performed as described (Grundhoff & Ganem, 2001),

with a limited number of cycles to avoid the plateau stage of

the PCR (Fig 2A). In three out of four cases, v-FLIP

monocistronic transcript (470 bp) expression was enhanced

following TPA treatment (Fig 2A see BCBL1, BC1 and BC2).

The monocistronic v-FLIP transcript could also be detected in

one uncultured PEL patient samples studied (Fig 1A right

panel, PH551). As a control, GAPDH levels were assessed

(Fig 1B). These results demonstrated that the quasi-undetect-

able induction of v-FLIP expression after TPA treatment could

not account by itself for the inhibition of MYB mRNA

transcription following chemical exposure. We thus performed

a v-GPCR Northern-blot analysis on the same RNA extracts to

test the v-GPCR induction after TPA treatment. As expected,

v-GPCR mRNA was detected only in TPA-treated cells. The

level of v-GPCR induction was variable between the cell lines

(Fig 2C), with BC2 being the most inducible cell line tested.

To date, studies on v-GPCR expression have been per-

formed on PEL cell lines (Cesarman et al, 1996; Kirshner et al,

1999; Chiou et al, 2002), and only once used an uncultured

PEL sample, for which v-GPCR expression was detected in

2–5% of the cells (Chiou et al, 2002). The lack of detection of

v-GPCR expression by Northern blotting in our uncultured

PEL samples (Fig 2C, right panel) is therefore consistent with

the probable limited number of cells expressing v-GPCR.

Altogether, these results suggest that in uncultured patients

PEL cells, MYB downregulation is due to v-FLIP expression,

while in PEL cell-lines, both v-FLIP and v-GPCR play a role.

Inhibition of MYB functions by KSHV v-FLIP and v-GPCR

ª 2007 The AuthorsJournal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 487–501 491

(A)

(B)

(C)

(D)

(E)

(F)

MYB mRNA

MYB mRNA

TPA Jurk

at

Jurk

atPH

809

PH 5

51PH

895

PH 7

18PH

820

– + – + – + – + – +

PEL cell lines

BC1 BC2 BC3 BBG1 BCBL1

PEL Patients

MYB mRNA siginal quantification

MYB mRNA siginal quantification

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

750

500

250

% o

ver

BC

2 si

gnal

afte

r G

AP

DH

nor

mal

izat

ion

% o

ver

Jurk

at s

igna

laf

ter

GA

PD

H n

orm

aliz

atio

n

01 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10GAPDH mRNA

GAPDH mRNA

B cells

0

50

100

0

50

100

0

50

100

KSHV–

DHL-4

patie

nt D

patie

nt A

patie

nt K

patie

nt Z

patie

nt B

DHL-9

BC2

697

Nalm

-6

KSHV–KSHV+

Fig 1. Northern Blot analysis of MYB expression in KSHV+ PEL cell lines before and after lytic cycle induction, KSHV+ PEL patient samples as well as

in KSHV) B-cell lines and KSHV) B-leukaemia patients. RNA was run, transferred to a nylon membrane and hybridised either with a MYB (A, D) or

a GAPDH probe (B, E). (A–C): lane 1: Jurkat; lanes 2–11: BC1, BC2, BC3, BBG1, BCBL1 without (lanes 2, 4, 6, 8, 10) or with (lanes 3, 5, 7, 9, 11) TPA

treatment (20 ng/ml). Lane 12: Jurkat, lanes 13–17: PEL patients PH809, PH551, PH895, PH718, PH820. (D–F): lane 1: DHL-4; lane 2: Nalm-6; lane

3: 697; lane 4: BC2; lane 5: DHL-9; lane 6: Patient B; lane 7: Patient Z; lane 8: Patient K; lane 9: Patient D; Lane 10: Patient A. (C) and (F):

quantification of the MYB signal over those of Jurkat (C) or BC2 (F). MYB RNA quantifications were corrected for the GAPDH signal. (A, B): Lanes

1–9 and 12–17 were run on the same gel.

