Hepatitis C virus core protein regulates p300/CBP co-activation function. Possible role in the regulation of NF-AT1 transcriptional activity

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Virology 328 (20

Hepatitis C virus core protein regulates p300/CBP co-activation function.

Possible role in the regulation of NF-AT1 transcriptional activity

Marta Gomez-Gonzaloa, Ignacio Benedictoa, Marta Carreteroa, Enrique Lara-Pezzia,

Alejandra Maldonado-Rodrıgueza, Ricardo Moreno-Oterob,

Michael M.C. Laic, Manuel Lopez-Cabreraa,*

aUnidad de Biologıa Molecular, Hospital Universitario de la Princesa, 28006 Madrid, SpainbUnidad de Hepatologıa, Hospital Universitario de la Princesa, 28006 Madrid, Spain

cDepartment of Molecular Microbiology and Immunology, University of Southern California, Keck School of Medicine, Los Angeles, CA 90033-1054, USA

Received 10 March 2004; returned to author for revision 25 March 2004; accepted 30 June 2004

Available online 21 August 2004

Abstract

Hepatitis C virus (HCV) core is a viral structural protein; it also participates in some cellular processes, including transcriptional

regulation. However, the mechanisms of core-mediated transcriptional regulation remain poorly understood. Oncogenic virus proteins often

target p300/CBP, a known co-activator of a wide variety of transcription factors, to regulate the expression of cellular and viral genes. Here

we demonstrate, for the first time, that HCV core protein interacts with p300/CBP and enhances both its acetyl-transferase and transcriptional

activities. In addition, we demonstrate that nuclear core protein activates the NH2-terminal transcription activation domain (TAD) of NF-AT1

in a p300/CBP-dependent manner. We propose a model in which core protein regulates the co-activation function of p300/CBP and activates

NF-AT1, and probably other p300/CBP-regulated transcription factors, by a novel mechanism involving the regulation of the acetylation state

of histones and/or components of the transcriptional machinery.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Hepatitis C virus; Core protein; p300/CBP; HAT activity; NF-AT; Transcriptional activation domain

Introduction

Hepatitis C virus (HCV) infection is the most frequent

cause of chronic liver disease in Western countries and 20–

30% of these patients will develop cirrhosis with the risk of

hepatocellular carcinoma. The genome of HCV is composed

of a positive-stranded RNA of 9.6 kb encoding a poly-

protein, of approximately 3010 amino acids, which is

processed by viral and cellular proteases to render 10

proteins (Suzuki et al., 1999). HCV core protein spans the

first 191 amino acids of the polyprotein (Lai and Ware,

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

doi:10.1016/j.virol.2004.06.044

* Corresponding author. Unidad de Biologıa Molecular, Hospital

Universitario de la Princesa, C/Diego de Leon, 62, 28006 Madrid, Spain.

Fax: +34 91 5202374.

E-mail address: [email protected] (M. Lopez-Cabrera).

2000). This immature core protein is further processed at its

COOH-terminal region to render the mature form of 173

amino acids, which shapes the viral nucleocapsid (Yasui

et al., 1998). Analysis of the subcellular localization of core

protein has provided interesting but discrepant results. It has

been shown that immature (1–191) and mature (1–173) core

proteins localize in the cytoplasm, associated with endo-

plasmic reticulum, mitochondria and/or lipid droplets

(Barba et al., 1997; Liu et al., 1997; Marusawa et al.,

1999; Okuda et al., 2002). In addition, other authors have

also described a nuclear localization of the mature and

COOH-terminal truncated forms of core protein (Sabile

et al., 1999; Watashi et al., 2001; Yamanaka et al., 2002b;

Yasui et al., 1998). There are three nuclear localization

signals in the NH2-terminal region of core protein that could

be involved in nuclear translocation (Chang et al., 1994). On

04) 120–130

M. Gomez-Gonzalo et al. / Virology 328 (2004) 120–130 121

the other hand, the hydrophobic COOH-terminal domain of

core appears to be responsible for its cytoplasmic retention

(Liu et al., 1997). In some instances, the function of core

and its subcellular localization have been associated

(Watashi et al., 2001; Yamanaka et al., 2002a).

Besides its structural function, core protein also regulates

different cellular processes. In this context, it has been

shown that core protein expression activates several signal

transduction pathways, leading to activation of transcription

factors such as Elk-1, AP1, Sp1, and Egr-1 (Giambartolomei

et al., 2001; Kato et al., 2000; Lee et al., 2001; Shrivastava

et al., 1998). Other transcription factors, such as p53 and

RXR, are activated by core via mechanisms involving

protein–protein interactions (Otsuka et al., 2000; Tsutsumi

et al., 2002). Modulation of these and other cellular

components by core protein alters the gene expression

pattern and affects important cellular processes, including

apoptosis, cell growth, and transformation (Cho et al., 2001;

Hahn et al., 2000; Machida et al., 2001; Moriya et al., 1998;

Ray et al., 1998, 2000; Ruggieri et al., 1997; Zhu et al.,

1998).

