www.elsevier.com/locate/yviro

Virology 322 (2004) 51–60

Hepatitis C virus NS5A mediated STAT3 activation requires

co-operation of Jak1 kinase

Bhaswati Sarcar,a Asish K. Ghosh,a Robert Steele,a Ranjit Ray,b and Ratna B. Raya,b,*

aDepartment of Pathology, Saint Louis University, St. Louis, MO 63104, USAbDepartment of Internal Medicine, Saint Louis University, St. Louis, MO 63104, USA

Received 15 November 2003; returned to author for revision 9 December 2003; accepted 5 January 2004

Abstract

Hepatitis C virus (HCV) is a major etiologic agent for chronic hepatitis worldwide and often leads to cirrhosis and hepatocellular

carcinoma. However, the mechanism for development of chronic hepatitis or hepatocarcinogenesis by HCV remains unclear. Signal

transducers and activators of transcription (STATs) family proteins function as the downstream effectors of cytokine signaling and play a

critical role in cell growth regulation. In many cancers including liver, STAT3 is often constitutively activated, although the mechanism of

persistent activation of STAT3 is unknown. The nonstructural protein 5A (NS5A) encoded from the HCV genome has shown cell growth

regulatory properties. In this study, we have observed that HCV NS5A activates STAT3 phosphorylation, which in turn translocates into the

nucleus. In vivo activation of STAT3 was also observed in the liver of transgenic mice expressing HCV NS5A. Introduction of NS5A in

hepatoma cells modulated STAT3 downstream molecules Bcl-xL and p21 expression. To determine if STAT3 activation by NS5A could

induce STAT3 mediated gene expression, a luciferase reporter construct based on a synthetic promoter was used to transfect hepatoma cells.

Activation of endogenous cellular STAT3 by HCV NS5A induced luciferase gene expression through STAT3 specific binding elements. Our

subsequent studies suggested that NS5A forms a complex with Jak1 and recruits STAT3 for activation. Taken together, our results suggested

that NS5A activates STAT3 through co-operation of Jak1 kinase and activated STAT3 may contribute to HCV-mediated pathogenesis.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Hepatitis C virus; STAT3; Hepatoma cells

Introduction (Clarke, 1997; Grakoui et al., 1993; Hijikata et al., 1991).

Hepatitis C virus (HCV) is a major etiologic agent for

chronic hepatitis worldwide and may lead to the develop-

ment of hepatocellular carcinoma. However, the mechanism

for development of chronic hepatitis or hepatocarcinogene-

sis by HCV remains unclear. HCV genome contains a linear,

positive-strand RNA molecule of approximately 9500

nucleotides (Choo et al., 1989). Many HCV genomes have

been cloned and the sequence divergence indicates several

genotypes and a series of subtypes for this virus (Simmonds,

2001). The HCV genome encodes a single polyprotein

precursor of approximately 3000 amino acids which is

cleaved by both host and viral proteases to generate at least

10 individual proteins, Core, E1, E2, p7, nonstructural

protein (NS) 2, NS3, NS4A, NS4B, NS5A, and NS5B

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

doi:10.1016/j.virol.2004.01.008

* Corresponding address. Department of Pathology, Saint Louis

University, 4th Floor, 1402 South Grand Boulevard, St. Louis, MO

63104. Fax: +1-314-771-3816.

E-mail address: rayrb@slu.edu (R.B. Ray).

HCV NS5A exists as two phosphoproteins p56 and p58 that

are phosphorylated at serine residues after the mature NS5A

protein and is released from the polyprotein (Kaneko et al.,

1994; Tanji et al., 1995). NS5A modulates cell cycle and

immunoregulatory genes (Ghosh et al., 1999a, 2000a;

Majumder et al., 2001; Polyak et al., 2001) and promotes

murine fibroblasts to transformed phenotype (Gale et al.,

1999; Ghosh et al., 1999a). Because NS5A does not bind to

the DNA directly, it probably exerts its cell growth regula-

tory effect through other protein(s). In fact, NS5A down-

regulates the p21 gene by sequestering p53 in the cytoplasm

(Lan et al., 2002; Majumder et al., 2001). NS5A also exerts

anti-apoptotic activity in mammalian cells (Gale et al., 1999;

Ghosh et al., 2000b). NS5A physically associates with PKR

(Gale et al., 1999), human vesicle associated protein

hVAP33 (Tu et al., 1999), growth factor receptor-binding

protein 2 (Tan et al., 1999), a novel transcription factor

SRCAP (Ghosh et al., 2000a), and a nuclear import ma-

chinery component karyopherin h3 protein (Chung et al.,

2000). These results together suggest that HCV NS5A

B. Sarcar et al. / Virology 322 (2004) 51–6052

protein is likely to play an important role in virus–host

interaction.

