1 large isoform of hepatitis delta antigen activates serum

8/17/00 Kato

1

Large Isoform of Hepatitis Delta Antigen Activates

Serum Response Factor-Associated Transcription

Short title: LHDAg activates SRF-associated transcription

Tadashi Goto, Naoya Kato*, Suzane Kioko Ono-Nita, HideoYoshida,

Motoyuki Otsuka, Yasushi Shiratori, and Masao Omata

Department of Gastroenterology, Faculty of Medicine,

University of Tokyo

*To whom correspondence should be addressed:

Department of Gastroenterology, Faculty of Medicine,

University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan

PHONE: +81-3-3815-5411 ext. 33070

FAX: +81-3-3814-0021

E-mail: [email protected]

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on August 28, 2000 as Manuscript M002947200 by guest on February 7, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

8/17/00 Kato

2

SUMMARY

Hepatitis delta virus (HDV) infection sometimes causes severe and

fulminant hepatitis as a coinfection or superinfection along with the hepatitis B

virus. To elucidate the underlying mechanism of injury caused by HDV, we

examined whether two isoforms of the hepatitis delta antigen (HDAg) had any

effect on five well-defined intracellular signal transduction pathways: serum

response factor (SRF)-, serum response element (SRE)-, nuclear factor κB-,

activator protein 1-, and cyclic AMP response element-dependent pathways.

Reporter assays revealed that large HDAg (LHDAg) activated the SRF- and SRE-

dependent pathways. In contrast, small HDAg (SHDAg) did not activate any of

five pathways. LHDAg enhanced the transcriptional ability of SRF without

changing its DNA-binding affinity in an electrophoretic mobility shift assay. In

addition, LHDAg activated a rat SM22α promoter containing SRF-binding site

and a human c-fos promoter containing SRE. In conclusion, LHDAg, but not

SHDAg, enhances SRF-associated transcriptions. Despite structural similarities

between the two HDAgs, there are significant differences in their effects on

intracellular signal transduction pathways. These results may provide clues that

will aid in the clarification of functional differences between LHDAg and SHDAg

and the pathogenesis of delta hepatitis.

by guest on February 7, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

3

INTRODUCTION

Hepatitis delta antigen (HDAg) was discovered as an antigen localized in

the nuclei of hepatocytes in a patient who had already been infected with the

hepatitis B virus (HBV) (1). HDAg is the only known protein encoded by the

hepatitis delta virus (HDV), which is a defective human pathogen whose

transmission requires the helper function of HBV (2). The two isoforms of HDAg,

large and small forms (3, 4), are identical except for additional 19 residues located

at the C-terminus of the large form. The small form of HDAg (SHDAg) consists

of 195 amino acids (aa) (molecular weights; 24 kDa), and the large form of HDAg

(LHDAg) consists of 214 aa (27 kDa) (5, 6). Both forms of HDAgs have common

functional domains: an N-terminal coiled-coil domain responsible for

oligomerization (7), a central domain responsible for a nuclear localization signal

(8, 9), and a central helix-turn-helix domain responsible for binding to the RNA

genome (10, 11). Despite these structural similarities, the two HDAgs play

complementary roles in HDV replication; SHDAg is required for genome

replication (12), but LHDAg acts as an inhibitor of replication (13). LHDAg is

also required for HDV assembly (14, 15); the formation of HDV viral particles

requires isoprenylation at the C-terminus of the LHDAg (16), although the

mechanism of LHDAg on this packaging remains unclarified.

It has been shown that SHDAg suppresses the gene expression of HBV (17).

Nevertheless, HDV infection often causes severe chronic hepatitis and liver

cirrhosis as a superinfection of chronic HBV carriers and fulminant hepatitis as a

coinfection with HBV (18). It was also reported that SHDAg possessed a

cytotoxic effect on infected hepatocytes, while LHDAg was thought to reduce this


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

4

effect (19). The reason why HDV coinfection or superinfection with HBV causes

hepatic injury, however, remains unclear. Therefore, we investigated whether the

two isoforms of HDAg had any effect on five well-defined intracellular signaling

pathways: serum response factor (SRF)-, serum response element (SRE)-, nuclear

factor κB (NF-κB)-, activator protein 1 (AP-1)-, and cyclic AMP response

element (CRE)-dependent pathways.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

5

EXPERIMENTAL PROCEDURES

Construction of HDAgs expressing plasmids

The HDV sequence used in this study was derived from pSVLD3 (kindly

provided by Dr. J. Taylor, Fox Chase Cancer Center, PA), containing a trimer of

unit-length HDV cDNA (12, 20). Both LHDAg and SHDAg regions were

amplified by polymerase chain reaction using the following primers having a ScaI

restriction site (underlined) (nucleotide positions according to the HPDGEN

sequence (21) are shown in parentheses): sense primer 5’-AAA AGT ACT ACC

ATG AGC CGG TCC GAG TCG AGG A-3’ (1598-1577) and antisense primer

5’-AAA AGT ACT TCA CTG GGG TCG ACA ACT CTG GGG A-3’ (954-978)

for the LHDAg region, and 5’-AAA AGT ACT CTA TGG AAA TCC CTG GTT

TCC CCT GA-3’ (1011-1036) for the SHDAg region. Each amplified fragment

was digested with ScaI and then cloned into pCXN2 (kindly provided by Dr. J.

Miyazaki, University of Osaka, Japan), a mammalian expression plasmid having a

β-actin based CAG promoter (22) to generate pCXN2-DLm and pCXN2-DS

expressing SHDAg. Because pCXN2-DLm contained the nucleotide A at position

1012 according to the sequence of HPDGEN (21), we substituted G for A to make

a construction of LHDAg expressing plasmid pCXN2-DL using site-directed

mutagenesis (Quick change site-directed mutagenesis kit, Stratagene, La Jolla,

CA).