Specific transcription involves the assembly of a multi-

protein complex at the gene enhancer/promoter regions,

which is composed of specific transcription factors, general

transcription machinery and co-activators such as p300/CBP

among others. p300/CBP functions as a bridge due to its

ability to interact both with transcription factors and the

basal transcription machinery (Chan and La-Thangue, 2001;

Goodman and Smolik, 2000; Shikama et al., 1997). In

addition, p300/CBP has histone acetyltransferase activity,

which results in relaxation of chromatin structure to

facilitate the transcription process (Chan and La-Thangue,

2001). More recently, it has been described that other

proteins involved in transcription may also be targets of this

acetyltransferase activity (Barlev et al., 2001; Boyes et al.,

1998; Imhof et al., 1997; Lu et al., 2003; Martinez-Balbas et

al., 2000). Many regulatory proteins of oncogenic viruses

target p300/CBP to control the expression of viral and

cellular genes (Goodman and Smolik, 2000); however, this

mechanism of transcriptional regulation has not been

explored so far for HCV core protein.

Although HCV is mainly hepatotropic, it has been shown

that this virus may also infect cells of the immune system

(Lerat et al., 1998; Sung et al., 2003). Thus, it is conceivable

that HCV may also induce in these cells the expression of

molecules involved in the immune and inflammatory

responses. In this regard, it has been shown that core protein

activates the IL-2 promoter and nuclear factor of activated T

cells (NF-AT)-dependent transcription in T lymphocytes

(Bergqvist and Rice, 2001), by a mechanism involving

cytosolic calcium mobilization and requiring the C-terminal

hydrophobic portion of core (Bergqvist et al., 2003).

NF-AT is a family of transcription factors that includes

four related members, NF-AT 1–4. In resting cells, NF-AT

proteins are phosphorylated in the NF-AT homology region

and retained in the cytoplasm. When cells are activated by

calcium mobilization stimuli, the calcium/calmodulin-

dependent phosphatase calcineurine is activated and

dephosphorylates NF-AT proteins (Rao et al., 1997). Then,

NF-AT proteins translocate to the nucleus where they bind

to their specific DNA sequences either alone or coopera-

tively with transcription factors of the AP-1 family to form

composite NF-AT:AP-1 sites (Rao et al., 1997). Once NF-

AT proteins have targeted their recognition DNA sequen-

ces, they are further functionally regulated by interactions

with transcriptional co-activators and by posttranslational

modifications (Avots et al., 1999; Garcıa-Rodrıguez and

Rao, 1998, 2000; San-Antonio et al., 2002).

NF-AT1 is the founding member of the NF-AT family

and is expressed in several cells of the immune system as

well as in other nonimmune tissues (Rao et al., 1997). In

addition, NF-AT1 is the only family member that is

expressed in resting T lymphocytes (Lyakh et al., 1997). It

contains a strong acidic transcription activation domain

(TAD) in the first 100 amino acids of the protein, not

conserved in other family members (Rao et al., 1997),

which is bound and regulated by the co-activator p300/CBP

(Garcıa-Rodrıguez and Rao, 1998).

The aim of this study was to determine whether HCV

core was a p300/CBP-regulating protein. Herein, we show

that nuclear core protein interacts with p300/CBP, regulates

both its transcriptional and histone acetyltransferase activi-

ties, and cooperates with calcium-mobilizing stimuli to

further induce NF-AT-mediated transcription and the acti-

vity of NF-AT1-TAD, apparently through the activation of

p300/CBP function.

Results

Direct interaction of core protein with the co-activator

p300/CBP

The co-activators p300 and CBP are highly related

proteins that present conserved domains and functions, thus

hereafter they will be described as p300/CBP, unless

otherwise specified. To determine whether core protein

interacts with p300/CBP, a GST-based pull-down assay

was performed. The fusion protein GST-core 1–191, but not

GST, interacted with endogenous p300 from a cellular lysate

of HEK293 cells (Fig. 1A). To study the in vivo interaction

of these proteins, HEK293 cells were co-transfected with the

expression vector pEF-core 1–153, encoding nuclear core

protein (data not shown), along with pCI-Flag-p300, encod-

ing Flag-tagged p300, or the control empty vector. Lysates

from transfected cells were immunoprecipitated using an

anti-p300 mAb and immunoblotted with anti-core or anti-

Flag mAbs. The amount of core protein in the precipitates

increased dramatically when cells were co-transfected with

both expression plasmids (Fig 1B).

To identify the region of p300/CBP involved in the

interaction with core protein and to determine whether the

Fig. 1. Direct interaction of core protein with the co-activator p300/CBP. (A) Pull down assay with GST-core 1–191 or GST and a cellular lysate of HEK293

cells. Bound proteins were subjected to Western blot analysis using an anti-p300 mAb. Coomassie brilliant blue staining of 1/10 of the GST proteins used

shows full-length and degradation products of the chimeric protein (bottom). (B) Cellular lysates of HEK293 cells co-transfected with 5 Ag of pEF-core 1–153

and 5 Ag of pCI-Flag-p300 or a control vector were immunoprecipitated with an anti-p300 mAb. Immunoprecipitates and cellular lysates were analyzed using

anti-core mAb and anti-Flag M2 mAb. (C) Pull-down assay with different GST-CBP constructs and in vitro translated 35S-labeled core protein 1–191 (top).