Signal transducers and activators of transcription (STATs)

are a family of latent cytoplasmic transcription factors that

are activated in response to various extracellular polypeptide

ligands, including cytokines and growth factors (Darnell et

al., 1994; Schindler and Darnell, 1995). STATs have an

amino-terminal protein–protein interaction domain, a DNA

interaction domain, a SH2 domain, and a single tyrosine

phosphorylation site. Phosphorylation of this tyrosine resi-

due stabilizes the association of two STAT monomers

through the interaction between the phosphotyrosine and

SH2 domain (Bowman et al., 2000; Bromberg and Darnell,

2000; Chatterjee-Kishore et al., 2000; Darnell, 1997; Schin-

dler and Darnell, 1995). These activated STAT dimers

translocate into the nucleus, where they bind to their cognate

DNA response elements. In addition to their normal roles in

cell signaling, a relationship between the activation of STATs

and oncogenesis has been observed (Bowman et al., 2000;

Darnell, 1997). Many oncoproteins can activate specific

STATs, and the activated STAT protein participates in

oncogenesis by stimulating cell proliferation and preventing

Fig. 1. Activation of STAT3 by HCV NS5A. (Panel A) Hep-5A (lane 1) and Hep

Western blot analysis using phospho-STAT3 (shown on the left) or STAT3 (shown

(approximately 85 kDa) was ascertained from the migration of standard protein mo

transgenic mice. Liver extracts were prepared from the two normal (lanes 1 and 2)

performed using specific antibodies. The molecular weight of specific protein ban

markers (Invitrogen). (Panel C) NS5A modulates Bcl-xL and p21 expression. Hep

blot using specific antibodies. The blots were reprobed with antibody to actin for

apoptosis. The activated JAKs also induce STAT activation

by a two-step mechanism. Initially, JAKs phosphorylate

receptor tyrosine residues, which in turn become docking

sites for the recruitment of cytoplasmic STAT proteins.

Subsequently, the recruited STAT proteins are directly

phosphorylated by the receptor-associated JAKs. Activated

STATs then dimerize and translocate to the nucleus, where

they bind to specific promoter sequences of target genes

and induce transcription (Darnell, 1997). In this study, we

have investigated how NS5A activates STAT3 in hepatoma

cells. Our results suggest that NS5A induces Jak1 phos-

phorylation via physical interaction, which in turn activates

STAT3.

Results

NS5A activates STAT3 in hepatocytes

Constitutive activation of STAT3 often occurs in human

tumors, which is correlated with proliferation and anti-

apoptosis (Bromberg and Darnell, 2000). We examined

G2-neo (lane 2) and cell lysates were subjected to SDS-PAGE followed by

on the right) specific antibody. The molecular weight of STAT3 protein band

lecular weight markers (Invitrogen). (Panel B) STAT3 is activated in NS5A

and four NS5A transgenic (lanes 3–6) mice and Western blot analysis was

d was ascertained from the migration of standard protein molecular weight

G2-neo (lane 1) or Hep-5A (lane 2) cell lysates were analyzed by Western

comparison of protein level.

Fig. 2. HCV NS5A facilitates localization of STAT3 into the nucleus. HepG2-neo or Hep-5A cells were transiently transfected with wild-type (wt) STAT3-Flag

and mutant STAT3-Flag (mutated at tyrosine 705) plasmid DNAs. After 48 h of transfection, cells were fixed and incubated with a monoclonal antibody to

Flag. Unrelated monoclonal antibody of same isotype was used as a negative control. Cytoplasmic localization of wt STAT3 was observed in empty vector-

transfected HepG2 cells (panel A), whereas wt STAT3 translocated into the nucleus of Hep-5A cells (panel D). Cytoplasmic localization of mutant STAT3 was

observed in both cell lines (panels B and E), whereas control antibody did not display a detectable fluorescence (panels C and F). Photomicrographs (�60)

were taken with a confocal microscope (Bio-Rad 1024). In a different set of experiment, transiently expressed wt STAT3-Flag was examined for subcellular

localization. Cells were stained with anti-Flag antibody (panels G and H) and with DAPI for nuclear staining (panels J–L). Cells exhibited cytoplasmic

localization of STAT3 in HepG2-neo (panels G and J) and nuclear localization in Hep-5A (panels H and K) cells. Nuclear staining of endogenous STAT3 with

specific antibody was also observed (panels I and L) when vector-transfected HepG2 cells were treated with IL-6 (20 ng/ml) for 6 h as a positive control.