For an electrophoretic mobility shift assay (EMSA), pCXN2-FlagDL,

which expresses Flag-tagged LHDAg, was constructed by adding the Flag

sequence into the 5’ terminal of the LHDAg region.

For evaluation of the transcriptional activation of LHDAg, pM-GAL4DL,

which expresses LHDAg protein fused to the yeast GAL4 DNA binding domain


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

6

(DNA-BD), was constructed by subcloning the LHDAg region into the pM vector,

a mammalian expression plasmid encoding the GAL4 DNA-BD gene (Clontech,

Palo Alto, CA).

All cloned plasmids were purified using the Endofree plasmid kit (Qiagen,

Hilden, Germany) and sequenced using an autosequencer (PE Applied

Biosystems, Foster City, CA) by the dye-termination method as described

previously (23) to confirm the integration of HDAg genes.

Construction of Elk1- and SRF-expressing plasmids

Using the T7Elk 1-428 (kindly provided by Dr. R. Treisman, Imperial

Cancer Research Fund, UK) (24) as a template, the full-length Elk1 region (1-428

aa) was amplified by polymerase chain reaction with Elk1 region-specific primers

having an XhoI restriction site. Amplified products were digested with XhoI and

then cloned into the XhoI site of pCXN2 to generate pCXN2-Elk1.

pFA-GALElk (Stratagene) expresses the activation domain of Elk1 (307-

427 aa) fused to the GAL4 DNA-BD. A plasmid expressing SRF (10-508 aa)

fused to the GAL4 DNA-BD, pSG5-GALSRF (10-508), was a gift from Dr. M.

Fujii (Kanazawa University, Japan) (25).

Reporter plasmids of intracellular signal transduction pathways

Five reporter plasmids containing the Photinus pyralis (Firefly)-luciferase

reporter gene driven by a basic promoter element (TATA box) plus a defined

inducible cis-enhancer element were utilized (PathDetect cis-reporting systems,

Stratagene, La Jolla, CA). Each Firefly-luciferase gene in the reporter plasmid

was controlled by the following synthetic enhancer sequences (the binding site for

the transcription factor is capitalized): 5 repeats of the binding sites for SRF


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

7

(gtCCATATTAGGac, pSRF-Luc), 5 repeats of SRE

(AGGATgtCCATATTAGGacatct, pSRE-Luc), 5 repeats of the binding sites for

NF-κB (tGGGGACTTTCCgc, pNF-κB-Luc), 7 repeats of the binding sites for

AP-1 (TGACTAA, pAP1-Luc), and 4 repeats of CRE (agccTGACGTCAgag,

pCRE-Luc). A control plasmid expressing Renilla reniformis (Seapansy)-

luciferase driven by the herpes simplex virus thymidine kinase promoter, pRL-TK,

was used to correct the efficiency of transfection (Promega, Madison, WI). As a

positive control for the activation of SRF-associated pathways, pFC-PKA

(Stratagene), which expresses the catalytic subunit of cAMP-dependent protein

kinase (protein kinase A; PKA) driven by a CMV promoter, was utilized.

In addition to these reporter plasmids which contain synthetic promoters,

SM22α-Luc, a luciferase reporter plasmid with a rat smooth muscle-specific gene

SM22α promoter (-1507 to +32) containing the binding sites for SRF, generally

called the CArG box (kindly provided by Dr. M. E. Lee, Brigham and Women’s

Hospital, MA) (26), and HF456, a luciferase reporter plasmid with a human c-fos

promoter (-456 to +47) containing SRE (kindly provided by Dr. T. Ishikawa,

University of Tokyo, Japan) (27), were utilized. Specific mutations were

introduced into the SM22α-Luc and HF456 plasmids using site-directed

mutagenesis (Stratagene). The specific base changes were chosen based on their

ability to disrupt SRF binding. As for the SM22α-Luc, the CArG box 1 at bp -162

to bp -153 was converted from CCAAATATGG to AAAAATATGG (SM22α

m1-Luc); the CArG box 2 at bp -283 to bp -266 was converted from

CCATAAAAGGTTTTTCCC to CCATAAAAAATTTTTCCC (SM22α m2-Luc).

Combinations of mutations were generated by performing mutagenesis reactions

on a template already containing a mutation (SM22α m1m2-Luc). As for the

HF456, the CArG box at bp -314 to bp -305 was converted from CCATATTAGG


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

8

to CCATATTATT (HF456 m1). The sequences and nucleotide positions are

according to the previous reports (28, 29). Altered nucleotides are indicated in

bold type. All mutations were verified by sequencing.

pFR-Luc (Stratagene) has a Firefly-luciferase gene controlled by five yeast

GAL4 upstream activation sequences, which was utilized as a reporter to examine

transcriptional activation of GAL4 DNA-BD fused Elk1 or SRF by HDAg.

Cell culture

HeLa cells (human cervical carcinoma cell line), HuH-7 cells (human

hepatocellular carcinoma cell line), and HepG2 cells (hepatoblastoma cell line)

were obtained from the Riken cell bank (Tsukuba science city, Japan). Cells were

cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco BRL,

Gaitherburg, MD) supplemented with 10% fetal bovine serum (FBS) at 37 oC in

5% CO2.

Transfection and luciferase assays

Approximately 4 x 105 HeLa, HuH7, or HepG2 cells were plated into a 6-

well tissue culture plate (Iwaki Glass, Chiba, Japan) 24 hours before transfection.