One microliter of 35S-labeled core protein was run as a control (input). Coomassie brilliant blue staining shows 1/10 of the GST proteins used (middle).

Schematic representation of the CBP constructs employed and summary of the core-binding activity observed (bottom). p300/CBP domains: RID, nuclear

hormone receptors interaction domain; CH1/2/3, cysteine/histidine rich domains 1/2/3; KIX, kinase inducible domain/CREB-binding domain; Br,

bromodomain; HAT, histone acetyltransferase domain.

M. Gomez-Gonzalo et al. / Virology 328 (2004) 120–130122

interaction could be direct, a GST-based pull-down assay

was performed, employing different GST-CBP constructs

and in vitro-translated 35S-core 1–191 protein. GST-fused

sequences 1098–1877, 1098–1620, and 1679–1858 of CBP

interacted with 35S-core 1–191, whereas no interaction was

observed with the NH2-terminal fragment of CBP spanning

amino acids 1 to 1099 (Fig. 1C). This result suggests that

the core–CBP interaction is direct. However, the possibility

that a protein of reticulocyte lysate, employed for in vitro

translation, is mediating the interaction cannot be excluded.

Sequence alignment of p300 and CBP showed that the

regions of CBP involved in the interaction with core

protein are highly conserved in p300 protein (data not

shown).

Core protein induces the transcriptional and histone

acetyltransferase activities of p300/CBP

The transcriptional activity of the co-activator p300/CBP

may be studied by the Gal4 reporter system using a chimeric

M. Gomez-Gonzalo et al. / Virology 328 (2004) 120–130 123

Gal4-CBP construct (Chawla et al., 1998). To determine

whether the core-p300/CBP interaction affected the tran-

scriptional activity of the co-activator, Jurkat cells were co-

transfected with the reporter plasmid pGal4-Luc along with

the expression vectors pGal4-CBP and pEF-core 1–153, or

the control plasmids. Co-expression of nuclear core protein

increased the transcriptional activity of Gal4-CBP, with

activations up to fivefold over the values obtained in the

absence of the viral protein (Fig. 2A). These results suggest

that nuclear core protein interacts with p300/CBP and

induces its transcriptional activity.

Given that the region of p300/CBP that interacted with

core contained the histone acetyltransferase domain (HAT)

(Fig. 1C), the effect of core protein on the HAT activity

was analyzed. To this end, HEK293 cells were co-

transfected with the expression vectors pCI-Flag-p300

and pEF-core 1–153 or the control empty vector pEF1a,

and then Flag-p300 was immunoprecipitated with anti-

Flag mAb and used for in vitro acetylation assay. As

Fig. 2. Core protein induces the transcriptional and histone acetyltransferase

activities of p300/CBP. (A) Jurkat cells were co-transfected with 0.5 Ag of

pGAL4-Luc along with 1 Ag of either pGal4-CBP or pRSV-Gal4-DBD and

2 Ag of pEF-core 1–153 or pEF1a. Luciferase activity is represented as

mean fold induction over the value obtained in cells transfected with pRSV-

Gal4-DBD and pEF1a and only the values of pGal4-CBP-transfected cells

are shown. Values shown in this and following luciferase activity figures

represent the mean fold induction F SE of at least three independent

experiments. (B) Cellular lysates of HEK293 cells co-transfected with 5 Agof pCI-Flag-p300 and 5 Ag of either pEF-core 1–153 or pEF1a were

immnunoprecipitated with anti-Flag M2 mAb and immunoprecipitates were

either employed in in vitro acetylation assays along with purified histones

and [14C]-acetyl coenzyme A (top) or subjected to Western blot analysis

using an anti-Flag M2 mAb (bottom).

shown in Fig. 2B, the ability of Flag-p300 to acetylate

histones was increased when core 1–153 was co-

expressed. Similar levels of Flag-p300 were immunopre-

cipitated both in the presence and absence of core protein

(lower panel).

Nuclear forms of core protein induce NF-AT-dependent

transcription

The ability of core protein to induce the IL-2 promoter

in cooperation with different stimuli is abolished by

deletion of its COOH-terminal domain (Bergqvist and

Rice, 2001), which suggests that core function is dependent

of its subcellular localization. To investigate whether this

domain of core was required for cooperative activation of

NF-AT-dependent transcription, Jurkat cells were co-trans-

fected with pNF-AT-Luc, containing three copies of the

composite NF-AT/AP-1 site of the human IL-2 enhancer

element, along with expression vectors coding for full-

length core (pEF-core 1–191) or COOH-terminal truncated

core proteins (pEF-core 1–173 and pEF-core 1–153),

which localize predominantly in the cytoplasmic extract

and in the nuclear insoluble fraction, respectively (data not

shown). The transfected cells were stimulated either with

PMA, to activate only the AP-1 element of the composite

NF-AT/AP-1 site, or with PMA plus calcium ionophore

(hereafter PMA + Io), which provides a full stimulus for

this composite site. As expected, PMA + Io induced the

reporter plasmid pNF-AT-Luc up to 30-fold, whereas PMA

alone was not sufficient to activate this construct (Fig 3A).