Photomicrographs (�40) for this set of experiment were taken with an Olympus BX51 microscope. In another set of experiment, cells expressing NS5A or

vector alone were transiently transfected with STAT3-RFP and intracellular STAT3 localization was examined after 48 h of transfection. Nuclear localization of

STAT3 was observed in Hep-5A cells (panel M), whereas cytoplasmic expression of STAT3 was noted in HepG2-neo cells (panel N). Photomicrographs (�60)

were taken with a confocal microscope (Bio-Rad 1024).

B. Sarcar et al. / Virology 322 (2004) 51–60 53

B. Sarcar et al. / Virology 322 (2004) 51–6054

whether NS5A activates the endogenous STAT3 in stable

HepG2 cells expressing NS5A (Hep-5A cells). For this

purpose, cell lysates were analyzed by Western blot using

specific antibodies. Hep-5A cells expressed a higher level of

phosphorylated STAT3 as compared to cells expressing

empty vector (HepG2-neo) (Fig. 1, panel A). However, total

STAT3 expression level was similar in both HepG2-neo and

Hep-5A cells. We have also investigated whether HCV

NS5A protein activates the phosphorylation of STAT3

expression in transgenic mouse liver. Mouse liver was

collected in cold perfusion buffer (50 mM phosphate buffer,

pH 7.4, 120 mM NaCl, and 10 mM EDTA) and a small

portion was homogenized in 2 ml ice-cold glycerol buffer

using a Dounce homogenizer as previously described

(Majumder et al., 2002). Liver homogenates were subjected

Fig. 3. NS5A enhances transcriptional activity of the STAT3-regulated promote

reporter (3 Ag) and increasing concentrations of the CMV NS5A expression vecto

plasmid DNA (6 ug) was used as a negative control. Plasmid DNA expressing v

constant in each transfection by adding empty vector DNA. Luciferase assay was p

each set of experiments, and relative luciferase activity is presented. (Panel B) Huh

reporter plasmid DNA (3 Ag) and luciferase activity was measured after 48 h of tran

and relative luciferase activity is presented with standard deviation from triplicate

to Western blot analysis using specific antibodies for phos-

pho-STAT3 and STAT3 expression. Phosphorylation of

STAT3 was observed from the liver extracts of four NS5A

transgenic mice (Fig. 1, panel B, lanes 3–6), but not from

two normal littermates (lanes 1–2). The blot was reprobed

with a rabbit antibody to STAT3, and comparable level of

specific protein was observed from all mice liver. We also

examined the expression of p21 and Bcl-xL (downstream

molecules of STAT3) in Hep-5A and HepG2-neo cells.

Densitometric scanning of the results from Western blot

analysis suggested an approximately 3.5-fold increase of

Bcl-xL and approximately 3-fold reduction of p21 expres-

sion (Fig. 1, panel C). Results from these set of experiments

suggest that HCV NS5A expression increases phosphoryla-

tion of STAT3 level and alters Bcl-xL and p21 expression.

r. (Panel A) HepG2 cells were transiently co-transfected with pLucTKS3

r (1, 3, and 6 Ag). Carboxy-terminal deletion mutant of NS5A (NS5A1– 332)

-Src (4 Ag) was used as a positive control. The amount of DNA was kept

erformed at 48 h post-transfection. Triplicate transfection was performed in

7, Huh7-NS5A, and Rep-1 cells were separately transfected with pLucTKS3

sfection. Triplicate transfections were performed in each set of experiments,

analysis. Basal value was arbitrarily set at 1.

Fig. 4. Jak1 kinase inhibitor, AG490, inhibits NS5A induced activation of

Jak1 kinase. Huh7 cells were transduced with Adh-gal (lanes 1 and 2) or

Ad-NS5A (lanes 3 and 4) and treated with 50 AM of AG490 (lanes 2 and 4).