Using the Effectene transfection reagent (Qiagen, Hilden, Germany), cells were

transiently cotransfected with 0.2 µg of reporter plasmids and 0.2 µg of pCXN2,

pCXN2-DL, or pCXN2-DS.

For evaluation of the transcriptional ability of Elk1 or SRF, cells were

transiently cotransfected with 0.15 µg reporter plasmids, 0.05 µg pFA-GALElk or

pSG5-GALSRF, and 0.2 µg pCXN2, pCXN2-DL, or pCXN2-DS. For evaluation

of the transcriptional ability of LHDAg, 0.2 µg reporter plasmids and 0.2 µg pM

vector or pM-GAL4DL were used. For the assay using HF456 containing the c-


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

9

fos promoter, HeLa cells were cultured in DMEM supplemented with 0.5% FBS

to give low background.

Forty-eight hours after transfection, whole cell lysates were examined for

luciferase activity (PicaGene dual Sea Pansy system, Toyo ink, Tokyo, Japan)

with a luminometer (Lumat LB9507, EG&G Berthold, Bad Wildbad, Germany).

Firefly-luciferase activity was normalized for transfection efficiency based on

Seapansy-luciferase activity. The luciferase activity of the cells which were

transfected with the reporter plasmid plus pCXN2 was set arbitrarily at 1.0, and

then the relative luciferase activity was compared to this established value. Assays

were performed at least in triplicate.

Concentration of cells transiently transfected with plasmids

As only a small percentage of the cells were transfected by a transient

transfection method, we utilized the MACSelect system (Miltenyi Biotec,

Germany) to concentrate transiently plasmid-transfected cells. The concentration

of the plasmid-transfected cells was achieved by magnetically isolating the

transfected cells via a surface marker, a truncated mouse H-2 k molecule, which

was expressed from the cotransfected plasmid, pMacsKk. HeLa cells were

cotransfected with pCXN2, pCXN2-FlagDL, or pFC-PKA together with pMacsKk.

After 36 hours, cells were treated with 0.05% trypsin and dispersed by being

pipetted into single-cell suspensions after addition of 100 µl FBS. The cells were

resuspended with 600 µl of PBE buffer (phosphate-buffered saline supplemented

with 0.5% bovine serum albumin and 2 mM EDTA) containing 80 µl of

micromagnetic beads conjugated with a monoclonal antibody against mouse H-2 k

and incubated for 15 min at room temperature. Then, magnetically labeled cells

were recovered by the magnetic separation column and used for an electrophoretic


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

10

mobility shift assay (EMSA).

Electrophoretic mobility shift assay (EMSA)

Annealed oligonucleotides for the CArG box (5’-GGATGTCCATATTA

GGACATC-3’) were end-labeled with [α-32P] ATP using T4 polynucleotide

kinase. Nuclear extracts were obtained from HeLa cells transfected with pCXN2,

pCXN2-DL, or pFC-PKA as previously described (30). The protein concentration

of nuclear extracts was measured by a Micro BCA protein assay reagent kit

(Pierce, Chester, UK), then adjusted to give equal concentration. Four micrograms

of the nuclear extracts were incubated with 0.035 pmol of the CArG box-

radiolabeled probe. Anti-SRF antibody (Santa Cruz Biotechnology, Santa Cruz,

CA) were used for the supershift assay. The CArG box-unlabeled competitor was

added at a 100-fold molar excess to confirm site specific binding. The binding

reaction was performed at room temperature for 30 minutes in a 10 µl mixture

consisting of 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol,

50 mM NaCl, and 0.5 µg of poly(dI-dC)·(dI-dC). DNA-protein complexes were

then loaded onto a chilled 4% nondenaturing acrylamide gel. Gel electrophoresis

was executed in 0.25 x Tris borate-EDTA at 4oC. The gel was dried, and

autoradiography was performed using a Fujix bio-imaging analyzer BAS 2000

(Fuji Photo Film, Tokyo, Japan).

Western blotting analysis

LHDAg and SHDAg expression was confirmed by Western blotting (ECL-

plus, Amersham, Buckinghamshire, UK) using the soluble protein extracts of

HeLa cells which were transfected with pCXN2, pCXN2-DL, or pCXN2-DS (31).

HDAg expression was examined using serum obtained from a patient with


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

11

chronic HDV infection. Using the soluble protein extracts of HeLa cells

transfected with pCXN2-Elk1 and pCXN2-DL cultured in DMEM supplemented

with 0.5% FBS, the phosphorylation of Elk1 was examined by anti-Elk1 antibody

and anti-phospho-specific Elk1 (Ser 383) antibody [PhosphoPlus Elk1 (Ser 383)

antibody kit, New England Biolabs, Beverly, MA] according to the

manufacturer’s instructions.

Statistics

Data are expressed as the means + S.D. Statistical analysis was performed

using the t test. A p-value of less than 0.05 was considered statistically significant.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

12

RESULTS

Activation of SRF- and SRE-dependent signal transduction pathways by

LHDAg

Each of the five reporter plasmids (pSRF-Luc, pSRE-Luc, pNF-κB-Luc,

pAP1-Luc, or pCRE-Luc) was transiently cotransfected into HeLa cells with

pCXN2, pCXN2-DL, or pCXN2-DS. LHDAg activated the SRF- and SRE-

dependent signal transduction pathway at a value 4.0 + 1.2 (mean + SD)-fold and

2.5 + 1.0-fold higher than the control, respectively (Fig. 1A). There was no

significantly increased activation in the remaining three pathways by LHDAg. In

addition, SHDAg did not activate any of the five pathways (Fig. 1A). Expression

of LHDAg and SHDAg were confirmed by Western blotting at the expected size:

LHDAg at a molecular weight of 27 kDa, and SHDAg at a molecular weight of

24 kDa (5).