Only core protein 1–191 cooperated with PMA treatment,

with activations of eight- to ninefold. In contrast, both full-

length and truncated core proteins (core 1–173 and core 1–

153) exerted a cooperative effect with PMA + Io in the

activation of the NF-AT enhancer (Fig. 3A). No effect of

core protein was observed in the absence of cellular

activation (data not shown). In summary, these results

indicate that nuclear accumulation of core protein,

observed even in core 1–191-transfected cells, synergize

with PMA + Io to further induce NF-AT-dependent

transcription.

To analyze whether core protein was able to induce the

NF-AT1 transactivation domain (TAD) activity once this

factor had targeted its recognition sequences, a Gal4-derived

reporter system was used. Jurkat cells were co-transfected

with the chimeric vectors pGal4-NF-AT1 (1–415), pGal4-

NF-AT1 (1–171), or the parental vector pRSV-Gal4-DBD,

along with the reporter plasmid pGal4-Luc and either pEF-

core 1–191 or the control vector pEF1a. Both chimeric

proteins Gal4-NF-AT1 (1–415) and (1–171) contained the

NH2-terminal TAD of NF-AT1 and activated the reporter

plasmid pGal4-Luc (Fig. 3B). As expected, core 1–191 did

not stimulate transcription when co-transfected with the

control plasmid pRSV-Gal4-DBD (Fig. 3B). In contrast,

the transcription of pGal4-Luc was induced by the full-

length core protein when co-transfected with both Gal4-

Fig. 3. Nuclear forms of core protein induce NF-AT-dependent transcription. (A) Jurkat cells were transfected with 1 Ag of pNF-AT-Luc along with 2 Ag of

pEF-core 1–191, pEF-core 1–173, pEF-core 1–153, or the empty vector pEF1a. Then, cells were either left untreated or stimulated with PMA or PMA/Io.

Luciferase activity is represented as mean fold induction over the value obtained in untreated cells transfected with pEF1a. (B) Jurkat cells were co-transfected

with 0.5 Ag of pGAL4-Luc along with 1 Ag of either pGal4-NF-AT1 1–415, pGal4-NF-AT1 1–171 or pRSV-Gal4-DBD, and 2 Ag of pEF-core 1–191 or pEF1a.Luciferase activity is represented as mean fold induction over the value obtained in cells transfected with pRSV-Gal4-DBD and pEF1a. (C) Luciferase assay

similar to that described in B in which expression vectors employed were pGal4-NF-AT1 1–171 and pEF-core 1–191, pEF-core 1–173, pEF-core 1–153, or

pEF1a. The luciferase activity is represented as mean fold induction by core proteins over the value obtained in cells transfected with pGal4-NF-AT1 1–171

and pEF1a. *P b 0.05, by using repeated measures ANOVA test. (D) Luciferase assay similar to that described in C in which expression vectors employed

were pGal4-NF-AT1 1–171 and pEF-core 1–153 or pEF-core 1–153-ER. **P b 0.05, by using paired t test. The subcellular distribution of core 1–153 and core

153-ER proteins in HEK293 cells is also shown. (E) Jurkat cells were co-transfected with 0.5 Ag of pGAL4-Luc along with 1 Ag of pGal4-NF-AT1 1–171, 2 Agof either pEF-core 1–153 or pEF1a, and 2 Ag of either pE1A or the empty control vector. Luciferase activity is represented as mean fold induction by core

protein over the values obtained in cells transfected with pGal4-NF-AT1 1–171 and pEF1a F E1A.

M. Gomez-Gonzalo et al. / Virology 328 (2004) 120–130124

NF-AT1 chimeric proteins, suggesting that the viral protein

was able to induce the NF-AT1-TAD activity (Fig. 3B).

Interestingly, when the truncated forms of core (1–173 and

1–153) were included in the Gal4 reporter assays, statisti-

cally significant higher transactivation abilities of these

mutants, compared with core 1–191, were observed (Fig.

3C). In addition, a construct containing core 1–153 fused

to an endoplasmic reticulum signal peptide (core 1–153-

ER), which showed a partial decrease of nuclear accumu-

lation, displayed a statistically significant parallel reduction

of its ability to induce the NF-AT1-TAD (Fig. 3D). To

analyze whether the effect of nuclear core protein on NF-

AT1 transcription depended on p300/CBP function, the

Gal4 reporter assay was performed in the presence of the

p300/CBP-inhibiting adenoviral protein E1A. As shown in

Fig. 3E, E1A protein inhibited the core-protein-mediated

induction of Gal4-NF-AT1 (1–171) transcriptional activity,

suggesting that p300/CBP might mediate transactivation

function of core.

Core protein interacts indirectly with the NH2-terminal

transactivation domain of NF-AT1

To analyze whether core protein was able to interact with

the NH2-terminal transactivation domain of NF-AT1 (NF-

AT1-TAD), a GST-based pull-down assay was performed.