V. Lacoste et al

ª 2007 The Authors492 Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 487–501

MYB protein level is low in PEL cell lines and inuncultured PEL samples

As mRNA levels might not always be reflected at the protein

levels, we also performed a series of MYB Western-blot

analyses with protein extracts from the different PEL cell-lines

that were treated, or not, with TPA, as well as with PEL patient

samples (Fig 3A and B). As a control, we also treated KSHV)

DHL-9 B cells and did not notice any significant change in

MYB expression (Fig 3A lane 1 versus 2), confirming that TPA

treatment had no effect by itself on the MYB protein level.

When compared to Jurkat cells, and in common with the

Northern-blot and RT-PCR results, very low levels of MYB

were detected in the PEL cell lines, with the exception of BC2

(Fig 3A). In all cell lines, MYB protein level became undetect-

able after TPA treatment (Fig 3A). MYB protein level was also

very low in the uncultured PEL patients’ samples when

compared to Jurkat cells (Fig 3B). As a control, MYB

expression was determined in KSHV-uninfected B-cell lines

as well as in non-KSHV B-leukaemia fresh samples (Fig 3C).

MYB protein level was always perfectly correlated with MYB

mRNA level (Fig 3C). Interestingly, all PEL cell-lines tested

expressed lower amounts of MYB than uninfected B-cell lines

or B-leukaemia samples, confirming the role of KSHV in the

downregulation of MYB expression in PEL cells.

Both latent v-FLIP and early lytic v-GPCR proteins caninhibit MYB transactivation through their NF-jBinducing activity

Given the fact that MYB levels are inversely correlated to

v-FLIP expression in latently-infected PEL cells and to both

v-FLIP and v-GPCR in TPA-treated PEL cells, we next wanted

to determine whether ectopic expression of v-FLIP and/or

v-GPCR was sufficient for repressing MYB transcriptional

activity in human lymphoid cells through NF-jB activation. As

previously described (Chaudhary et al, 1999; Schwarz &

Murphy, 2001; Liu et al, 2002; Matta et al, 2003), transient

transfection of a v-FLIP Flag-epitope tagged expression vector

in Jurkat cells first confirmed the ability of this ectopically

expressed protein to activate a NF-jB reporter construct

(mean 17Æ5-fold; Fig 4A). As a control, expression of the

phosphorylation-resistant mutant of IjBa (IjBaS32/36A) pre-

vented NF-jB activation (Fig 4A). v-FLIP expression was

confirmed by Western-blot (Fig 4B). We then assessed the

effect of v-FLIP expression on MYB activity using a Myb

responsive element (MRE) luciferase reporter construct

(Fig 4C) (Nicot et al, 2000a). Jurkat cells express high levels

of MYB protein and, therefore, do not require the addition of

ectopically expressed MYB. As expected, the repression of MYB

activity was only observed in the presence of v-FLIP (Fig 4C).

(A)

(B)

(C)

PEL cell lines

1 2 3 4 5 6 7 8 9 10 11 12 13

PH 8

09PH

551

PH 8

95PH

718

PH 8

20

PH 809

Jurka

t

Jurk

at

PH 551

PH 895

PH 718

PH 820

14 15

1 2

BCBL1 BC1 BC2 BC3

3 4 5 6 7 8 9 10 11 12 13 14 15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

PEL patients

TPA+–+–+–+–

TPA+–+–+–+–

+–RT

470 bp(monocistronic)

1200 bp(bicistronic)

BCBL1Jurk

at

BC2 BC1 BC3

V-FLIP mRNA

GAPDH mRNA

518 bp

v-GPCR mRNA

Fig 2. Expression of v-FLIP and v-GPCR in PEL cell lines before and after lytic cycle induction and in PEL patient samples. RT-PCR analysis of (A)

v-FLIP and (B) GAPDH RNA in PEL cell lines and in PEL patient samples. Lanes 1, 2: Jurkat; lanes 3–10: BCBL1, BC2, BC1, BC3 without (lanes 3, 5,