Cell line independent and dose-dependent activation of SRF-dependent pathway

by LHDAg

We investigated whether LHDAg activated the SRF-dependent pathway in

other cell lines. Results similar to those in HeLa cells were obtained in HuH-7

cells (4.3 + 2.4-fold higher than control) and HepG2 cells (3.3 + 1.0-fold) (Fig.

1B). In addition, LHDAg activated the SRF-dependent signal in a dose-dependent

manner in HeLa cells (Fig. 1C).

No additional effect of SHDAg on the activation of the SRF-dependent pathway

by LHDAg

Since LHDAg and SHDAg play complementary roles in genome viral


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

13

replication, we examined whether SHDAg had influence on SRF-dependent

transcription by LHDAg. Although HeLa cells were transiently transfected with

pSRF-Luc and pCXN2-DL in combination with various amounts of pCXN2-DS,

the presence of SHDAg had no influence on activation of the SRF-dependent

pathway by LHDAg (Fig. 2).

Enhancement of transcriptional ability of SRF by LHDAg

Because LHDAg is a nuclear protein, we examined whether LHDAg itself

possessed transactivation activity. pM-GAL4DL, a GAL4 DNA-BD-LHDAg

fusion protein expression plasmid, was cotransfected into HeLa cells with pFR-

Luc. The GAL4 DNA-BD-LHDAg fusion protein, however, did not activate

transcription from pFR-Luc (1.0 + 0.1-fold higher than control), suggesting that

LHDAg itself had no transcriptional ability in mammalian cells.

To elucidate how LHDAg activates the SRF-dependent signal transduction

pathway, we examined the influence of LHDAg on: 1) transcriptional ability of

SRF by a reporter assay, 2) SRF binding to SRF binding site, the CArG box

[CC(A/T)6GG], by EMSA, and 3) a rat SM22α promoter containing two CArG

boxes.

Transcriptional activation of SRF: we initially examined whether LHDAg

enhanced the transcriptional activation of SRF. HeLa cells were transiently

transfected with pFR-Luc, pSG5-GALSRF(10-508) and pCXN2-DL. The cells

were then assayed for the luciferase activity. As shown in Fig. 3A, the

transcriptional ability of SRF increased by approximately 2.5-fold with LHDAg.

LHDAg did not increase SRF binding to the CArG box: next, we examined

whether LHDAg modified SRF binding to the CArG box by EMSA using nuclear

extracts of HeLa cells transfected with pCXN2, pCXN2-FlagDL or pFC-PKA and


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

14

concentrated by MACSelect system. After concentration by the MACSelect

system, we could confirm that more than 70% of collected cells were transfection

positive by immunostaining with anti-Flag M2 antibody (Upstate Biotechnology,

Lake Placid, NY) (data not shown). LHDAg did not influence SRF binding to the

CArG box, however binding increased by the catalytic subunit of PKA (Fig. 3B).

Moreover, LHDAg was not contained in the DNA-protein complex because

mobility of DNA-SRF complex did not change in the presence of Flag-tagged

LHDAg and no supershifted band was observed by adding anti-Flag M2 antibody

(data not shown).

The effect of HDAgs on the rat SM22 promoter: we examined whether

LHDAg activated a rat promoter containing the CArG box. By use of SM22α-Luc,

the reporter plasmid with a SM22α promoter containing two CArG boxes,

luciferase activity increased by a value 3.3 + 0.5-fold higher than the control with

LHDAg (Fig. 4B), while there was no increase with SHDAg (data not shown). To

determine whether this activation by LHDAg was mediated by the CArG box, we

mutated two CArG boxes, either alone or combinations, and then measured

luciferase activity with LHDAg or without LHDAg. Mutations of each CArG box,

which abolish SRF binding, were introduced into the indicated elements in the

context of the wild type reporter construct (Fig. 4A), and their influence were

examined in transient transfection assays. As shown in Fig. 4B, the effect of

LHDAg on the SM22α promoter were decreased when the CArG boxes were

mutated, indicating that activation of the SM22α promoter by LHDAg is

dependent on enhancement of the transcriptional ability of SRF.

LHDAg activates the SRE-dependent pathway through SRF, not Elk1

LHDAg activated not only the SRF-dependent pathway but also the SRE-


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

15

dependent pathway. SRE is composed of the CArG box bound SRF and nearby

Ets motif bound ternary complex factors (TCFs) such as Elk1. Elk1, regulated by

mitogen-activated protein kinase (MAPK) (32-34), is phosphorylated by MAPK

at a cluster of Serine/Threonine motifs located at its C-terminus. Phosphorylation

at these sites, particularly Ser 383, is critical for transcriptional activation (24, 35).

Therefore, we examined the influence of LHDAg on: 1) transcriptional activation

of Elk1 protein by a reporter assay, 2) phosphorylation of Elk1 protein by

Western blotting, and 3) the human c-fos promoter containing SRE.

Transcriptional activation and phosphorylation of Elk1 protein: HeLa cells

were transiently transfected with pFR-Luc, pFA-GALElk, and pCXN2-DL. Cells

were then assayed for luciferase activity. The transcriptional ability of ELK1 was

not increased by LHDAg (Fig. 3C). Western blotting analysis of Elk1 also

indicated that LHDAg did not enhance the phosphorylation of Elk1 (Fig. 3C),

thereby suggesting that LHDAg has no effect on the transcriptional ability of

Elk1.