After 48 h, cell lysates were analyzed by Western blot using specific

antibodies. Phosphorylation of Jak1 kinase was inhibited in presence of

AG490. When these samples were analyzed for phospho-STAT3, similar

results were observed (middle panel). Expression of total STAT3 remained

unaltered as seen from reprobed blot (bottom panel). The molecular weight

of specific protein band was ascertained from the migration of standard

protein molecular weight markers (Invitrogen).

B. Sarcar et al. / Virology 322 (2004) 51–60 55

NS5A facilitates translocation of STAT3 into the nucleus

To examine the localization of phospho-STAT3 in the

presence of NS5A, indirect immunofluorescence was per-

formed. HepG2 or Hep-5A cells were transiently trans-

fected with wild-type (wt) STAT3-Flag and mutant STAT3-

Flag (mutated at tyrosine 705) plasmid DNAs. After 48 h of

transfection, cells were fixed and incubated with a mono-

clonal antibody to Flag. Unrelated monoclonal antibody of

the same isotype was used as a negative control. STAT3

translocated into the nucleus of Hep-5A cells (Fig. 2, panel

D), whereas cytoplasmic localization of STAT3 was ob-

served in empty vector-transfected HepG2 cells (panel A).

Predominant perinuclear localization of mutant STAT3 was

observed in both the cell lines (panels B and E). Detectable

fluorescence was not observed from the control antibody of

same isotype (panels C and F). We further examined the

nuclear staining with DAPI (panels J–L) to confirm the

subcellular localization of STAT3 in Hep-5A cells. Our

results demonstrated cytoplasmic localization of STAT3 in

HepG2-neo cells (panels G and J) and nuclear localization

of STAT3 in Hep-5A cells (panels H and K). When we

transiently expressed the STAT3-RFP (Bild et al., 2002),

nuclear localization of STAT3 was observed in Hep-5A

cells (panel M); however, cytoplasmic localization of

STAT3 was observed in HepG2-neo cells (panel N). We

have also treated the HepG2-neo cells with IL-6 (20 ng/ml)

as a positive control for nuclear localization as described

earlier (Barre et al., 2003). STAT3 localization was ob-

served after 6 h of IL-6 treatment (panels I and L), as

expected. These results suggest that NS5A activates phos-

phorylation of STAT3, which in turn, accumulates in the

nucleus. To examine whether HCV NS5A physically asso-

ciates with STAT3 for activation, a co-immunoprecipitation

experiment was performed. Cells were co-transfected with

CMV Flag STAT3 plasmid DNA and different concentra-

tions of CMV NS5A to examine in vivo complex forma-

tion. However, after several attempts, we did not observe a

direct association between NS5A and STAT3 in HepG2 or

Huh7 cells. Thus, our results suggest that HCV NS5A

facilitates translocation of activated STAT3 into the nucleus,

although a direct interaction of NS5A and STAT3 could not

be determined.

NS5A enhances transcriptional activity of STAT3-regulated

promoter

We next performed an in vitro transient transfection

assay using STAT3-regulated reporter plasmid DNA. The

STAT3 reporter pLucTKS3 was constructed from pLucTK

by inserting seven copies of an annealed oligonucleotide

corresponding to a STAT3-specific binding site from the

human C-reactive protein (CRP) gene and was used as a

reporter plasmid for determining transcriptional activity of

the STAT3 protein (Turkson et al., 1998). Cells were co-

transfected with STAT3 reporter construct pLucTKS3 and

CMV NS5A. After 48 h of transfection, cells were harvested

using the reporter lysis buffer, and luciferase activity was

measured. HCV NS5A enhanced luciferase expression by 4-

to 5-fold in HepG2 cells (Fig. 3, panel A). However,

carboxy-terminal deletion mutant of NS5A (CMV

NS5A1–332) did not display an increase of luciferase activ-

ity. The v-src expression vector was used as a positive

control and enhancement of luciferase activity was ex-

hibited, as expected. Similar results were also obtained from

Huh7 cells in an in vitro reporter assay (data not shown). To

examine whether similar effects are observed in presence of

other HCV proteins, we used Huh7 cells expressing NS5A

(Huh-NS5A) or full-length replicon (Rep-1). Enhancement

of luciferase activity was observed when we used stable cell

lines expressing NS5A or Rep-1 (Fig. 3, panel B), sug-

gesting that NS5A activates STAT3 in the presence of

other HCV proteins. These results suggest that HCV

NS5A enhances transcriptional activity of STAT3 regulated

promoter.