HEK293 cells were transfected with pEF-core 1–191 and

cellular lysates were used in pull-down assays with different

HA-tagged NF-AT1 fragments fused to GST. As shown in

Fig. 4A, core protein interacted with the first 68 amino

acids of NF-AT1, which are located within the NH2-

terminal TAD of this transcription factor. No interaction

was observed when the control proteins GST or GST-HA

were employed in the pull-down assay. A similar binding

was observed when the pull-down assay was performed

with GST-core 1–191 or 1–153 constructs and cellular

lysates of HEK293 cells transfected with pEF-HA-NF-AT1

(1–384) (Fig. 4B). To determine whether core–NF-AT1

interaction was direct, a pull-down assay was performed

Fig. 4. Core protein interacts indirectly with the NH2-terminal transactivation domain of NF-AT1. (A) Pull-down assays using different GST-HA-NF-AT1

constructs and cellular lysates of HEK293 cells transfected with pEF-core 1–191. Coomassie brilliant blue staining shows 1/10 of the GST proteins used

(bottom). (B) Pull-down assay in which GST-core 1–191 and 1–153 were assayed to bind to HA-NF-AT1 1–384 expressed in HEK293 cells. (C) Pull-down

assay with GST-HA-NFAT1 1–384 or GST and in vitro translated 35S-labeled core protein 1–191, either in the presence or absence of the cross-linker DSP. One

microliter of 35S-labeled core protein was run as a control (input).

M. Gomez-Gonzalo et al. / Virology 328 (2004) 120–130 125

using in vitro-translated 35S-labeled core protein 1–191 and

GST-NF-AT1 (1–384). As shown in Fig. 4C, in vitro-

translated core protein did not bind to NF-AT1 unless the

binding was forced in the presence of the thiol-cleavable

cross-linker DSP. In agreement with these results, the binding

of core to NF-AT1 could not be observed in vivo by co-

immunoprecipitation assays (data not shown). Taken as a

whole, these results suggested that the induction of the

transcriptional activity NF-AT1-TAD by core protein was not

mediated by a direct interaction between these two proteins.

Deacetylases inhibitors induce the NF-AT-dependent

transcription and mask nuclear core protein function

To analyze whether acetylation might regulate NF-

AT1-dependent transcription, the activity of Gal4-NF-AT1

(1–171) was assessed in the presence of increasing

amounts of the deacetylases inhibitor sodium butyrate.

The activity of NF-AT1-TAD increased in a dose-depend-

ent manner by sodium butyrate (Fig. 5A, left panel).

Similar activation of NF-AT1-TAD was observed with

another deacetylases inhibitor, trichostatin A (Fig. 5A,

central panel). In addition, sodium butyrate further induced

the expression of the reporter plasmid pNF-AT-Luc over

the values obtained with PMA + Io stimulation (Fig. 5A,

right panel). These results suggest that NF-AT1-dependent

transcription is regulated by acetylation of histones and/or

components of the transcriptional machinery.

Interestingly, when the effect of nuclear core 1–153 was

analyzed on sodium butyrate-induced Gal4-NF-AT1 (1–171)

activity, a gradual mask of core function was observed as

the concentration of sodium butyrate increased (Fig. 5A,

left panel). It is important to note that the cooperative

effect of core and sodium butyrate in the activation of NF-

AT1-TAD was synergistic at low concentration of the

inhibitor (0.5mM) and tended to be additive as the

concentration of sodium butyrate increased. In addition,

core 1–153 hardly increased TSA-induced Gal4-NF-AT1

(1–171) activity (Fig. 5A, central panel) or the expression

of pNF-AT-Luc induced with PMA + Io and sodium

butyrate (Fig. 5A, right panel). To rule out that the

apparent loss of core function was due to a reduction of

core protein expression or to a change in its subcellular

localization, Jurkat cells were transfected with pEF-core 1–

153 and treated with sodium butyrate. Similar expression

levels and subcellular localization of core 1–153 were

observed both with and without sodium butyrate (data not

shown). In addition, core–CBP interaction was maintained

in the presence of sodium butyrate and only a marginal

decrease in core–NF-AT1 interaction was observed, which

would not explain the mask of core protein function by

deacetylase inhibitors (Fig. 5B). Interestingly, the coopera-

tive activity of PMA and core 1–191 was not blocked by

sodium butyrate (Fig. 5C), suggesting that the masking

effect of the deacetylases inhibitors was specific for the

nuclear function of core. These results, in addition to those

Fig. 5. Deacetylases inhibitors induce the NF-AT-dependent transcription and mask nuclear core protein function. (A) Jurkat cells were co-transfected with 0.5

Ag of pGAL4-Luc along with 1 Ag of pGal4-NF-AT1 1–171 and 2 Ag of pEF1a or pEF-core 1–153. Cells were either left untreated or stimulated with

increasing amounts of the deacetylases inhibitor sodium butyrate (left) or with another deacetylases inhibitor, trichostatin A (TSA) (middle). Luciferase activity

is represented as mean fold induction over the value obtained in untreated cells. Jurkat cells were also co-transfected with 1 Ag of pNF-AT-Luc along with 2 Agof the control vector pEF1a or pEF-core 1–153 and either left untreated or stimulated with sodium butyrate and/or PMA/Io (right). Luciferase activity is

represented as mean fold induction over the value obtained in PMA/Io-treated cells. Numbers above the graphics indicate the mean fold induction by core

protein over the values obtained in cells transfected with pGal4-NF-AT1 1–171 and pEF1a F deacetylase inhibitors (left and middle) or the mean fold

induction by core protein over the values obtained in PMA/Io-treated cells F sodium butyrate (right). (B) Pull-down assays using GST-HA-NF-AT1 and GST-

CBP constructs and cellular lysates of HEK293 cells transfected with pEF-core 1–153 and either left untreated or stimulated with 2.5 mM sodium butyrate for

16 h. (C) Jurkat cells were co-transfected with 1 Ag of pNF-AT-Luc along with 2 Ag of pEF-core 1–191 or pEF1a. Cells were either left untreated or stimulated

with sodium butyrate and/or PMA. Luciferase activity is represented as mean fold induction over the value obtained in PMA-treated cells.