7, 9) or with (lanes 4, 6, 8, 10) TPA treatment. Lanes 11–15: PH809, PH551, PH895, PH718, PH820. Lane 1 no reverse transcriptase added. (C)

Northern-blot analysis of v-GPCR RNA in PEL cell lines and in PEL patient samples. lane 1: Jurkat; lanes 2–9: BCBL1, BC1, BC2, BC3, without (lanes

2, 4, 6, 8) or with (lanes 3, 5, 7, 9) TPA treatment (20 ng/ml); lane 10: Jurkat; lanes 11–15: PH809, PH551, PH895, PH718, PH820. Lanes 10–15 were

run on the same gel.

Inhibition of MYB functions by KSHV v-FLIP and v-GPCR

ª 2007 The AuthorsJournal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 487–501 493

The impact of v-FLIP on the MRE construct was also assessed

in the presence of the IjBaS32/36A dominant negative mutant.

In that case, v-FLIP could no longer repress MYB transcrip-

tional activity, demonstrating that the observed effect was not

simply due to the presence of the viral protein but rather to its

NF-jB inducing activity (Fig 4C). These results confirm that

v-FLIP-mediated NF-jB activation is involved in the repres-

sion of transcription activity of MYB both in latently KSHV

infected PEL cell lines and in PEL patient samples.

Transient transfection of a v-GPCR expression vector in

Jurkat cells also leads to the activation of the NF-jB reporter

construct (Fig 5A). As a control, the expression of v-GPCR was

confirmed by Western-blot (Fig 5B). Similar to v-FLIP, v-GPCR

expression repressed MYB functions (Fig 5C) and v-GPCR

effects were prevented by the IjBaS32/36A mutant (Fig 5C).

v-FLIP and v-GPCR repress the endogenous MYBpromoter

To validate the effects of v-FLIP on the endogenous MYB

promoter, Jurkat cells were transduced with a lentiviral vector

expressing v-FLIP. Consistent with the above results, the MYB

protein levels strongly decreased in Jurkat cells that had been

transduced with the v-FLIP retroviral vector (Fig 6A). These

results indicate that v-FLIP expression is sufficient for trans-

repressing the human MYB promoter in lymphoid cells. As

a control, expression of the v-FLIP mRNA was demonstrated

by RT-PCR in the v-FLIP transduced cells (Fig 6B).

We also evaluated the levels of MYB protein expression in

Jurkat cells that were stably transfected with pKSHV-GPCRwt-

EGFP or pKSHV-GPCRD5-EGFP expressing vectors (Fig 7).

Jurkat cells transfected with the GPCRD5 vector, which cannot

activate the NF-jB pathway (Schwarz & Murphy, 2001),

expressed the MYB protein, whereas MYB was not detected in

the cells that have been transfected with the GPCRwt plasmid

(Fig 7A). A GFP control Western-blot demonstrated the

presence of the GFP-v-GPCR proteins in both cell lines (Fig 7B).

MYB expression induces ORF 50 transactivation

Treatment of KSHV infected cells with chemicals such as TPA

induces the lytic replication (Renne et al, 1996; Sarid et al,

(A)

(B)

(C)

1

β-tubulin

MYB

β-tubulin

MYB

β-tubulin

MYB

TPA – + – + – + – + – + –BCBL1

PEL cell lines

DHL-9 Jurka

tC8166

BC1 BC3 BBG1 BC2+

PEL patients

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 2 3

DHL-9

BCBL1

BC1BC3

BBG1BC2

Nalm-6

697Patie

nt N

PH 809Jurkat

PH 820PH 895

Patient K

Patient B

Patient Z

697BCBL1

BC2

4

B-cell lines and B-leukemias vs. PEL cell lines

2 3 4 5 6 7 8 9 10 11 12 13 14 15

Fig 3. Downregulation of MYB expression in KSHV-infected PEL cell lines as well as in PEL patients but not in cells that are free of KSHV infection.

