JOURNAL OF VIROLOGY, July 2006, p. 7052–7059
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 14
TORC1 and TORC2 Coactivators Are Required for Tax Activation of
the Human T-Cell Leukemia Virus Type 1 Long Terminal Repeats
Yeung-Tung Siu,1† King-Tung Chin,1† Kam-Leung Siu,1Elizabeth Yee Wai Choy,1
Kuan-Teh Jeang,2and Dong-Yan Jin1*
Department of Biochemistry, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong,1and Laboratory of
Molecular Microbiology, National Institute of Allergy and Infectious Diseases, 9000 Rockville Pike, Bethesda, Maryland 20892-04602
Received 15 January 2006/Accepted 28 April 2006
Human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription from the long
terminal repeats (LTR). Mechanisms through which Tax activates LTR have been established, but coactivators
of this process remain to be identified and characterized. Here we show that all three members of the TORC
family of transcriptional regulators are coactivators of Tax for LTR-driven expression. TORC coactivation
requires CREB, but not ATF4 or other bZIP factors. Tax physically interacts with TORC1, TORC2, and
TORC3 (TORC1/2/3), and the depletion of TORC1/2/3 inhibited Tax activity. TORC coactivation can be
further enhanced by transcriptional coactivator p300. In addition, coactivators in the p300 family are required
for full activity of Tax independently of TORC1/2/3. Thus, both TORC and p300 families of coactivators are
essential for optimal activation of HTLV-1 transcription by Tax.
Human T-cell leukemia virus type 1 (HTLV-1) is etiologi-
cally associated with adult T-cell leukemia, an aggressive and
fatal malignancy of CD4?T lymphocytes (32). HTLV-1 en-
codes a 40-kDa oncoprotein, Tax, which initiates the process of
leukemogenesis (10, 16). Tax is a potent transcriptional acti-
vator of the viral long terminal repeats (LTR) as well as a
subset of cellular genes, including various cytokine genes and
proto-oncogenes (10, 23). The mechanisms through which Tax
activates the viral LTR have been well studied. Thus, we un-
derstand that Tax acts as a homodimer (18, 39) that interacts
with CREB and contacts a stretch of DNA to activate the three
21-bp repeats, also known as Tax-responsive elements (TRE),
on HTLV-1 LTR (24, 29, 41). Optimal activation of LTR by
Tax requires the HTLV-1 core promoter CREB and the 21-bp
repeats (6). However, the molecular details before and after
the formation of the ternary complex remain largely unknown.
Coordinated activation of transcription requires both DNA-
binding activators, such as Tax and CREB, and coactivators
which act through chromatin modification and/or the stimula-
tion of preinitiation complex formation (36). Several transcrip-
tional coactivators that are histone or factor acetyltransferases,
including CREB-binding protein (CBP), p300, and P/CAF,
have been shown to play roles in Tax-mediated transcription
(11, 12, 17, 22, 27). Because Tax activation of the LTR is
potent and Tax can influence multiple steps of transcription
both prior and subsequently to TATA-binding factor recruit-
ment (6), it likely interacts with additional coactivators.
A new family of CREB coactivators, termed transducers of
regulated CREB activity (TORCs), has recently been identi-
fied and characterized (2, 8, 14, 37). Currently there are three
members, TORC1, TORC2, and TORC3 (TORC1/2/3), in this
family. All three activate CREB-dependent transcription (8,
14) but are differentially expressed and regulated by upstream
signals such as AMP-activated protein kinase and LKB1 (26,
38). A recent study has suggested that TORC3 also serves to
enhance Tax activation of HTLV-1 LTR (25). However,
whether TORC3 is specifically required and whether Tax
might generally interact with other TORC factors remain un-
clear. In particular, it will be of interest to understand whether
TORC1 and TORC2 are also involved in mediating the action
In this study, we investigated the contributory roles of
TORC1, TORC2, and TORC3 in Tax activation of HTLV-1
LTR. We also explored the requirement for CREB in TORCs’
coactivator function and in Tax-TORC interaction. We found
that all three TORCs provide essential coactivator function for
Tax activation of the HTLV-1 LTR and that Tax directly binds
TORCs. In addition, we demonstrated that the p300 coactiva-
tor cooperates with TORCs and is required for their full ac-
tivity in the activation of HTLV-1 LTR. Our work presents a
new mechanistic facet to Tax-dependent regulation of gene
MATERIALS AND METHODS
Plasmids. Human cDNAs containing complete coding regions for TORC1,
TORC2, and TORC3 were derived from expressed sequence tag clones (IMAGE
clone identification no. 4938995, 6188068, and 6470060) obtained from RZPD
Deutsches Ressourcenzentrum fu ¨r Genomforschung GmbH (Berlin, Germany).
Eukaryotic expression plasmids for TORC1, TORC2, and TORC3 were based
on pcDNA3.1/V5 (Invitrogen) or pEGFP (Clontech). The expression plasmid for
myc-tagged CREB was constructed by inserting both a myc tag (via HindIII and
BamHI) and the CREB gene (via EcoRI and EcoRV) into pcDNA3.1/V5-His6B
(Invitrogen). Expression vectors for Gal-CREB, Gal-ATF4, Gal-LZIP, and Gal-
CREB-H were derived from pM (Clontech). Reporter plasmids pLTR-Luc and
pGal-Luc as well as expression plasmids for Tax, Gal-Tax, A-CREB, A-ATF4,
A-LZIP, and A-CREB-H have been previously described in detail (5, 6, 18–21).
Tax expression plasmid pIEX is driven by a cytomegalovirus promoter (19).