M. Gomez-Gonzalo et al. / Virology 328 (2004) 120–130126

shown in Figs. 2 and 3, suggest that nuclear core protein

might activate NF-AT1-dependent transcription by activa-

tion of the acetyltransferase activity of p300/CBP, which

results in the acetylation of histones and/or nonidentified

component(s) of the transcriptional complex.

Discussion

During the last few years, acetylation has emerged as an

important posttranslational modification to regulate the

function of proteins involved in transcriptional control,

including general and specific transcription factors (Barlev et

al., 2001; Boyes et al., 1998; Imhof et al., 1997; Lu et al.,

2003; Martinez-Balbas et al., 2000). The co-activator

p300/CBP has intrinsic histone acetyltransferase (HAT)

activity, which regulates not only the chromatin structure

but also the activity of numerous proteins of the transcription

complex. It has been shown that the HAT activity of p300/

CBP may be regulated by different cellular factors and by

several viral proteins (Ait-Si-Ali et al., 1998; Deng et al.,

2001; Hamamori et al., 1999; Soutoglou et al., 2001; Zhao

et al., 2003). Therefore, this co-activator should not be

considered as a mere component of the transcriptional

complex, whose only regulatory function is to bridge

specific transcription factors with the basal transcriptional

machinery.

In this study, we demonstrate that HCV core protein

interacts both in vivo and in vitro with p300/CBP and

induces its transcriptional and HAT activities. Furthermore,

we demonstrate that core protein activates the transcriptional

activity of the p300/CBP-regulated protein NF-AT1, once

this transcription factor has targeted its recognition sequen-

ces in the nucleus.

M. Gomez-Gonzalo et al. / Virology 328 (2004) 120–130 127

Other viral proteins known to induce NF-AT1 activity

through a nuclear localization are Tat of HIV-1 (Macian and

Rao, 1999) and HBx of HBV (Carretero et al., 2002). These

two viral proteins induce the NF-AT1-TAD activity by a

direct protein–protein interaction. In contrast, the interaction

of core with NF-AT1 did not appear to be direct. In an

attempt to identify the mechanism by which core protein

induces the NF-AT1-TAD activity, we employed the

adenoviral protein E1A 12S, which inhibits the activity of

p300/CBP by interacting with and inhibiting its HAT

activity (Goodman and Smolik, 2000; Hamamori et al.,

1999) and our results suggested that core protein might

enhance NF-AT1 activity by regulating p300/CBP function

and the acetylation state of histones and/or components of

the transcriptional machinery. First, the HAT activity of

p300 is increased in the presence of core protein. And

second, there is a gradual mask of nuclear, but not of

cytoplasmic, core function in the presence of deacetylases

inhibitors. Given that the acetylation state of cellular

proteins is the result of counteracting activities achieved

by acetyltransferases and deacetylases, an increase in

acetylation level may be obtained by stimulating acetyl-

transferases or by inhibiting deacetylases. Therefore, it can

be hypothesized that core protein is not able to further

increase the acetylation level induced by deacetylases

inhibitors, being the effect of core protein masked by these

agents.

In agreement with our results, it has been recently

reported that the expression of core protein induces the

hyperacetylation of p53 at Lys373 and Lys382 residues,

which are specific targets for p300/CBP HAT activity,

leading to enhanced DNA-binding activity of this tran-

scription factor (Kao et al., 2004). Interestingly, the Lys320

residue of p53, which is acetylated by PCAF, is not affected

by core expression (Kao et al., 2004). Our results provide

evidence, for the first time, that both NF-AT-dependent

transcription and NF-AT1-TAD activity are regulated by

acetylation. It would be worth to analyze whether NF-AT1

and/or other proteins involved in the transcriptional complex

built from NF-AT1 could be the target of this posttransla-

tional modification induced by core protein.

Previous studies have shown that core protein synergize

strongly with incomplete stimuli, which do not mobilize

calcium, to activate IL-2 promoter and NF-AT-dependent

transcription in T lymphocytes, suggesting that core protein

may compensate the calcium signal (Bergqvist and Rice,

2001). In agreement with this observation, expression of

full-length core protein induces an increase in the levels of

cytosolic calcium and spontaneous calcium oscillations in

T cells (Bergqvist et al., 2003). The effect of core on

calcium mobilization appears to be mediated by the

insertion of core in the endoplasmic reticulum (ER)

membrane, through its COOH-terminal hydrophobic

sequence, which causes calcium leakage (Bergqvist et al.,

2003). Herein, we show that both full-length and C-

terminal truncated versions of core protein, which do not

interact with the ER membrane, are able to cooperate with

calcium-mobilizing stimuli to further induce NF-AT-

dependent transcription and to increase the activity of

NF-AT1-TAD. We propose a novel mechanism for

activation of NF-AT1, and probably other transcription

factors, by core, involving the interaction of nuclear core

with and activation of the co-activator p300/CBP. Our

results point to core protein as a new p300/CBP HAT-

inducing protein and add new insight to understand the

transcriptional regulation by HCV in the infected cells.