The effect of HDAgs on a human c-fos promoter: by use of HF456, a reporter

plasmid with a c-fos promoter containing SRE, the luciferase activity increased by

a value 1.8 + 0.3-fold higher than the control with LHDAg (Fig. 4C), while it did

not increase with SHDAg (data not shown). To determine whether this activation

by LHDAg was mediated by the CArG box, we mutated the CArG box and then

measured luciferase activity with LHDAg or without LHDAg. Mutation of the

CArG box was introduced into the indicated element in the context of the wild

type reporter construct (Fig. 4A) and the influence was examined. As shown in

Fig. 4C, the effect of LHDAg on the c-fos promoter was decreased when the

CArG box was mutated. Since LHDAg had no effect on Elk1, this activation of

the c-fos promoter occurred through the transcriptional activation of SRF by


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

16

LHDAg. These results suggest that activation of the SRE-dependent signal by

LHDAg is dependent on enhancement of the transcriptional ability of SRF, not

Elk1.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

17

DISCUSSION

In this study, LHDAg clearly activated SRF-associated pathways by

enhancing the transcriptional ability of SRF without changing its DNA-binding

affinity. We confirmed this LHDAg-induced activation not only by using

plasmids with synthetic enhancers, but also by using plasmids with the rat SM22α

promoter and the human c-fos promoter as well as a plasmid with the yeast GAL4

upstream activation sequence. SRF was identified as a critical factor involved in

mediating serum and growth factor-induced transcription from the c-fos proto-

oncogene (36). The SRF binding site in the promoters of immediate-early genes c-

fos and pip92 (37) is called SRE, which is composed of two elements, the CArG

box (CCATATTAGG) and the nearby Ets motif (AGGAT). SRF binds to the

CArG box as a dimer whereas Elk1 can not do so by itself, but it binds to the Ets

motif by making a ternary complex with SRF (34, 36). Although the activation of

c-fos SRE through the MAPK cascade is mainly dependent on Elk1 (32-34), SRF

can solely activate c-fos SRE in response to serum growth factors and intracellular

activation of heterotrimeric G protein (29).

Several viral transforming proteins such as human T-cell leukemia virus

type I (HTLV-I) activator protein Tax, the polyomavirus middle-T antigen, and

HBV X protein (HBx) target c-Fos induction (25, 38-40). Tax and the

polyomavirus middle-T antigen enhance c-Fos induction through SRF (38, 39).

Tax directly binds to SRF to activate transcription. On the other hand, middle-T

antigen activates transcription via Rac activation in the cytoplasm, but the

pathway from Rac to SRF is unknown. Although LHDAg is a nuclear protein, we

could not observe an interaction between LHDAg and SRF by

immunoprecipitation (data not shown). It has been shown that stimulation by


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

18

growth hormone, angiotensin II, and HMG-I induce the transcriptional activation

of SRF by enhancing binding activity of SRF to the CArG box (26, 41, 42).

LHDAg, however, did not increase SRF binding to the CArG box, while PKA

enhanced the binding (Fig. 3B). Although the mechanism underlying activation of

the SRF-dependent signaling pathway has not been completely elucidated, there is

mention that PKA is required for SRF nuclear import (43). PKA has also been

shown to activate the CRE-dependent pathway. Therefore, this evidence together

with a lack of activation of the CRE-dependent pathway by LHDAg (Fig.1A)

leads to the conclusion that SRF activation by LHDAg seems to be PKA-

independent. These results suggest that LHDAg activates SRF-associated

transcription through a novel mechanism.

In the case of HBx, results similar to those of LHDAg have been noted (44).

Although HBx can not bind double strand DNA directly and does not act as a

transcriptional activator (45), it can transactivate many cellular genes. HBx

activates transcription through its interaction with a variety of transactivators

(46,47) and transcription factors such as TFIIB, TFIIH, and RNA polymerase

subunit 5 (48,49). Therefore, a possible mechanism underlying the enhancement

of transcriptional activation of SRF by LHDAg could be that LHDAg functions as

a co-transactivator. Since HDAgs have no RNA polymerase activity, HDV

requires host cellular proteins such as RNA polymerase II for replication of its

genome (50,51). Since both HDAgs are known to participate in HDV replication,

it is not difficult to speculate that HDAgs may recruit a transcription factor or

RNA polymerase II related proteins for its viral replication. In fact, HDAg

interacts with nuclear proteins such as DIPA, nucleolin, and karyopherin alpha2

(52-54). Overexpression of nucleolin (nucleolin expressing plasmid pCGN-Nu

was kindly provided by Dr. S. C. Lee, National Taiwan University, Taiwan (55))


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

19

activated the SRF-dependent pathway at a value two times higher than that with

LHDAg alone (data not shown). Although there is no evidence that DIPA or

karyopherin alpha2 mediates transcription, nucleolin has been implicated in

rDNA transcription, rRNA maturation, ribosome assembly, and nucleo-

cytoplasmic transport (56). Therefore, it is possible that nucleolin plays an

important role in the enhancement of transcriptional activation of SRF by

LHDAg.

Recently, Wei et al. showed that LHDAg, but not SHDAg, could activate

transcription from several promoters, including the AP-1 binding site (57). Their

findings are consistent with our results that only LHDAg can activate

transcription, although Wei et al. did not investigate the mechanism of how

LHDAg activates the pathway. Although transfection of 0.2 µg pCXN2-DL did

not activate the AP-1-dependent pathway, transfection of 0.4 µg pCXN2-DL

activated the pathway by approximately 2.5-fold higher than control (data not

shown). Thus, LHDAg may be a potent activator of the AP-1-dependent pathway

when expressed in a relatively large amount. On the other hand, Lo et al.

demonstrated that both LHDAg and SHDAg inhibited SP1-activated and basal

RNA polymerase II transcription (58). Although we did not examine the effect of

HDAgs on SP-1-associated or basal RNA polymerase II transcription, it may be

possible that LHDAg inhibits this transcription by withholding the key

transcriptional factors necessary for the activation of SRF-dependent pathways.