A-CREB, A-ATF4, A-LZIP, and A-CREB-H were constructed by fusing a
designed acidic amphipathic extension onto the N terminus of the leucine zipper
region (1). A-CREB was a gift from Charles Vinson (National Cancer Institute,
Maryland) and contains 274 to 341 amino acids of mouse CREB (1). A-ATF4
* Corresponding author: Mailing address: Department of Biochem-
istry, The University of Hong Kong, 3/F Laboratory Block, Faculty of
Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong. Phone:
852-2819-9491. Fax: 852-2855-1254. E-mail: firstname.lastname@example.org.
† These authors contributed equally to this work.
contains 304 to 352 amino acids of human ATF4 (6). A-LZIP contains 175 to 223
amino acids of human LZIP (21). A-CREB-H contains 267 to 312 amino acids
of human CREB-H (5).
The short hairpin RNA (shRNA) expression vector pSHAG-1 (34) was kindly
provided by Greg Hannon (Cold Spring Harbor Laboratory, New York). The
expression plasmid for E1A-12S (31) was from James Lundblad (Oregon Health
and Science University, Oregon). Expression plasmid for p300 (30, 40) was
originally obtained from Xiang-Jiao Yang (McGill University Health Center,
Montre ´al, Canada) and provided to us by Zengguo Wu. Expression vectors for
p300?HAT (33) and p300DY (15) mutants were provided by Y. Nakatani
(Dana-Farber Cancer Institute, Massachusetts) and Tso-Pang Yao (Duke Uni-
versity Medical Center, North Carolina), respectively.
Reporter assay. HeLa or 293T cells were cultured in Dulbecco’s modified
Eagle’s medium and transfected using GeneJuice transfection reagent (Nova-
gen). Jurkat cells were propagated in RPMI 1640 medium and transfected by
electroporation (20). Cells were harvested 48 h after transfection. Luciferase
activity was determined as described previously (5, 13) using dual-luciferase
reagents (Promega). Transfection efficiencies were normalized to a control plas-
mid (pRLSV40 from Promega) expressing Renilla luciferase.
Coimmunoprecipitation. HeLa cells grown in a 100-mm petri dish were har-
vested in 0.5 ml of immunoprecipitation buffer (25 mM HEPES, pH 7.5, 0.3 M
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1% Triton X-100,
0.1% sodium dodecyl sulfate, 20 mM ?-glycerophosphate, 1 mM sodium vana-
date, and 1 mM phenylmethylsulfonyl fluoride). V5-tagged TORC protein was
immunoprecipitated from the cleared lysate by overnight incubation at 4°C with
rabbit anti-V5 (Sigma). Immunocomplex was precipitated by using protein A
agarose (Invitrogen) and washed three times with immunoprecipitation buffer
before being resuspended in sample buffer (60 mM Tris-Cl, 2% sodium dodecyl
sulfate, 6% glycerol, 1% ?-mercaptoethanol, and 0.002% bromophenol blue).
RNA interference (RNAi) knockdown of TORCs. The shRNA expression cas-
settes comprising U6 promoter, shRNA (sense-loop-antisense), and termina-
tion signal T6were PCR amplified and inserted into the vector by TA cloning.
The three shRNAs targeting TORC1/2/3, namely shTORC1, shTORC2, and
shTORC3, target nucleotides 1709 to 1729 of TORC1 (GACTCGCAGCAACT
GGGATAC), nucleotides 452 to 472 of TORC2 (CAGCGAGATCCTCGAAG
AATG), and nucleotides 1779 to 1807 of TORC3 (GTCACTTAACATGTTG
AGTCCATCCAGGC), respectively. For the knockdown experiment, 5 million
Jurkat cells were electroporated at 960 ?F and 300 V with plasmids in serum-free
RPMI 1640 medium.
RESULTS AND DISCUSSION
TORC proteins are coactivators of Tax. TORC3 was re-
cently shown to stimulate Tax activation of HTLV-1 LTR (25).
TORC1 and TORC2 (TORC1/2) share only ?30% amino acid
identity with TORC3 (14). In addition, TORC1/2 transcripts
are found in thymus, CD4?lymphocytes, and Molt4 leukemic
cells (8). Actually, TORC2 is most abundantly expressed in
thymus (8). Thus, we asked whether TORC1/2 might be phys-
FIG. 1. TORC proteins are transcriptional coactivators of HTLV-1 Tax. (A) Expression of TORCs in HeLa cells. Cells were transfected with
Tax expression plasmid pIEX alone (lanes 1 and 5) or with pIEX plus pcDNA3.1-V5-TORC1/2/3 (lanes 2 to 4 and 6 to 8). Cell lysates were
analyzed by Western blotting with rabbit anti-V5 (Invitrogen) or mouse anti-Tax (11). The expression of TORCs did not affect the activity of the
cytomegalovirus promoter. (B to D) Cooperation of TORCs and Tax in the activation of HTLV-1 LTR. HeLa cells (2 ? 105per well) were
transfected with reporter plasmid pLTR-Luc (1 ?g) plus the indicated combinations of expression plasmids. A fixed amount of Tax expression
plasmid pIEX (500 ng) and escalating amounts of expression plasmids (60 to 250 ng) for TORCs were used. (E to G) Cooperation of TORCs and
Tax in the activation of TRE. HeLa cells (2 ? 105per well) were transfected with reporter plasmid pTRE-Luc (1 ?g) plus the indicated
combinations of expression plasmids. A fixed amount of Tax expression plasmid pIEX (500 ng) and escalating amounts of expression plasmids (60
to 250 ng) for TORCs were used. (H to J) Stimulation of Gal-Tax-mediated transcriptional activation by TORCs. HeLa cells (2 ? 105per well)
were cotransfected with reporter plasmid pGal-Luc (1 ?g) and expression plasmids for Gal-Tax (500 ng) and TORCs (60 to 250 ng). Cells were
harvested 48 h after transfection, and relative luciferase activities (RLA) in arbitrary units were calculated from the readouts of firefly luciferase
normalized to the readouts of Renilla luciferase. The results represent three independent experiments, and the error bars indicate standard
VOL. 80, 2006 REQUIREMENT OF TORC PROTEINS FOR HTLV-1 TRANSCRIPTION7053
iological coactivators in the context of Tax activation of
We expressed V5-tagged TORC1, TORC2, and TORC3
(TORC1/2/3) in HeLa cells (Fig. 1A) and noted that, in the
absence of Tax, all three TORC proteins activated basal tran-
scription from HTLV-1 LTR in a dose-dependent manner
(Fig. 1B to D, compare groups 2 through 4 to group 1). In
addition, TORC1/2/3 also cooperated with Tax in activating
the LTR (Fig. 1B to D, compare groups 6 to 8 to group 5).