(A–C) Sixty micrograms of total proteins were resolved on Tris-glycine 16%, transferred onto PVDF membranes and probed with an antibody

specific for the MYB protein. Comparable protein loading was verified using an antibody specific for the housekeeping gene product b-tubulin.

Results are representative of two independent experiments. (A): PEL cell lines, lane 1 and 2: DHL-9 B cells without ()) or with (+) TPA treatment;

lane 3: Jurkat; lane 4: C8166 (HTLV-1+); lanes 5–14: BCBL1, BC1, BC3, BBG1, BC2 without (lanes 5, 7, 9, 11, 13) or with (lanes 6, 8, 10, 12, 14) TPA

treatment. (B): PEL patients, lane 1: Jurkat; lanes 2–4: PH809, PH820, PH895. (C): B-cell lines and B-leukaemias versus PEL cell lines, lane 1: DHL-9;

lane 2: BCBL1; lane 3: BC1; lane 4: BC3; lane 5: BBG1; lane 6: BC2; lane 7: Nalm-6; lane 8: 697; lane 9: Patient N; lane 10: Patient K; lane 11: Patient B,

lane 12: Patient Z; lane 13: 697; lane 15: BCBL1; lane 15: BC2. Lanes 1–8 were run on the same gel.

V. Lacoste et al

ª 2007 The Authors494 Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 487–501

1998). The KSHV immediate early protein RTA, which is

induced by such treatment, has been shown to drive the lytic

cycle to completion (Lukac et al, 1998). It was also shown that

RTA can activate its own promoter (Deng et al, 2000). To

investigate whether MYB inhibition could play any role during

the KSHV cycle, cells were transfected with an Rta promoter-

luciferase construct (pRP-luc) with or without a MYB

expression vector (Fig 8). MYB significantly activated the Rta

promoter, in the absence of any RTA expression (Fig 8, lane 1

versus 2), suggesting that decreasing MYB expression may also

prevent the induction of the lytic cycle.

(A)

(B)

(C)

I-κBS32/36A

v-FLIP

0

25 000

50 000

75 000

1 2

IB: anti-Actin

IB: anti-FlagVec

tor

vFLIP

Jurkat

v-FLIP

I-κBS32/36A

MRE-luc

NF-κB-lucIu

c un

its

0

25 000

50 000

75 000Iu

c un

its

– + +

– – +

– + +

– – +

Fig 4. Repression of the MYB promoter by the KSHV v-FLIP protein

through the Myb responsive elements (MRE) in lymphoid cells. (A)

Jurkat cells were transfected with v-FLIP vector in the presence of a

NF-jB-luc construct. (B) Western-blot of the v-FLIP protein in Jurkat

cells. Jurkat cells were transfected with either an empty vector or a

vector-expressing Flag-epitope tagged v-FLIP. After 4 h, the cells were

treated with PHA and TPA. Fifty micrograms of total extract were

resolved on SDS-PAGE 10%. Lane 1: empty vector, lane 2: v-FLIP

expressing vector. (C) Jurkat cells were transfected with v-FLIP vector

in the presence of a MRE-luc construction. (A) and (C): When needed,

the dominant negative IjBaS32/36A was transfected. Twenty-four hours

after transfection, cells were harvested, and luciferase assays performed.

Luciferase activity is expressed as light units. Results represent the

average value of three independent experiments.

(A)

(B)

(C)

NF-κB-luc

MRE-luc

v-GPCR –

75 000

50 000

25 000

0

Iuc

un

its

75 000

50 000

25 000

0

Iuc

un

its

Vector

v-GPCR

IB: anti v-GPCR N

1 2

IB: anti ββ-tubulin

Jurkat

+

v-GPCRI-κBS32/36A

– +– –

+ + +

Fig 5. Repression of the MYB promoter by the KSHV v-GPCR protein

through the Myb responsive elements (MRE) in lymphoid cells. (A)

KSHV v-GPCR wild type (wt) protein activates NF-jB in Jurkat cells.