LHDAg activated a rat SM22α promoter having a CArG box. The CArG

box has been identified as a constituent in the promoters of a number of muscle-

specific genes, including SM22α (28), α-smooth muscle actin (α-SMA) (59), and

the cardiac and skeletal muscle actin (60, 61). LHDAg may enhance the

transcription of these muscle-specific genes containing a CArG box. For example,


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

20

α-SMA is expressed by activated hepatic stellate cells, a kind of hepatic

sinusoidal cell that causes hepatic fibrosis (62). Therefore, there may be a

possibility that LHDAg upregulates transcription of α-SMA through SRF, causes

activation of hepatic stellate cells, and induces hepatic fibrosis. LHDAg also

activated the c-fos promoter through SRF. A number of experiments have

suggested that c-Fos plays a critical role in the response to growth factors and that

aberrant production of c-Fos protein can lead to oncogenesis. Interestingly,

hepatitis delta patients with primary hepatocellular carcinoma were younger than

patients infected by HBV alone (63). It is tempting to speculate that LHDAg may

promote cell proliferation through the induction of c-Fos protein.

In summary, LHDAg, but not SHDAg, activates SRF-associated pathways.

This observation may provide clues to help clarify the functional differences

between LHDAg and SHDAg and the pathogenesis of delta hepatitis.

Acknowledgments-We would like to thank Drs. Taylor J, Miyazaki J, Treisman R,

Lee ME, Lee SC, Ishikawa T, and Fujii M for plasmids.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

21

REFERENCES

1. Rizzetto, M., Canese, M. G., Arico, S., Crivelli, O., Trepo, C., Bonino, F., and

Verme, G. (1977) Gut. 18, 997-1003

2. Rizzetto, M. (1983) Hepatology 3, 906-912

3. Wang, K.-S., Choo, Q.-L., Weiner, A.-J., Ou, J.-H., Najarian, R. C., Thayer, R.

M., Mullenbach, G. T., Denniston, K. J., Gerin, J. L., and Houghton, M. (1986)