Because the three 21-bp repeats (or TREs) in HTLV-1 LTR
are recognized by Tax and CREB (39, 41), we asked whether
these TRE enhancers would also be targeted by TORCs. In-
deed, TORC1/2/3 were capable of activating transcription
from TRE alone and they cooperated with Tax in the activa-
tion of TRE (Fig. 1E to F, compare groups 2 through 4 to
group 1 and compare groups 6 through 8 to group 5). Thus, the
transcriptional activity of TORCs is mediated through TRE.
To verify the coactivator function of TORCs on Tax, we
repeated the experiments in the context of a fusion protein
comprising the Gal4 DNA binding domain and Tax (Gal-Tax)
and a luciferase reporter construct under the control of Gal4-
binding enhancer elements (pGal-Luc). In this setting, the
escalation of the amount of TORC1/2/3 also led to substantial
potentiation of Tax-dependent transcription (Fig. 1H to J,
compare groups 6 through 8 to group 5). We also appreciated
that TORCs had a weak general coactivator activities on pGal-
Luc in the absence of Gal-Tax (Fig. 1H to J, compare groups
2 through 4 to group 1). Hence, TORC proteins preferentially
coactivate Tax and HTLV-1 LTR.
Association of TORCs and Tax. TORC3 has previously been
shown to interact through its N-terminal coiled-coil domain
with Tax (25). This finding agrees with our prediction that Tax
preferentially interacts with a subset of cellular coiled-coil pro-
teins, including mitotic checkpoint protein MAD1 and I?B
kinase regulatory subunit IKK-?/NEMO (7, 13, 19, 20). To
investigate whether TORC1/2 also form a complex with Tax in
cells, we precipitated them from cultured HeLa cells (Fig. 2A,
lanes 3, 4, 7, 8, 11, and 12). In cells expressing both Tax and
TORC1/2/3, the ?-V5/myc precipitates also contained Tax
(Fig. 2B, lanes 4, 8, and 12). By contrast, the Tax-TORC
complex was not found in mock-, Tax-, or TORC-transfected
cells (Fig. 2B, lanes 1 to 3, 5 and 6, and 9 to 11). Our results
suggest that all three TORCs can complex with Tax in cultured
CREB is required for Tax/TORC-induced activation of LTR.
While CREB is needed for optimal activation of HTLV-1 LTR
by Tax (6), other bZIP proteins, including ATF4 and LZIP can
also contribute to Tax activation of LTR (6, 9, 35). Two im-
portant questions are currently unanswered. First, is CREB
uniquely essential for the transcriptional activation by Tax and
TORCs? Second, can TORC proteins also serve as coactiva-
tors for ATF4, LZIP, and other bZIP factors?
To address these questions, we employed a group of four
dominant inactive bZIP proteins (A-CREB, A-ATF4, A-LZIP,
and A-CREB-H) that were constructed using the same strat-
egy of fusing an acidic peptide with the leucine zipper region
(1). The specific dominant inhibitory activities of these mutants
have been previously verified (1, 6). Particularly, the expression
of A-CREB leads to a dramatic suppression of Tax activation
of LTR, with more modest reduction observed with A-ATF4
and A-LZIP (6). When we carried out similar sets of experi-
ments using TORC1/2/3, we found that only A-CREB com-
pletely inhibited the transcriptional activity of LTR (Fig. 3A to
C, compare groups 3 to 5 to group 2), while A-ATF4, A-LZIP,
and A-CREB-H did not significantly affect TORC-dependent
activation (Fig. 3A to C, groups 6 through 17), with only small
inhibitions observed at the highest doses (Fig. 3A, groups 16
and 17, and B, groups 9 and 17). Consistent with this, A-CREB
could also erase the transcriptional activity from Tax plus
TORC1/2/3, whereas the other three dominant inactive bZIP
FIG. 2. Association of Tax with TORCs. HeLa cells were transfected with the indicated combinations of expression plasmids. Lysates of cells
expressing V5-tagged TORC1 (lanes 3 and 4), myc-tagged TORC2 (lanes 7 and 8), V5-tagged TORC3 (lanes 11 and 12), and Tax (lanes 2, 4, 6,
8, 10, and 12) were immunoprecipitated (i.p.) with anti-V5 (?-V5) or anti-myc (?-myc) (Santa-Cruz), and the precipitates were analyzed by
Western blotting with rabbit anti-V5 (panel A, lanes 1 to 4 and 9 to 12), rabbit anti-myc (panel A, lanes 5 to 8), or mouse anti-Tax (?-Tax) (B).