Materials and methods

Cell culture and plasmid constructs

The human T lymphoma cell line Jurkat and HEK

(human embryonic kidney) 293 cells were grown as

previously described (Carretero et al., 2002).

The expression vector pEF1a has been previously

described as pcDEF (Zhu et al., 1998). The expression

vectors pEF-core 1–191, 1–173, and 1–153 were generated

by subcloning, into pEF1a, PCR fragments encoding amino

acids 1–191, 1–173, and 1–153 of core protein, respec-

tively, from HCV genotype 1b. The expression vector pEF-

core 1–153-ER was obtained by fusing in COOH-terminus

of core 1–153 the endoplasmic reticulum signal peptide

GWSCIILFLVATATGAHS from vector pEF/myc/ER (Invi-

trogen, The Netherlands). The PCR fragment encoding

amino acids 1 to 191 of core protein was cloned into

pcDNA3.1 vector (Invitrogen), and this vector was

employed for in vitro synthesis of core protein. The

plasmids pGST-core 1–191 and 1–153 were constructed

by subcloning into pGEX-2T (Amersham Bioscience, UK),

fragments coding for core 1–191 or 1–153. The plasmid

pCI-Flag-p300, encoding full-length Flag-tagged p300, and

the vectors pGST-CBP 1–1099, 1098–1877, 1098–1620,

and 1679–1858, coding for the indicated CBP protein

amino acid sequences fused to the GST protein, were kindly

provided by Dra. A. Aranda (Instituto de Investigaciones

Biomedicas, Madrid, Spain). The plasmid pGal4-CBP,

coding for a human/mouse CBP chimera fused to the

DNA binding domain (DBD) of yeast Gal4 protein, has

been previously described (Chawla et al., 1998). The

expression vector pE1A, encoding the wild-type E1A 12S

adenoviral protein, was kindly provided by Dr. J.C. Lacal

(Instituto de Investigaciones Biomedicas). The plasmids

pRSVGal4-DBD, pGal4-NF-AT1 1–415, and pGal4-NF-

AT1 1–171 have already been described (Luo et al., 1996)

and were kindly provided by Dr. A. Rao (Harvard Medical

School, Boston, MA). The plasmid pEF-HA-NF-AT1 1–384,

encoding a C-terminal deleted HA-tagged murine NF-AT1,

has been described elsewhere (Carretero et al., 2002). The

plasmids pGST-HA, pGST-HA-NF-AT1 1–68, and pGST-

HA-NF-AT1 1–57 have been previously described (Carre-

tero et al., 2002). The construct pGST-HA-NF-AT1 1–384

M. Gomez-Gonzalo et al. / Virology 328 (2004) 120–130128

was generated by cloning into pGEX-4T-2 (Amersham) a

PCR fragment encoding amino acids 1–384 of murine HA-

tagged NF-AT1. The reporter construct pNF-AT-Luc con-

tains three tandem copies of a composite NF-AT/AP-1

binding site of the human IL-2 promoter fused to the

minimal IL-2 promoter (Durand et al., 1988) and was

provided by Dr. G. Crabtree (Stanford University Medical

School, Stanford, CA). The reporter plasmid pGal4-Luc,

bearing five copies of the Gal4-binding sites upstream of the

luciferase gene, was a kind gift from Dr. R. Perona (Instituto

de Investigaciones Biomedicas, Madrid, Spain).

Transfection and luciferase assay

Jurkat cells were transfected with the indicated plasmids,

employing Lipofectin (Gibco-BRL, Gaithersburg, MD)

according to manufacturer’s recommendations. At 24 h after

transfection, cells were either left untreated or stimulated

with PMA, Io, sodium butyrate, and/or trichostatin A (TSA)

for 12 h. Then, cells were harvested and cellular extracts

were assayed for luciferase activity using a Lumat LB9501

luminometer (Berthold, Wildbad, Germany).

Nuclear and cytoplasmic extracts

Jurkat cells were transfected, by electroporation, with 40

Ag of expression vector pEF-core 1–191, 1–173, or 1–153

using an electrical pulse of 0.28 kV and 1200 micro-

Faradays (AF). At 36 h after transfection, cells were either

lysed with buffer Laemmli or separated into cytoplasmic

and nuclear soluble fractions, employing the NE-PER

Nuclear and Cytoplasmic Extraction Reagents from Pierce

(Rockford, IL). After extraction of the nuclear soluble

fraction, the remaining insoluble material was resuspended

in Laemmli buffer and it is called nuclear-insoluble fraction.

Purity of the different fractions was determined by checking

in these extracts the presence of the cytoplasmic marker

tubulin and of the nuclear marker Sp1, by Western blot

assay, employing an anti-tubulin monoclonal antibody from

Sigma (St. Louis, MO) and an anti-Sp1 polyclonal antibody

from Santa Cruz (Santa Cruz, CA). The presence of core

protein in the different fractions was determined by Western

blot analysis using an anti-core monoclonal antibody (mAb)

from Affinity BioReagents (Goleen, CO).