Nature 323, 508-514

4. Makino, S., Chang, M.-F., Shieh, C.-K., Kamahora, T., Vannier, D. M.,

Govindarajan, S., and Lai, M. M. C. (1987) Nature 329, 343-346

5. Bergmann, K. F., and Gerin, J. L. (1986) J. Infect. Dis. 154, 702-706

6. Imazeki, F., Omata, M., and Ohto, M. (1990) J. Viol. 64, 5594-5599

7. Wang, J.-G., and Lemon, S.M. (1993) J. Virol. 67, 446-454

8. Xia, Y.-P., Yeh, C.-T., Ou, J.-H., and Lai, M. M. C. (1992) J. Virol. 66, 914-

921

9. Chang, M.-F., Chang, S. C., Chang, C.-I., Wu, K., and Kang, H.-Y. (1992). J.

Virol. 66, 6019-6027

10. Chang, M. F., Baker, S. C., Soe, L. H., Kamahora, T., Keck, J. G., Makino, S.,

Govindarajan, S., and Lai, M. M. C. (1988). J. Virol. 62, 2403-2410

11. Lin, J. H., Chang, M.-F., Baker, S.C., Govindarajan, S., and Lai, M. M. C.

(1990) J. Virol. 64, 4051-4058

12. Kuo, M. Y. P., Chao, M., and Taylor, J. (1989) J. Virol. 63, 1945-1950

13. Taylor, J. (1993) Prog. Clin. Biol. Res. 382, 1-4

14. Chao, M., Hsieh, S.-Y., and Taylor, J. (1990) J. Virol. 64, 5066-5069

15. Chang, F.-L., Chen, P.-J., Tu, S.-J., Wang, C.-J., and Chen, D.-S.(1991) Proc.

Natl. Acad. Sci. USA 88, 8490-8494


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

22

16. Glenn, J. S., Watson, J. A., Havel, C. M., and White, J. M. (1992) Science 256,

1331-1333

17. Wu, J.-C., Chen, P.-J., Kuo, M.-Y., Lee, S.-D., Chen, D.-S., and Ting, L.-P.

(1991) J. Virol. 65, 1099-1104

18. Hoofnagle, J. H. (1989) J. Am. Med. Assoc. 261, 1321-1325

19. MacNaughton, T. B., Gowans, E. J., Jilbert, A. R., and Burrell, C. J. (1990)

Virology 177, 692-698

20. Bichko, V., Barik, S., and Taylor, J. (1997) J. Virol. 71, 512-518

21. Kuo, M. Y. .P., Goldberg, J., Coates, L., Mason, W. S., Gerin, J., and Taylor, J.

(1988) J. Virol. 63, 1855-1861

22. Niwa, H., Yamamura, K. and Miyazaki, J. (1991) Gene 108, 193-199

23. Togo, G., Toda, N., Kanai, F., Kato, N., Shiratori, Y., Kishi, K., Imazeki, F.,

Makuuchi, M., and Omata, M. (1996) Cancer Res. 56, 5620-5623

24. Marais, R., Wynne, J., and Treisman, R. (1993) Cell 73, 381-393

25. Fujii, M., Chuhjo, T., Minamino, T., Nomura, M., Miyamoto, K., and Seiki,

M. (1995) Oncogene 11, 7-14

26. Chin, M. T., Pellacani, A., Wang, H., Lin, S. S. J., Jain, M. K., Perrella, M. A.,

and Lee, M.-E. (1998) J. Biol. Chem. 273, 9755-9760

27. Ishikawa, T., Igarashi, T., Hata, K., and Fujita, T. (1999) Biochem. Biophys.

Res. Commun. 254, 566-571

28. Kemp, P. R., Osbourn, J. K., Grainger, D. J., and Metcalfe, J. C. (1995)

Biochem. J. 310, 1037-1043

29. Hill, C.S., and Treisman, R. (1995) EMBO J. 14, 5037-5047

30. Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989) Nucleic

Acids Res. 17, 6419

31. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: A


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

23

Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring

Harbor, NY

32. Hill, C. S., Marais, R., John, S., Wynne, J., Dalton, S., and Treisman, R.

(1993) Cell 73, 395-406

33. Gille, H., Kortenjann, M., Thomae, O., Moomaw, C., Slaughter, C., Cobb, M.

H., and Shaw, P. E. (1995) EMBO J. 14, 951-962

34. Treisman, R. (1994) Curr. Opin. Geneti Dev. 4, 96-101

35. Kortenjann, M., Thomae, O., and Shaw, P. E. (1994) Mol. Cell. Biol. 14,

4815-24

36. Treisman, R., and Ammerer, G. (1992) Curr. Opin. Geneti Dev. 2, 221-226

37. Latinkic, B. V., and Lau, L. F. (1994) J. Biol. Chem. 269, 23163-23170

38. Fujii, M., Tsuchiya, H., Chuhjo, T., Akizawa, T., and Seiki, M. (1992) Genes

Dev. 6, 2066-2076

39. Urich, M., Senften, M., Shaw, P. E., and Ballmer-Hofer, K. (1997) Oncogene

14, 1235-1241

40. Avantaggiati, M. L., Natoli, G., Balsano, C., Chirillo, P., Artini, M., DeMarzio,

E., Collepardo, D., and Levrero, M. (1993) Oncogene 8, 1567-1574

41. Liao, J., Hodge, C., Meyer, D., Ho, P. S., Rosenspire, K., and Schwartz, J.

(1997) J. Biol. Chem. 272, 25951-25958

42. Hautmann, M. B., Thompson, M. M., Swartz, E. A., Olson, E. N., and Owens,

G. K. (1997) Circ. Res. 81, 600-610

43. Gauthier-Rouviere, C., Vandromme, M., Lautredou, N., Cai, Q. Q., Girard, F.,

Fernandez, A., and Lamb, N. (1995) Mol. Cell. Biol. 15, 433–444

44. Lin, Y., Tang, H., Nomura, T., Dorjsuren, D. Hayashi, N., Wei, W., Ohta, T.,

Roeder, R., and Murakami, S. (1998) J. Biol. Chem. 273, 27097-27103

45. Yen, B. T. S. (1996) J. Biomed. Sci. 3, 20-30


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

24

46. Maguire, H. F., Hoeffler, J. P., and Siddiqui, A. (1991) Science 252, 842-844

47. Lin, Y., Nomura, T., Yamashita, T., Dorjsuren, D., Tang, H., and Murakami,

S. (1997) Cancer Res. 57, 5137-5142

48. Haviv, I., Vaizel, D., and Shaul, Y. (1996) EMBO J. 15, 3413-3420

49. Cheong, J. H., Yi, M., Lin, Y., and Murakami S. (1995) EMBO J. 14, 143-150

50. MacNaughton, T. B., Gowans, E. J., McNamara, S. P., and Burrell, C. J.

(1991) Virology 184, 387-390

51. Fu, T.-B., and Taylor, J. (1993) J. Virol. 67, 6965-6972

52. Brazas, R., and Ganem, D. (1996) Science 274, 90-94

53. Lee, C. H., Chang, S. C., Chen, C. J., Chang, M. F. (1998) J. Biol. Chem. 273,

7650-7656

54. Chou, H. C., Hsieh, T. Y., Sheu, G. T., Lai, M. M. (1998) J. Virol. 72, 3684-

3689

55. Yang, T., H., Tsai, W., H., Lee, Y., M., Lei, H., Y., Lai, M., Y., Chen, D., S.,

Yeh, N., H., and Lee, S., C. (1994) Mol. Cell. Biol. 14, 6068-6074

56. Ginisty, H., Sicard, H., Roger, B., and Bouvet, P. (1999) J. Cell Sci. 112, 761-

772

57. Wei, Y., and Ganem, D. (1998) J. Virol. 72, 2089-2096

58. Lo, K., Sheu, G.-T., and Lai, M.M.C. (1998) Virology 247, 178-188.

59. Min, B. H., Foster, D. N., and Strauch, A. R. (1990) J. Biol. Chem. 265,

16667-16675

60. Muscat, G. E., Gustafson, T. A., and Kedes, L. (1988) Mol. Cell. Biol. 8,

4120–4133

61. Walsh, K. (1989) Mol. Cell. Biol. 9, 2191–2201

62. Friedman SL. (1993) N. Engl. J. Med. 328, 1828-35

63. Oliveri, F., Brunetto, M. R., Baldi, M., Piantino, P., Ponzetto, A., Forzani, B.,


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

25

Smedile, A., Verme, G., and Bonino, F. (1991) Prog. Clin. Biol. Res. 364, 217-

222


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

26

The abbreviations used are:

HDAg, hepatitis delta antigen; HBV, hepatitis B virus; HDV, hepatitis delta virus;

SHDAg, small hepatitis delta antigen; LHDAg, large hepatitis delta antigen; SRF,

serum response factor; SRE, serum response element; NF-κB, nuclear factor κB;

AP-1, activator protein 1; CRE, cyclic AMP response element; EMSA,

electrophoretic mobility shift assay; DNA-BD, DNA binding domain; PKA,

protein kinase A; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine

serum; TCFs, ternary complex factors; MAPK, mitogen-activated protein kinase;

α-SMA, α-smooth muscle actin; HTLV1, human T-cell leukemia virus type 1;

HBx, hepatitis B virus X protein.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

27

FIGURE LEGEND

Fig. 1. LHDAg activates SRF-associated signal transduction pathways.