Expression vector for myc-tagged TORC2 was derived from pcDNA4 (Invitrogen). Immunoglobulin H (IgH), heavy chain of immunoglobulin G.
?, absence of indicated protein; ?, presence of indicated protein.
7054 SIU ET AL.J. VIROL.
proteins had minimal effects (Fig. 3D to F, compare groups 6
through 17 to groups 2 through 5). Moreover, the coactivator
function of TORC1/2/3 in the context of Gal-Tax and pGal-
Luc was also blunted by A-CREB (Fig. 3G). To address
whether CREB is required after the recruitment of Tax to the
promoter, we tested the activity of Gal-Tax in the presence of
A-CREB. We observed that Gal-Tax activity was abrogated by
only A-CREB but not other dominant inactive bZIP proteins
FIG. 3. TORC coactivation with Tax is specific for CREB. (A to F) CREB is required for transcriptional activities of TORC and Tax plus
TORC. HeLa cells (2 ? 105per well) were transfected with reporter plasmid pLTR-Luc (1 ?g) plus the indicated combinations of expression
plasmids. Escalating amounts (60 to 250 ng) of expression vectors for dominant inactive forms of CREB, ATF4, LZIP, and CREB-H were used.
(G and H) CREB is essential for TORC coactivation of Gal-Tax and for Gal-Tax activity. HeLa cells (2 ? 105per well) were transfected with
reporter plasmid pGal-Luc (1 ?g) plus the indicated combinations of expression plasmids. Escalating amounts (60 to 250 ng) of expression vector
for dominant inactive form of CREB (A-CREB) were used. (I to K) TORC proteins are CREB-specific transcriptional coactivators. HeLa cells
were cotransfected with reporter plasmid pGal-Luc and expression plasmids for TORC1 (I), TORC2 (J), and TORC3 (K) as well as expression
plasmids for Gal (groups 1 to 4), Gal-CREB (groups 5 to 8), Gal-ATF4 (groups 9 to 12), Gal-LZIP (groups 13 to 16), and Gal-CREB-H (groups
17 to 20). The same amount (500 ng) of Gal plasmids and escalating amounts (60 to 250 ng) of TORC plasmids were used. The results represent
three independent experiments, and the error bars indicate standard deviations. RLA, relative luciferase activity.
VOL. 80, 2006 REQUIREMENT OF TORC PROTEINS FOR HTLV-1 TRANSCRIPTION7055
(Fig. 3H, compare groups 3 through 5 to groups 7 through 9, 11
through 13, and 15 through 17). Thus, we interpret CREB, but
not ATF4 or other bZIP factors, to be responsible for Tax/
TORC-mediated activation of HTLV-1 LTR.
We also addressed whether TORC proteins act as coactiva-
tors for bZIP factors, such as ATF4 (9, 35), LZIP (21, 42), and
CREB-H (5). We observed that fusion proteins Gal-CREB,
Gal-ATF4, Gal-LZIP and Gal-CREB-H could activate pGal-
Luc to different degrees (Fig. 3I to K, compare groups 5, 9, 13,
and 17 to group 1). However, the expression of TORC1/2/3
enhanced the transcriptional activity of only Gal-CREB, not
Gal-ATF4, Gal-LZIP, or Gal-CREB-H (Fig. 3I to K, compare
groups 5 through 8 to groups 9 through 20). Taken together,
TORC coactivators are active with only CREB, not other
CREB-related bZIP proteins.
TORC coactivators were newly identified from genome-
wide screening of transcriptional coregulators specific for
CREB (8, 14). Our present work, together with a recent report
on TORC3 (25), established that TORCs are also needed for
the transcriptional activity mediated by Tax. We noted that
TORC3 overexpressed in HeLa cells exhibited a relatively
strong transcriptional activity on pGal-Luc compared to those
of TORC1 and TORC2 (Fig. 1J compared to H and I and Fig.
3G and K compared to I and J). In contrast, TORC1/2/3
showed comparable coactivator activity on pLTR-Luc and
pTRE-Luc (Fig. 1B to G). Further experiments conducted with
recombinant TORC proteins are required to clarify whether
any of the three TORC proteins is particularly active in inter-
acting with Tax and/or stimulating transcription.
Our findings support the notion that the TORC-mediated
coactivation of Tax is specific for CREB (Fig. 3). Thus, Tax
plus CREB requires TORCs, while Tax plus other cellular
transcription activator, may utilize a different set of coactiva-
tors. We suggest that Tax plus activator plus coactivators ulti-
mately determines the specificity of the ternary complex for the
viral LTR or for cellular promoters.
Consistent with our previous finding that CREB is preferred
over ATF4, c-Jun, and other bZIP proteins for Tax activation
of HTLV-1 LTR (6), here we showed that only CREB, not
ATF4 or other bZIP factors, is required for the recruitment
and activity of TORCs (Fig. 3). We further demonstrated that
TORCs exert coactivator function on only CREB, not other
CREB-related bZIP factors (Fig. 3). The recruitment and ac-
tivation of TORCs by CREB could explain the preference for
CREB by Tax in the activation of HTLV-1 LTR.
TORC coactivators are required for Tax activation of
HTLV-1 LTR in T lymphocytes. Above we characterized the
role of TORC coactivators in Tax-mediated LTR transcription
in cultured HeLa cells. To ask whether similar requirements
can be shown in suspension T lymphocytes that are susceptible
to physiological infection of HTLV-1, we used RNAi knock-
down to investigate the roles of TORC coactivators. We de-
signed three shRNAs targeting TORC1/2/3 (shTORC1/2/3).