Jak1 is essential for STAT3 activation induced by NS5A in

hepatoma cells

Our initial examination suggested that NS5A does not

physically associate with STAT3 in HepG2 or Huh7 cells.

Jak kinases have been shown to phosphorylate STATs for

translocation into the nucleus and modulate various genes

involved in cell growth (Bowman et al., 2000). We exam-

ined whether NS5A mediated STAT3 activation in HepG2

or Huh7 cells is inhibited in the presence of a JAK1 kinase

inhibitor, AG490, by Western blot analysis. AG490 is

selective for JAK family kinases and reported not to inhibit

Src, Lck, Lyn, Btk, and Syk kinases (Zhang et al., 2000).

Cells were infected with nonreplicating adenovirus express-

ing NS5A (Ad-NS5A) or h-galactosidase (Adh-gal). Trans-

B. Sarcar et al. / Virology 322 (2004) 51–6056

duced cells were treated with 50 AM of AG490 for every 12

h, and cells were lysed 48 h postinfection for Western blot

analysis using a rabbit antibody to phospho-Jak1 kinase.

Cells transduced with Ad-NS5A displayed a higher level of

phospho-Jak1 expression, which was inhibited in the pres-

ence of AG490. On the other hand, Adh-gal control virustransduced cells did not exhibit significant phosphorylation

of Jak1 kinase in the presence or absence of AG490 (Fig. 4).

When we examined the status of phospho-STAT3 by

Western blot analysis using specific antibody, activation

of STAT3 was observed in Ad-NS5A infected cells, which

Fig. 5. STAT3-mediated gene regulation is inhibited by AG490. (Panel A) Huh7

negative control, CMV NS5A or v-Src as a positive control. Cells were treated w

h post-transfection. Luciferase activity was measured as described in Fig. 3 legend

treated with DMSO (0.5%) or 50 AM of AG490. Cell extracts were prepared afte

were co-transfected with pLucTKS3 reporter, and CMV NS5A with or without

transfection. Triplicate transfections were performed in each set of experiments, and

as error bars. Basal value was arbitrarily set at 1.

was also inhibited in the presence of AG490. However,

total STAT3 expression remained unaltered in each lane

(bottom panel). We further investigated the role of Jak1

kinase in STAT3-mediated gene regulation induced by

NS5A. For this purpose, we transiently transfected cells

with a CMV NS5A expression vector and the STAT3

responsive reporter plasmid, pLucTKS3. Transfected cells

were treated with AG490 for every 12 h for 48 h. As shown

in Fig. 5A, AG490 treatment inhibited NS5A induced

STAT3-mediated reporter gene expression. Zhang et al.

(2000) have shown that Src phosphorylates Jak1 for acti-

cells were co-transfected with pLucTKS3 reporter, and empty vector as a

ith 50 AM of AG490 or DMSO (0.5%) and cell extracts were prepared 48

. (Panel B) Rep-1 cells were transfected with pLucTKS3 reporter gene and

r 48 h of transfection and luciferase activity was measured. (Panel C) Cells

Jak1 kinase plasmid DNA. Luciferase assay was performed at 48 h post-

relative luciferase activity is presented. Standard deviations are represented

B. Sarcar et al. / Virology 322 (2004) 51–60 57

vation of STAT3 and Jak1 phosphorylation can be specif-

ically inhibited by treatment with AG490. We treated Src-

transfected cells with AG490 to use as a positive control for

inhibition of Jak1 phosphorylation. Inhibition of STAT3

activation was also observed in Huh7 cells expressing HCV

full-length replicon (Rep-1) in the presence of AG490 (Fig.

5, panel B). An in vitro reporter assay was performed using

pLucTKS3, NS5A, and Jak1 plasmid DNAs to further

examine whether NS5A-mediated STAT3 activation is al-

tered in the presence of Jak1 kinase. Our results suggested

that co-expression of Jak1 and NS5A further induces

STAT3 activation (at least 2-fold) as compared to NS5A

or Jak1 kinase alone (Fig. 5, panel C).

Does NS5A physically associate with Jak1?