A, The effect of HDAgs on various synthetic promoters. Reporter

plasmids, pSRF-Luc, pSRE-Luc, pNF-κB-Luc, pAP1-Luc, and pCRE-Luc were

cotransfected into HeLa cells with pCXN2, pCXN2-DL, or pCXN2-DS. The

results are expressed as the fold of luciferase activity above that induced from the

reporter plus pCXN2 plasmid. Plasmid expressing seapansy luciferase was used

as an internal control for transfection efficiency. Data shown are the average +

S.D. of more than three independent experiments. Western blotting shows the

expression of LHDAg (lane 1) and SHDAg (lane 2). The left bar indicates the

position at 27 and 24 kDa and the expected size of LHDAg and SHDAg,

respectively.

B, Activation of SRF by LHDAg in various cell lines. HeLa, HuH7, and

HepG2 cells were transfected with pSRF-Luc and the pCXN2 or pCXN2-DL.

Cells were assayed for luciferase activity. The results are expressed as the fold of

luciferase activity above that induced in the absence of HDAg.

C, LHDAg activates SRF-dependent pathway in a dose-dependent

manner. Increasing amounts of LHDAg expressing plasmid (0, 0.05, 0.1, 0.2 µg)

were cotransfected into HeLa cells with pSRF-Luc. pCXN2 was added to each

transfection to keep the total amount of DNA constant (0.4 µg). * p<0.05 vs.

control.

Fig. 2. The presence of SHDAg has no influence on activation of the SRF-

dependent pathway by LHDAg.

0.1 µg of pSRF-Luc was transiently cotransfected into HeLa cells with 0.1


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

28

µg of pCXN2-DL and with: lanes 2-4, pCXN2-DS, 0.033, 0.1, 0.2 µg,

respectively. pCXN2 was added to each transfection to keep the total amount of

DNA constant (0.4 µg).

Fig. 3. LHDAg enhances transcriptional activation of SRF, but not Elk1.

A, LHDAg enhances transcriptional activation of SRF. pFR-Luc having

the firefly-luciferase gene controlled by the yeast GAL4 upstream activation

sequence, pRL-TK and either GAL4-SRF expressing plasmid or empty vector

were cotransfected into HeLa cells with pCXN2 or pCXN2-DL. The results of

luciferase assay are expressed as the fold of luciferase activity above that induced

from the reporter plus pCXN2 without GAL4-SRF expressing plasmid. * p<0.05

B, No increase in SRF binding to the CArG box by LHDAg. Nuclear

extracts of HeLa cells transfected only with pCXN2 (lane 2), or 0.3 (lane 3, 5, and

6) or 0.9 µg (lane 4) of pCXN2-DL were incubated with 0.035 pmol of the CArG

box-radiolabeled probe. Lane 5 showed a supershifted band with use of the anti-

SRF antibody. The CArG box-unlabeled competitor was added at a 100-fold

molar excess (lane 6). Nuclear extracts of HeLa cells transfected with pFC-PKA

(lane 1) were incubated with 0.035 pmol of the CArG box-radiolabeled probe.

C, LHDAg does not enhance transcriptional activation of Elk1. pFR-

Luc, pRL-TK and Gal4-Elk1 expressing plasmid was cotransfected into HeLa

cells with pCXN2 or pCXN2-DL. The results of luciferase assay are expressed as

the fold of luciferase activity above that induced from the reporter plus pCXN2

without GAL4-Elk1 expressing plasmid. The right upper Western blotting showed

Elk1 and phosphorylated Elk1. HeLa cells were transiently transfected with

pCXN2-Elk1 and pCXN2 or pCXN2-DL in DMEM supplemented with 0.5%

FBS. Forty-eight hours after transfection, whole cell lysates were harvested for


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8/17/00 Kato

29

Western blotting analysis. Elk1 and phosphorylated Elk1 were detected by anti-

Elk1 antibody and anti-phospho-specific Elk1 (Ser 383) antibody.

Fig. 4. The effect of LHDAg on the natural promoters.

A, Schematic representation of reporter constructs used. Site-specific

mutations that disrupt the CArG box (see “Experimental Procedures”) were

introduced into SM22α-luc and HF456 having a luciferase gene under the human

c-fos promoter.

B, The effect of LHDAg on the SM22 promoter. The indicated reporter

constructs were cotransfected into HeLa cells with pCXN2 or pCXN2-DL. The

results of luciferase assay are expressed as the fold of luciferase activity above

that induced from the SM22α-luc plus pCXN2 plasmid. * p<0.05 vs. control.

C, The effect of LHDAg on the c-fos promoter. HF456 having a

luciferase gene under the human c-fos promoter containing SRE, or HF456 m1

having mutated CArG box was cotransfected into HeLa cells with pCXN2 or

pCXN2-DL. A luciferase assay was performed and the fold of luciferase activity

is shown. * p<0.05 vs. control.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Yasushi Siratori and Masao OmataTadashi Goto, Naoya Kato, Suzane Kioko Ono-Nita, Hideo Yoshida, Motoyuki Otsuka,

Factor-Associated TranscriptionLarge Isoform of Hepatitis Delta Antigen Activates Serum Response

published online August 28, 2000J. Biol. Chem.

10.1074/jbc.M002947200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M002947200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M002947200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2000/08/28/jbc.M002947200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2000/08/28/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2000/08/28/jbc.M002947200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

1 large isoform of hepatitis delta antigen activates serum

Documents