When we expressed these shRNAs in Jurkat cells, a 35 to 70%
inhibition of exogenously expressed TORC1/2/3 was achieved
(Fig. 4A to C). These shRNAs could also effectively knock-
down TORC expression in HeLa cells as shown by Western
blotting (Fig. 4D). Since shTORCs (e.g., shTORC1) inhibited
only the desired isoform (e.g., TORC1), not the other two
paralogs (e.g., TORC2/3), our RNAi depletion of TORCs was
isotype specific (data not shown).
When each of the three shTORCs was expressed singularly,
we did not observe appreciable inhibition of Tax activation of
LTR (data not shown). This might be explained by incomplete
knockdown and/or redundant function of the three TORC
proteins. We thus expressed all three shTORCs simultaneously
in Jurkat cells and observed a moderate dose-dependent sup-
pressive effect on Tax activity (Fig. 4E, compare groups 3
through 5 to group 2). The specificity of this effect was sup-
ported by a control experiment in which the shRNA targeting
green fluorescent protein (GFP) failed to affect Tax activation
of the LTR (Fig. 4E, compare group 6 to group 2). These
results indicated that TORC coactivators may redundantly co-
operate with Tax and that simultaneous knockdown of all three
FIG. 4. TORC coactivators are required for optimal activation of
the HTLV-1 LTR by Tax. (A to C) Inhibition of TORC-dependent
transcriptional activity by shRNAs. Jurkat cells were transfected with
reporter plasmid pLTR-Luc (8 ?g), a fixed amount (2 ?g) of expres-
sion plasmids for TORC1 (A), TORC2 (B), and TORC3 (C), and
escalating amounts (2 to 10 ?g) of plasmids expressing shRNA against
TORC1, TORC2, or TORC3 (shTORC). (D) Depletion of TORCs by
shRNAs. HeLa cells were transfected with expression plasmids for
GFP-tagged TORC1 (lanes 1 to 3), TORC2 (lanes 4 to 6) and TORC3
(lanes 7 to 9) as well as empty shRNA expression vector (vec; lanes 1,
4 and 7), expression vector for shRNA targeting luciferase (shLuc;
lanes 2, 5, and 8), and expression plasmid for shRNA against TORC1,
TORC2, and TORC3 (shTORC1/2/3; lanes 3, 6, and 9), respectively.
Expression of GFP-TORC proteins (upper panels) and antitubulin
(tub; lower panels) was analyzed by Western blotting. (E) TORCs are
required for Tax activation in T lymphocytes. Jurkat lymphocytes were
electroporated with reporter plasmid pLTR-Luc (8 ?g), Tax expres-
sion plasmid pIEX (1 ?g), and increasing amounts (2 to 10 ?g) of a
mixture of plasmids expressing shRNAs targeting TORC1, TORC2,
and TORC3 (shTORC1 to -3). The shRNA against GFP (shGFP) was
also included as a control. Cells were harvested 48 h after electropo-
ration, and relative luciferase activities (RLA) were measured. The
results represent three independent experiments and the error bars
indicate standard deviations. ?, absence of indicated protein; ?, pres-
ence of indicated protein.
7056SIU ET AL.J. VIROL.
endogenous TORCs in T cells is needed for significant inhibi-
tion of Tax.
The incompleteness of the RNAi-induced silencing effect
prevents us from establishing the physiological roles of TORCs
and their exact function in the context of Tax and HTLV-1
LTR. In these regards, the creation of TORC-null mice could
provide new opportunities for functional studies of TORCs.
Nevertheless, our RNAi data did implicate functional redun-
dancies among the three TORC coactivators.
Both TORCs and p300 family coactivators are required for
Tax activation of LTR. p300 and related coactivators in the
same family have also been shown to play an important role in
Tax-induced transcriptional activation (11, 12, 17, 22, 27). On
the other hand, suppression of all three TORCs by RNAi
resulted in only a partial inhibition of Tax-mediated activation
of LTR (Fig. 4), suggesting that other coactivators might also
contribute to this process. In light of this, we set out to inves-
tigate the relationship between the p300 and TORC families of
coactivators in the context of Tax and HTLV-1 LTR. When we
coexpressed p300 and TORC1/2/3 in human 293T cells in the
absence of Tax, p300 was able to enhance TORC activity on
to C, compare groups 6 through 8 to group 5). Consistent with
previous reports (17, 23), p300 could also coactivate Tax in this
experimental setting (Fig. 5A to C, compare groups 10 through
12 to group 9). In addition, in cells expressing p300, Tax and
TORC1/2/3 simultaneously, p300 further increased the tran-
scriptional activity mediated by Tax and TORC1/2/3 (Fig.
5A to C, compare groups 14 through 16 to group 13). Thus,
p300 and TORC1/2/3 act additively in mediating Tax acti-
vation of LTR.
Next, we examined the effect of adenovirus E1A-12S, a well-
characterized inhibitor of p300 and related coactivators, in-
cluding CBP and P/CAF (12, 29), on the activity of TORCs in
the activation of LTR. Generally consistent with previous find-
ings (12), E1A-12S could inhibit Tax activation of LTR (Fig.
5D to F, compare groups 10 through 12 to group 9). In con-
trast, it did not significantly influence TORC1/2/3-induced ac-
tivation of LTR in the absence of Tax (Fig. 5D to F, compare
groups 6 through 8 to group 5). Notably, E1A-12S substantially
repressed transcriptional activity when both Tax and TORC1/
2/3 were expressed (Fig. 5D to F, compare groups 14 through
16 to group 13). These results suggest that coactivators in the
p300 family are required for optimal activation of HTLV-1
LTR by Tax independently of TORCs.