To determine whether NS5A and Jak1 form a complex in

vivo, a co-immunoprecipitation assay was performed. Huh7

cells were co-transfected with myc-tagged Jak1 and CMV-

NS5A plasmid DNAs following vvT7 infection. Cell lysates

were immunoprecipitated with c-myc monoclonal antibody

for Jak1 kinase and subjected to Western blot analysis for

NS5A using specific antibody. Co-precipitation of Jak1 with

NS5A was evident from the specificity of the antibody and

Fig. 6. NS5A physically associates with Jak1 kinase. Huh7 cells were

infected with vvT7 and co-transfected with myc-tagged Jak1 and empty

vector (lane 1) or CMV NS5A (lane 2). After 24 h of transfection, cell

lysates were immunoprecipitated with a monoclonal antibody to c-myc and

immunoblotted with a monoclonal antibody to NS5A. Cells transfected

with myc-tagged Jak1 (lane 3) and NS5A (lane 4) were immunoblotted with

a monoclonal antibody to NS5A to examine the exogenous expression of

NS5A. The molecular weight of the NS5A protein was ascertained from the

migration of standard protein molecular weight markers (Invitrogen). The

blot was reprobed with a monoclonal antibody for detection of Jak1

(bottom panel). The positions of NS5A and immunoglobulin (Ig) heavy

chain (from experimental reagents) are shown.

the size of the NS5A protein (Fig. 6, lane 2). On the other

hand, cell lysates when similarly analyzed from myc-tagged

Jak1 and empty vector plasmid DNA as a negative control

did not exhibit precipitation of NS5A protein (Fig. 6, lane

1). The exogenous expression of NS5A from transfected cell

lysates was analyzed by Western blot (Fig. 6, lanes 3 and 4).

The blot when reprobed with a specific antibody to Jak1

displayed the Jak1 kinase protein band (bottom of panel).

However, endogenous Jak1 could not be detected as co-

precipitate with NS5A in Hep-5A cells, possibly for a low

level of Jak1 expression.

Discussion

STAT3 generally remains latent in the cytoplasm in an

unphosphorylated state (Schindler et al., 1992). Activation of

STAT3 requires tyrosine phosphorylation, which occurs

upon cytokine signaling or other stimuli (Wen et al.,

1995). Upon stimulation, STAT3 is phosphorylated at tyro-

sine residues and translocates into the nucleus, where it binds

cognate DNA sequences as dimers (Doria et al., 1995; Zhong

et al., 1994). Activation of STAT3 by NS5A in Huh7 cells

was observed earlier (Gong et al., 2001); however, the

mechanism of STAT3 activation is unknown. In this study,

we have shown that HCV NS5A activates STAT3 phosphor-

ylation, which in turn accumulates into the nucleus, although

a direct interaction between NS5A and STAT3 was not

observed. We have also shown that activation of STAT3

occurs in the presence of other HCV proteins using full-

length HCV replicon. Our results from transgenic mice

expressing HCV NS5A also suggested activation of STAT3

in mouse liver. Several tumor viruses are associated with

STAT activation. Epstein–Barr virus transformation leads to

uncontrolled activation of STATs (Weber-Nordt et al., 1996).

Kaposi’s sarcoma-associated herpesvirus ORF50 stimulates

transcriptional activity of STAT3 (Gwack et al., 2002). Rous

sarcoma virus v-src oncoprotein has been shown to phos-

phorylate and activate STAT3 (Yu et al., 1995; Cao et al.,

1996). The ability of these viruses to activate STATs is

closely related to viral oncogenic potential.

Jak kinases are large enzymes (120–140 kDa) that are

cytoplasmically pre-associated with signal-transducing cy-

tokine receptor subunits (Ihle, 1995). Upon cytokine-

induced receptor aggregation, Jaks are probably activated

by auto- and transphosphorylation. Tyrosine residues with-

in the cytoplasmic tail of the receptor are subsequently

phosphorylated by the kinases, providing docking sites for

SH2 domain containing signaling proteins including

STATs, tyrosine phosphatases, and suppressors of cytokine

signaling. Tyrosine-phosphorylated STATs homo- or heter-

odimerize and translocate to the nucleus where they bind

to specific DNA sequences in the promoter regions of their

respective target genes (Darnell et al., 1994; Heinrich et

al., 1998). We have demonstrated that NS5A interacts with

and activates Jak1 kinase, which in turn phosphorylates

B. Sarcar et al. / Virology 322 (2004) 51–6058

STAT3. Phosphorylation of STAT3 was also inhibited by a

specific Jak1 kinase inhibitor, further suggesting the in-

volvement of Jak1 kinase. NS5A does not possess tyrosine

kinase activity; therefore, the precise mechanism of NS5A-

mediated Jak1 phosphorylation remains to be elucidated.