HEK293 cells were transfected with 10 Ag of expression

plasmid pEF-core 1–153 or pEF-core 1–153-ER, using

FuGENE 6 transfection reagent (Roche, Mannheim, Ger-

many) and 24 h after transfection separated in subcellular

fractions as described above.

Purification of recombinant proteins and pull-down assays

GST fusion proteins were expressed in E. coli strain

BL21 and purified with glutathione-Sepharose 4B (Amer-

sham Pharmacia Biotech, Uppsala, Sweden) as previously

described (Carretero et al., 2002). Coomassie staining of

these preparations frequently showed a ladder pattern of

bands of degradation products or incompletely synthesized

polypeptides. Approximately 10 Ag of GST fusion proteins

immobilized on glutathione–Sepharose 4B were incubated

at 4 8C overnight with total cellular extracts from HEK293

cells or transfected HEK293 cells in lysis buffer containing

0.25% NP-40, 150 mM NaCl, 50 mM Tris–HCl pH 7.4, 4

Ag/ml aprotinin, 4 Ag/ml leupeptin, and 2 mM PMSF.

When indicated, transfected HEK293 cells were either left

untreated or stimulated with 2.5mM sodium butyrate for 16

h before lysis. Bound proteins were analyzed by Western

blot using anti-p300 mAb (Oncogene, Boston, MA), anti-

core mAb (Affinity BioReagents), or anti-HA 12CA5 mAb

(Roche). For direct interaction assays, GST proteins were

incubated with 15 Al of in vitro translated 35S-labeled core

protein 1–191, which was synthesized using TnT Coupled

Reticulocyte Lysate Systems (Promega, Madison, USA).

Binding was performed for 2 h at 4 8C in binding buffer

containing 0.25% NP-40, 150 mM NaCl, 50 mM Tris–HCl

pH 7.4, 4 Ag/ml aprotinin, 4 Ag/ml leupeptin and 2 mM

PMSF. When indicated, binding reactions were performed

in the presence of 30 mM thiol-cleavable cross-linker DSP

(dithiobis succinimidyl propionate) from Pierce. After

washing, bound proteins were fractionated by SDS-PAGE.

Radioactive signal was increased by incubating fixed gel

with Amplify reagent (Amersham Pharmacia Biotech) and

visualized by autoradiography.

Co-immunoprecipitation

HEK293 cells were co-transfected with 5 Ag of expres-

sion plasmid pEF-core 1–153 and 5 Ag of expression

plasmids pCI-Flag-p300 or the empty vector, using FuGENE

6 transfection reagent (Roche). Cells were lysed in buffer

containing 150 mM NaCl, 50 mM Tris–HCl pH 7.4, 0.25%

NP-40, 1 mM PMSF, 1 mM EGTA, 1 Ag/ml aprotinin, and 1

Ag/ml leupeptin, and precleared with protein A-Sepharose

(Amersham Pharmacia Biotech). Precleared lysates were

then immunoprecipitated with anti-p300 mAb from Onco-

gene. Bound proteins were separated on a SDS-polyacryla-

mide gel and analyzed by Western blotting using anti-Flag

M2 mAb (Sigma) and anti-core mAb.

Histone acetylation assays

HEK293 cells were co-transfected with 5 Ag of pCI-

Flag-p300 and 5 Ag of pEF-core 1–153 or pEF1a, using

FuGENE 6 transfection reagent. Cells were lysed and

cellular lysates were immunoprecipitated, as described in

the previous section, with an anti-Flag M2 mAb. Immu-

nocomplexes were then resuspended in acetylation buffer

containing 20 mM HEPES pH 7.4, 1 mM DTT, 10 mM

sodium butyrate, 1 Ag histones (type IIA from Sigma) and

0.5 ACi [14C]-acetyl coenzyme A (Amersham Pharmacia

Biotech) and reactions were performed at 37 8C for 1 h.

Then, samples were run in SDS-PAGE. Radioactive signal

M. Gomez-Gonzalo et al. / Virology 328 (2004) 120–130 129

was increased by incubating fixed gel with Amplify

reagent and visualized by autoradiography.

Acknowledgments

We are very grateful to Drs. A. Aranda, J.C. Lacal, A.

Rao, G. Crabtree, and R. Perona for providing critical

reagents that have made this work possible. The authors are

indebted to Drs. F. Rodrıguez and P. Cameno for critical

discussion and for statistical analysis of the data. This work

was supported by grants from the Ministerio de Ciencia y

Tecnologıa SAF 01/0305 and Fundacion I.C.O. to M.L.C.,

and SAF 2001/1414 to R.M.O. This study was also

supported by grant C03/02 from the Instituto de Salud

Carlos III (A.M.R. received a fellowship ascribed to this

grant). M.G.-G. was supported by grants 08.2/0003/2001

and 08.1/0030/2003 from the Comunidad Autonoma de

Madrid. E.L.-P. had a postdoctoral fellowship from the

Comunidad Autonoma de Madrid.

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