To determine the role of p300 in Tax/TORC-mediated tran-
FIG. 5. The role of p300 family coactivators in Tax/TORC-mediated activation of HTLV-1 LTR. (A to C) Additive action of TORC1/2/3 and
p300. 293T cells (1.5 ? 105per well) were transfected with reporter plasmid pLTR-Luc (250 ng) plus the indicated combinations of expression
plasmids. Escalating amounts of p300 expression plasmid (60 to 250 ng) were used with fixed amounts of plasmids for TORCs (15 ng) and Tax
(250 ng). (D to F) Influence of the transcriptional activity of Tax and TORCs by E1A-12S. HeLa cells (4 ? 104per well) were transfected with
reporter plasmid pLTR-Luc (250 ng) plus the indicated combinations of expression plasmids. Escalating amounts of E1A-12S plasmid (15 to 60
ng) were used with fixed amounts of TORC (15 ng) and Tax (250 ng) plasmids. (G to I) Influence of a dominant inactive p300 mutant on the activity
of Tax and TORCs. HeLa cells (4 ? 104per well) were transfected with reporter plasmid pLTR-Luc (250 ng) plus the indicated combinations of
expression plasmids. Escalating amounts of p300?HAT deletion mutant (60 to 250 ng) were used with fixed amounts of TORC (15 ng) and Tax
(250 ng) plasmids. The results represent three independent experiments, and the error bars indicate standard deviations. RLA, relative luciferase
VOL. 80, 2006 REQUIREMENT OF TORC PROTEINS FOR HTLV-1 TRANSCRIPTION7057
scriptional activation, we assessed the impact of p300?HAT, a
dominant inactive mutant of p300 lacking the acetyltransferase
domain (33, 40), on the activity of TORCs and Tax. The ex-
pression of p300?HAT did not significantly affect the activity
of TORC1/2/3 in the activation of LTR (Fig. 5G to I, compare
groups 6 through 8 to group 5), but it modestly inhibited the
activity of Tax both in the presence and in the absence of
TORC1/2/3 (compare groups 10 through 12 to group 9, and
compare groups 14 through 16 to group 13). Similar results
were also obtained with p300DY (15), another dominant neg-
ative mutant of p300 with an inactivating point mutation in the
acetyltransferase domain (data not shown). The repressive ac-
tivity of p300 mutants was not as dramatic as that with E1A-
12S, suggesting that, in addition to p300, other coactivators in
the same family that are inhibited by E1A-12S might also
contribute to the full activity of Tax. Taken together, coacti-
vators in both TORC and p300 families are required for Tax
activation of HTLV-1 LTR.
Emerging evidence suggests that TORCs represent another
regulatory point at which different stimuli converge (2, 26, 37,
38). Several lines of data support the notion that Tax serves to
trigger activation of TORCs, bypassing cellular regulatory
mechanisms. First, Tax physically interacts with TORCs (Fig.
2). Second, Tax activation of HTLV-1 LTR can bypass the
need for stimulation by cAMP/protein kinase A (27), which
also induces activation of TORCs (2, 8). Finally, Tax activation
of HTLV-1 LTR is potent (6), which is explained by constitu-
tive activation of a major group of transcriptional coactivators.
Exactly how TORCs activate transcription from HTLV-1
LTR remains to be understood. One mechanism by which
TORCs activate CREB-dependent transcription is through
TAFII130 (23). Tax has been shown to interact with TATA-
binding protein and TAFII28 (3, 4). Other TATA-binding pro-
tein-associated factors, such as TAFII250, have also been
shown to be important in Tax activation of HTLV-1 LTR (28).
Further experiments are required to clarify whether and how
TAFII130 might be particularly influential on Tax.
In summary, in this study we demonstrated the coactivator
function of TORC1/2 in the context of Tax and HTLV-1
LTR (Fig. 1 and 4), the association of Tax with TORC1/2 in
cultured cells (Fig. 2), and the requirement of CREB and
p300 family coactivators for Tax/TORC-dependent tran-
scription (Fig. 3 and 5). Our findings refine the model for
Tax action, in which Tax physically interacts with CREB,
TORCs, and coactivators of the p300 family to stimulate
transcription from HTLV-1 LTR.
We thank G. J. Hannon, J. R. Lundblad, Y. Nakatani, C. Vinson, Z.
Wu, X. J. Yang, and T. P. Yao for gifts of plasmid, Y.-P. Ching for
helpful comments and suggestions, and A. C. S. Chun for critical
reading of the manuscript.
D.-Y. Jin is a Leukemia and Lymphoma Society Scholar. This work
was supported by grants to D.-Y. Jin from the Hong Kong Research
Grants Council (projects HKU 7249/01 M, HKU 7294/02 M, and HKU
7683/05 M), Concern Foundation, and the National Natural Science
Foundation of China (Young Investigator Award 30029001).
1. Ahn, S., M. Olive, S. Aggarwal, D. Krylov, D. Ginty, and C. Vinson. 1998. A
dominate-negative inhibitor of CREB reveals that it is a general mediator of
stimulus-dependent transcription of c-fos. Mol. Cell. Biol. 18:967–977.
2. Bittinger, M. A., E. McWhinnie, J. Meltzer, V. Iourgenko, B. Latario, X. Liu,
C. H. Chen, C. Song, D. Garza, and M. Labow. 2004. Activation of cAMP
response element-mediated gene expression by regulated nuclear transport
of TORC proteins. Curr. Biol. 14:2156–2161.
3. Caron, C., R. Rousset, C. Be ´raud, V. Moncollin, J. M. Egly, and P. Jalinot.
1993. Functional and biochemical interaction of the HTLV-1 Tax1 transac-
tivator with TBP. EMBO J. 12:4269–4278.