Other transforming viruses have been shown to activate

STAT3 in cooperation with other kinases. Herpesvirus

saimiri Tip-484 activates STAT3 in cooperation with

p56lck (Lund et al., 1997). Human T-cell lymphotropic

virus type-1 transformed cells exhibit constitutive activa-

tion of the Jak–STAT pathway (Migone et al., 1995). Our

results suggest that NS5A forms complex with Jak1 kinase

and recruits STAT3 for activation. The activated STAT3

then translocates into the nucleus and modulates cell

growth regulatory genes Bcl-xL and p21, which may lead

to HCV mediated pathogenesis.

Many different cell lines and fresh isolates derived from

a variety of tumors express activated form of STAT3

(O’Shea et al., 2002). They are often activated upon

different stimuli and facilitate cellular expansion by trans-

activating genes encoding proteins to enhance cell surviv-

al. In fact, introduction of NS5A in hepatoma cells

enhances Bcl-xL expression and represses p21. We have

shown previously that NS5A associates and sequesters p53

into cytoplasm and downregulates p21 expression

(Majumder et al., 2001). Recently, Lin et al. (2002)

reported that transduction of p53 in human breast carcino-

ma cells inhibits phospho-STAT3 and enhances p21 ex-

pression. Therefore, it is possible that p53–NS5A complex

formation allows STAT3 to phosphorylate and translocate

into the nucleus for promotion of cell growth. HCV-

mediated promotion of cell growth may also involve

additional viral protein. Recently, STAT3 activation by

HCV core was reported in transgenic mice (Yoshida et

al., 2002). Co-expression of core and STAT3 was sug-

gested to decrease the dephosphorylation rate of phosphor-

ylated STAT3. Therefore, it is possible that expression of

core and NS5A together may have stronger oncogenic

potential than the individual proteins. Our results along

with others suggest that HCV evolves several mechanisms

to activate STAT3, which may contribute to virus-mediated

pathogenesis.

Materials and methods

Cell lines and reagents

Human hepatoma cell lines, HepG2 and Huh7, were

grown in DMEM containing 10% fetal bovine serum.

Expression vectors for STAT3-luc (pLucTKS3), STAT3,

STAT3-RFP, and v-src were generously provided by

Richard Jove (University of South Florida, FL); wild-type

and mutant STAT3-Flag was kindly provided by James

Darnell (Rockefeller University, NY); Jak1 cDNA was

kindly provided by James Ihle (St. Jude Children Hospital,

Memphis, TN); and Jak1-myc was kindly provided by

Robert Schriber (Washington University, St. Louis, MO).

Antibodies to Jak1, phospho-STAT3, and STAT3 were

obtained from Cell Signaling, MA. Monoclonal antibodies

to Flag and c-myc were obtained from Sigma (Missouri).

Jak1 kinase inhibitor, AG490, was obtained from Pharmin-

gen (California); protein A/G-agarose and enhanced chemi-

luminescence (ECL) detection kit were from Amersham

Pharmacia Biotech (Illinois). HCV full-length replicon from

Con1 strain was kindly provided by Ralf Bartenschlager,

University of Heidelberg, Germany.

Luciferase assay

Cells (5 � 105) were transfected with 3 Ag of reporter

plasmid pLucTSK3 and different doses of effector plasmid

DNAs using lipofectamine (Invitrogen, California). Empty

vector DNA was used as a negative control for the

luciferase assay. The total amount of DNA was kept

constant by addition of empty vector. Cells were lysed

using the reporter lysis buffer (Promega, Wisconsin), and

the luciferase activity was determined using a luminometer

(Optocomp II, MGM Instruments) as previously described

(Majumder et al., 2001).