(B) Western-blot analysis of v-GPCR-EGFP protein expression in

Jurkat cells. Sixty micrograms of total protein extract were resolved on

SDS-PAGE 12%. After electro-transfer, the immunoblot was incubated

with an anti-v-GPCR polyclonal serum. (C) KSHV v-GPCRwt protein

represses the MYB promoter through the MRE. (A) and (C): Jurkat

cells were transfected with the v-GPCRwt-EGFP vector in the presence

of (A) a NF-jB-luc or (C) a MRE-luc construct. Twenty-four hours

after transfection, cells were harvested, and luciferase assays performed.

Luciferase activity is expressed as light units. When necessary, the

dominant negative IjBaS32/36A was added. Results represent the mean

of at least two independent experiments.

Inhibition of MYB functions by KSHV v-FLIP and v-GPCR

ª 2007 The AuthorsJournal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 487–501 495

Discussion

Abnormalities in the MYB locus have been observed in several

acute T-cell leukaemias, B lymphomas, colon carcinomas,

melanomas, as well as myeloid leukaemias (Alitalo et al, 1984;

Pelicci et al, 1984; Barletta et al, 1987; Dasgupta et al, 1989;

Press et al, 1995). In these cases, this event is accompanied by

an amplification of MYB followed by its enhanced transcrip-

tion (Oh & Reddy, 1999). Interestingly, a study has demon-

strated that disrupting Myb expression with therapeutic intent,

either in vitro or in vivo in SCID mice can be successful

(reviewed in (Gewirtz, 1999)). Thus, it appeared that down-

regulation of Myb seems to preferentially kill leukaemic cells

(Calabretta & Gewirtz, 1991; Calabretta et al, 1991; Ratajczak

et al, 1992a). This paradigm is, however, not true for all types

of haematological cancers since, together with this present

report, we have now described a T-cell leukaemia/lymphoma,

(i.e. ATLL) (Nicot et al, 2000a, 2001) and a B-cell lymphoma

(i.e. PEL) where MYB expression and functions are repressed

via viral gene(s) expression without inducing cell death.

However, it is conceivable that certain KSHV proteins replace

MYB functions in PEL cells.

MYB protects normal and leukaemic cells against apoptosis.

Thus, it is intriguing that KSHV-mediated trans-repression of

MYB. It has been reported that v-FLIP expression is low in

early stage KS lesions but dramatically elevated in late-stage

lesions. This observation was associated with a reduction in

apoptosis in KS lesions (Sturzl et al, 1999). It would therefore

be of interest to determine whether MYB expression is

detectable in early stage KS and decreases as KS lesions

(A)

(B)

Western-blot

MYB

RT-PCR

1 2

RT – – + +

390 bp v-FLIP

v-FLIP

transd

uced

Control

v-FLIP

transd

uced

Control

v-FLIP

transd

uced

Control

β-tubulin

Fig 6. v-FLIP transrepresses the endogenous human MYB promoter.

(A) Western-blot analysis of MYB expression in Jurkat cells (lane 1) or

in Jurkat cells that have been transduced with a TRIP-v-FLIP vector

(lane 2). Forty-five micrograms of total cell extracts were resolved on

12%. SDS-PAGE After electro-transfer, the membrane was incubated

with a MYB polyclonal serum. Comparable protein loading was veri-

fied using a polyclonal serum specific for the housekeeping gene

product b-tubulin. (B) v-FLIP expression determined by RT-PCR in

transduced Jurkat cells. Five-hundred nanograms of total RNA

extracted from the transduced and the control cells were used as

template with the OneStep RT-PCR kit. The primers are described in

Table I. ), without reverse transcriptase; +, with reverse transcriptase.

(A)

(B)

(C)

Western-blot

MYB

v-GPCRΔ5

v-GPCRwt

GFP

1 2

β-tubulin

Fig 7. v-GPCRwt but not v-GPCRD5 proteins repress the endogenous

human MYB promoter in Jurkat cells. Fifty micrograms of proteins

were resolved on 12% Tris-glycine, transferred onto PVDF membranes

and probed with an antibody specific for (A) MYB, (B) GFP and (C)

b-tubulin. Lane 1: v-GPCRwt expressing Jurkat cells, lane 2: v-

GPCRD5 expressing cells.