4. Caron, C., G. Mengus, V. Dubrowskaya, A. Roisin, I. Davidson, and P.
Jalinot. 1997. Human TAFII28 interacts with the human T cell leukemia
virus type I Tax transactivator and promotes its transcriptional activity. Proc.
Natl. Acad. Sci. USA 94:3662–3667.
5. Chin, K. T., H. J. Zhou, C. M. Wong, J. M. F. Lee, C. P. Chan, B. Q. Qiang,
J. G. Yuan, I. O. L. Ng, and D. Y. Jin. 2005. The liver-enriched transcription
factor CREB-H is a growth suppressor protein underexpressed in hepato-
cellular carcinoma. Nucleic Acids Res. 33:1859–1873.
6. Ching, Y. P., A. C. S. Chun, K. T. Chin, Z. Q. Zhang, K. T. Jeang, and D. Y.
Jin. 2004. Specific TATAA and bZIP requirements suggest that HTLV-1
Tax has transcriptional activity subsequent to the assembly of an initiation
complex. Retrovirology 1:18.
7. Chun, A. C. S., Y. Zhou, C. M. Wong, H. Kung, K. T. Jeang, and D. Y. Jin.
2000. Coiled-coil motif as a structural basis for the interaction of HTLV type
1 Tax with cellular cofactors. AIDS Res. Hum. Retrovir. 16:1689–1694.
8. Conkright, M. D., G. Canettieri, R. Screaton, E. Guzman, L. Miraglia, J. B.
Hogenesch, and M. Montminy. 2003. TORCs: transducers of regulated
CREB activity. Mol. Cell 12:413–423.
9. Gachon, F., S. Thebault, A. Peleraux, C. Devaux, and J. M. Mesnard. 2000.
Molecular interactions involved in the transactivation of the human T-Cell
leukemia virus type 1 promoter mediated by Tax and CREB-2 (ATF-4). Mol.
Cell. Biol. 20:3470–3481.
10. Gatza, M. L., J. C. Watt, and S. J. Marriott. 2003. Cellular transformation by
the HTLV-1 Tax protein, a jack-of-all-trades. Oncogene 22:5141–5149.
11. Harrod, R., Y. Tang, C. Nicot, H. S. Lu, A. Vassilev, Y. Nakatani, and C. Z.
Giam. 1998. An exposed KID-like domain in human T-cell lymphotropic
virus type 1 Tax is responsible for the recruitment of coactivators CBP/p300.
Mol. Cell. Biol. 18:5052–5061.
12. Harrod, R., Y. L. Kuo, Y. Tang, Y. Yao, A. Vassilev, Y. Nakatani, and C. Z.
Giam. 2000. p300 and p300/cAMP-responsive element-binding protein asso-
ciated factor interact with human T-cell lymphotropic virus type-1 Tax in a
multi-histone acetyltransferase/activator-enhancer complex. J. Biol. Chem.
13. Huang, G. J., Z. Q. Zhang, and D. Y. Jin. 2002. Stimulation of IKK-?
oligomerization by the T-cell leukemia virus oncoprotein Tax. FEBS Lett.
14. Iourgenko, V., W. Zhang, C. Mickanin, I. Daly, C. Jiang, J. M. Hexham, A. P.
Orth, L. Miraglia, J. Meltzer, D. Garza, G. W. Chirn, E. McWhinnie, D.
Cohen, J. Skelton, R. Terry, Y. Yu, D. Bodian, F. P. Buxton, J. Zhu, C. Song,
and M. A. Labow. 2003. Identification of a family of cAMP response ele-
ment-binding protein coactivators by genome-scale functional analysis in
mammalian cells. Proc. Natl. Acad. Sci. USA 100:12147–12152.
15. Ito, A., C. H. Lai, X. Zhao, S. Saito, M. H. Hamilton, E. Appella, and T. P.
Yao. 2001. p300/CBP-mediated p53 acetylation is commonly induced by
p53-activating agents and inhibited by MDM2. EMBO J. 20:1331–1340.
16. Jeang, K. T., C. Z. Giam, F. Majone, and M. Aboud. 2004. Life, death, and
Tax: role of HTLV-1 oncoprotein in genetic instability and cellular trans-
formation. J. Biol. Chem. 279:31991–31994.
17. Jiang, H., H. Lu, R. L. Schiltz, C. A. Pise-Masison, V. V. Ogryzko, Y.
Nakatani, and J. N. Brady. 1999. PCAF interacts with tax and stimulates tax
transactivation in a histone acetyltransferase-independent manner. Mol.
Cell. Biol. 19:8136–8145.
18. Jin, D. Y., and K. T. Jeang. 1997. HTLV-1 Tax self-association in optimal
trans-activation function. Nucleic Acids Res. 25:379–388.
19. Jin, D. Y., F. Spencer, and K. T. Jeang. 1998. Human T-cell leukemia virus
type I oncoprotein Tax targets the human mitotic checkpoint protein MAD1.
20. Jin, D. Y., V. Giordano, K. V. Kibler, H. Nakano, and K. T. Jeang. 1999. Role
of adaptor function in oncoprotein-mediated activation of NF-?B: HTLV-1
Tax interacts directly with I?B kinase ?. J. Biol. Chem. 274:17402–17405.
21. Jin, D. Y., H. L. Wang, Y. Zhou, A. C. S. Chun, K. V. Kibler, Y. D. Hou, H.
Kung, and K. T. Jeang. 2000. Hepatitis C virus core protein-induced loss of
LZIP function correlates with cellular transformation. EMBO J. 19:729–740.