Co-immunoprecipitation

Cells were co-transfected with 2 Ag of CMV Flag

STAT3, or myc-tagged Jak1 plasmid DNA and CMV

NS5A, and cell lysates were prepared after 48 h using 0.3

ml lysis buffer (150 mM NaCl, 10 mM HEPES, pH 7.6,

0.1–1% Nonidet P-40, 5 mM EDTA) containing a cocktail

of protease inhibitors (aprotinin, leupeptin, pepstatin, and

phenylmethylsulphonylfluoride). In some cases, plasmid

DNAs were transiently expressed using vvT7 infection–

transfection system (Sen et al., 2003) and cells were lysed

after 24 h. Cell lysates were incubated with a monoclonal

antibody to Flag (M2) or myc (9E-10) epitopes for 4 h at

4 jC, followed by an overnight incubation with protein

A-Sepharose beads (Pharmacia). The immunoprecipitates

were separated by SDS-polyacrylamide gel electrophoresis

and electroblotted onto a nitrocellulose membrane. Immu-

noblotting was performed by incubation of the membrane for

1 h with a rabbit antibody to STAT3 or Jak1 or monoclonal

antibody to NS5A (Biogenesis, New Hampshire). Proteins

were detected by enhanced chemiluminescence.

Immunofluorescence study

HepG2 or Hep-5A (HepG2 cells stably expressing NS5A)

cells (Ghosh et al., 2000b) were transfected with wild-type or

mutant STAT3 with Flag tag. After 48 h of transfection, cells

were washed and fixed with 3.7% formaldehyde followed by

blocking with 3% BSA. Cells were incubated with mono-

clonal antibody to Flag epitope or unrelated monoclonal

antibody with same isotype (IgG1) or STAT3 specific

B. Sarcar et al. / Virology 322 (2004) 51–60 59

antibody for 1 h at room temperature. Cells were washed and

incubated with anti-rabbit Ig conjugated with Alexa 488

(Molecular Probes, Oregon) for 30 min at room temperature.

Finally, cells were rinsed and mounted for immunofluores-

cence microscopy (confocal microscope, Bio-Rad model

1024 or Olympus BX51). A parallel set of experiments

was performed with wild-type STAT3 for nuclear staining

with DAPI. For this set of experiment, photomicrographs

were taken with Olympus microscope.

Generation of recombinant adenovirus expressing NS5A

Recombinant adenovirus expressing NS5A (Ad-NS5A)

was generated as previously described (Ghosh et al.,

1999b). Briefly, NS5A was cloned into the pAdTrack-

CMV shuttle vector (He et al., 2002) under the control of

CMV promoter (kindly provided by Bert Vogelstein, John

Hopkins University, Maryland). The resulting AdTrack

CMV-NS5A plasmid DNA was linearlized with PmeI re-

striction enzyme and co-transformed into E. coli BJ5183

cells with pAdEasy adenoviral backbone plasmid DNA.

Recombinants were selected and confirmed by restriction

enzyme endonuclease digestion and sequence analysis.

Finally, linearized recombinant plasmid DNA was trans-

fected into an adenovirus packaging cell line (293 cells) for

generation of recombinant adenovirus (Ad-NS5A). The

integration and expression of the NS5A in the recombinant

adenovirus was verified by PCR. Ad-NS5A was purified by

CsCl density gradient (Ghosh et al., 2002) and used for this

study. Adh-gal (He et al., 2002) was used as negative

control.

Establishment of Huh7 cells stably expressing HCV

full-length replicon

Huh7 cells expressing full-length HCV replicon were

established as described earlier (Pietschmann et al., 2002).

Briefly, HCV full-length RNA was generated by in vitro

transcription from plasmid DNA containing Con1 sequen-

ces using T7 polymerase (Promega). Huh7 cells were

electroporated with 1 Ag of purified in vitro transcripts

containing HCV full-length RNA. Cells were selected with

G418 at a concentration of 500 Ag/ml. Approximately 4

weeks post-electroporation, small colonies were visible.

These colonies were characterized for the presence of

HCV RNA and protein expression. One of the colonies

was named as Rep-1 and used in this study.

Acknowledgments

We thank Ralf Bartenschlager, James Darnell, James

Ihle, Richard Jove, and Robert Schriber for providing us

the reagents for our study, and Richard Jove for helpful

suggestions. We also thank David Lagunoff for helping

us in immunofluorescence study and Adrish Sen for

initial work on this project. This research was supported

by PHS Grant AI45144 from the National Institutes of

Health.

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