0

50 000

Iuc

un

its

75 000pRP-luc

Contro

l

c-M

yb

25 000

Fig 8. MYB transactivates the Rta promoter in the absence of any

KSHV protein expression. 293T cells were transfected with the pRP-luc

construct (200 ng), with or without a MYB expression vector (200 ng).

Twenty-four hours after transfection, cells were harvested, and lucif-

erase assays performed. Luciferase activity is expressed as light units.

V. Lacoste et al

ª 2007 The Authors496 Journal Compilation ª 2007 Blackwell Publishing Ltd, British Journal of Haematology, 138, 487–501

develop. In addition, the KSHV genome encodes genes for at

least four other proteins: LANA, v-IL-6, v-IAP and v-Bcl-2,

which possess anti-apoptotic properties (Arvanitakis et al,

1997; Sarid et al, 1997; Bais et al, 1998; Friborg et al, 1999; Yu

et al, 1999; Mittnacht & Boshoff, 2000; Montaner et al, 2001;

Wang et al, 2002).

MYB participates in the regulation of the cell cycle and in

the cellular proliferation. It has been established for some time

that the exposure of peripheral blood mononuclear cells to

MYB antisense oligonucleotides blocked T-cell proliferation in

response to mitogens (Gewirtz et al, 1989). There are also viral

factors that directly affect the cell cycle as KSHV contains two

genes which encode (i) v-Cyclin that can drive cells into the

S phase in the absence of growth factor (Swanton et al, 1997)

and (ii) v-IL-6, a B-cell growth and differentiation factor

(Neipel et al, 1997).

The KSHV immediate early protein RTA, which is induced

by TPA treatment, has been shown to drive the lytic cycle to

completion (Lukac et al, 1998). It was also shown that RTA

can activate its own promoter (Deng et al, 2000). Our present

results clearly demonstrate that MYB expression is sufficient

for activating transcription from the Rta promoter in the

absence of any RTA protein expression. Repressing MYB

expression would therefore participate in maintaining the virus

in latency by preventing the transcriptional activation of Rta. It

will also limit the induction of cell death, as RTA has been

shown to be an apoptosis inducer (Nishimura et al, 2003).

In conclusion, while we have previously shown that

a human retrovirus, such as HTLV-1, encodes one protein,

Tax, which can repress and replace MYB functions, an

elaborated transforming virus, such as KSHV, encodes genes

for at least seven proteins (LANA, v-Cyclin, v-IL-6, v-IAP, v-

Bcl-2, v-FLIP and v-GPCR) whose expression could supplant

MYB functions. Although we cannot exclude that another

KSHV protein might also synergise with v-FLIP and v-GPCR

for repressing MYB functions during the late lytic stage of

KSHV cycle (Montaner et al, 2001; Samaniego et al, 2001), v-

FLIP is so far the only protein that can exert this effect in the

latent stage (An et al, 2002).

Acknowledgements

This work was supported by a grant from l’Association de

Recherche sur le Cancer (ARC) and from la Fondation de

France to RM and grants from NIH (CA85177 and CA124621)

to PMC. VL and CN were supported by a bourse Pasteur-

Weissman and la Ligue Nationale Contre le Cancer respect-

ively. RM was supported by a bourse Roux, from the Institut

Pasteur and by INSERM. We thank Dr D. Gonzalez-Dunia, J.S.

Seeler and C. Royer-Leveau for their help. We are indebted to

Drs P. Charneau, J. Golay, N. Rice, L. Boxer, P. Murphy and

W.C. Greene for generous gifts of reagents essential to the

realization of this study. We thank Pr O. Hermine for

providing us with some PEL fresh samples, and Dr Timothy

Stinear for his critical comments.

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