22. Kashanchi, F., J. F. Duvall, R. P. S. Kwok, J. R. Lundblad, R. H. Goodman,
and J. N. Brady. 1998. The coactivator CBP stimulates human T-cell lym-
photropic virus type 1 Tax transactivation in vitro. J. Biol. Chem. 273:34646–
23. Kashanchi, F., and J. N. Brady. 2005. Transcriptional and post-transcrip-
tional gene regulation of HTLV-1. Oncogene 24:5938–5951.
24. Kimzey, A. L., and W. S. Dynan. 1998. Specific regions of contact between
human T-cell leukemia virus type I Tax protein and DNA identified by
photocross-linking. J. Biol. Chem. 273:13768–13775.
25. Koga, H., T. Ohshima, and K. Shimotohno. 2004. Enhanced activation of
Tax-dependent transcription of human T-cell leukemia virus type I
(HTLV-1) long terminal repeat by TORC3. J. Biol. Chem. 279:52978–52983.
7058SIU ET AL.J. VIROL.
26. Koo, S. H., L. Flechner, L. Qi, X. Zhang, R. A. Screaton, S. Jeffries, S. Download full-text
Hedrick, W. Xu, F. Boussouar, P. Brindle, H. Takemori, and M. Montminy.
2005. The CREB coactivator TORC2 is a key regulator of fasting glucose
metabolism. Nature 437:1109–1111.
27. Kwok, R. P., M. E. Laurance, J. R. Lundblad, P. S. Goldman, H. M. Shih,
L. M. Connor, S. J. Marriott, and R. H. Goodman. 1996. Control of cAMP-
regulated enhancers by the viral transactivator Tax through CREB and the
co-activator CBP. Nature 380:642–646.
28. Lemasson, I., N. J. Polakowski, P. J. Laybourn, and J. K. Nyborg. 2004.
Transcription regulatory complexes bind the human T-cell leukemia virus 5?
and 3? long terminal repeats to control gene expression. Mol. Cell. Biol.
29. Lundblad, J. R., R. P. Kwok, M. E. Laurance, M. S. Huang, J. P. Richards,
R. G. Brennan, and R. H. Goodman. 1998. The human T-cell leukemia
virus-1 transcriptional activator Tax enhances cAMP-responsive element-
binding protein (CREB) binding activity through interactions with the DNA
minor groove. J. Biol. Chem. 273:19251–19259.
30. Ma, K., J. K. Chan, G. Zhu, and Z. Wu. 2005. Myocyte enhancer factor 2
acetylation by p300 enhances its DNA binding activity, transcriptional activity,
and myogenic differentiation. Mol. Cell. Biol. 25:3575–3582.
31. Madison, D. L., P. Yaciuk, R. P. Kwok, and J. R. Lundblad. 2002. Acetyla-
tion of the adenovirus-transforming protein E1A determines nuclear local-
ization by disrupting association with importin-?. J. Biol. Chem. 277:38755–
32. Matsuoka, M., and K. T. Jeang. 2005. Human T-cell leukemia virus type I at
age 25: a progress report. Cancer Res. 65:4467–4470.
33. Miyazato, A., S. Sheleg, H Iha, Y. Li, and K. T. Jeang. 2005. Evidence for
NF-?B- and CBP-independent repression of p53’s transcriptional activity by
human T-cell leukemia virus type 1 Tax in mouse embryo and primary
human fibroblasts. J. Virol. 79:9346–9350.
34. Paddison, P. J., A. A. Caudy, and G. J. Hannon. 2002. Stable suppression of
gene expression by RNAi in mammalian cells. Proc. Natl. Acad. Sci. USA
35. Reddy, T. R., H. Tang, X. Li, and F. Wong-Staal. 1997. Functional interac-
tion of the HTLV-1 transactivator Tax with activating transcription factor-4
(ATF4). Oncogene 14:2785–2792.
36. Roeder, R. G. 2005. Transcriptional regulation and the role of diverse coac-
tivators in animal cells. FEBS Lett. 579:909–915.
37. Screaton, R. A., M. D. Conkright, Y. Katoh, J. L. Best, G. Canettieri, S.
Jeffries, E. Guzman, S. Niessen, J. R. Yates III, H. Takemori, M. Okamoto,
and M. Montminy. 2004. The CREB coactivator TORC2 functions as a
calcium- and cAMP-sensitive coincidence detector. Cell 119:61–74.
38. Shaw, R. J., K. A. Lamia, D. Vasquez, S. H. Koo, N. Bardeesy, R. A. DePinho,
M. Montminy, and L. C. Cantley. 2005. The kinase LKB1 mediates glucose
homeostasis in liver and therapeutic effects of metformin. Science 310:1642–
39. Tie, F., N. Adya, W. C. Greene, and C. Z. Giam. 1996. Interaction of the
human T-lymphotropic virus type 1 Tax dimer with CREB and the viral
21-base-pair repeat. J. Virol. 70:8368–8374.
40. Yang, X. J., V. V. Ogryzko, J. Nishikawa, B. H. Howard, and Y. Nakatani.
1996. A p300/CBP-associated factor that competes with the adenoviral
oncoprotein E1A. Nature 382:319–324.
41. Zhao, L. J., and C. Z. Giam. 1991. Interaction of the human T-cell lympho-
trophic virus (HTLV) type I transcriptional activator Tax with cellular fac-
tors that bind specifically to the 21-base-pair repeats in the HTLV-1 en-
hancer. Proc. Natl. Acad. Sci. USA 88:11445–11449.
42. Zhou, H. J., C. M. Wong, J. H. Chen, B. Q. Qiang, J. G. Yuan, and D. Y. Jin.
2001. Inhibition of LZIP-mediated transcription through direct interaction
with a novel host cell factor-like protein. J. Biol. Chem. 276:28933–28938.
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