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Originally published In Press as doi:10.1074/jbc.M002947200 on August 28, 2000

J. Biol. Chem., Vol. 275, Issue 48, 37311-37316, December 1, 2000
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Large Isoform of Hepatitis Delta Antigen Activates Serum Response Factor-associated Transcription*

Tadashi Goto, Naoya KatoDagger, Suzane Kioko Ono-Nita, Hideo Yoshida, Motoyuki Otsuka, Yasushi Shiratori, and Masao Omata

From the Department of Gastroenterology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan

Received for publication, April 7, 2000, and in revised form, August 16, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hepatitis delta virus 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 hepatitis delta virus, 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 kappa B-, 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 SM22alpha 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hepatitis delta antigen (HDAg)1 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 masses, 24 kDa), and the large form consists 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, whereas LHDAg was thought to reduce this 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 kappa B (NF-kappa B)-, activator protein 1 (AP-1)-, and cyclic AMP response element (CRE)-dependent pathways.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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' () 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 beta -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 (QuikChange 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' terminus 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 (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 (), 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): five repeats of the binding sites for SRF (gtCCATATTAGGac, pSRF-Luc), five repeats of SRE (AGGATgtCCATATTAGGacatct, pSRE-Luc), five repeats of the binding sites for NF-kappa B (tGGGGACTTTCCgc, pNF-kappa B-Luc), seven repeats of the binding sites for AP-1 (TGACTAA, pAP1-Luc), and four repeats of CRE (agccTGACGTCAgag, pCRE-Luc). A control plasmid expressing Renilla reniformis (sea pansy)-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 cytomegalovirus promoter, was utilized.

In addition to these reporter plasmids that contain synthetic promoters, SM22alpha -Luc, a luciferase reporter plasmid with a rat smooth muscle-specific gene SM22alpha 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, Boston, 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 SM22alpha -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 SM22alpha -Luc, the CArG box 1 at base pair -162 to -153 was converted from CCAAATATGG to AAAAATATGG (SM22alpha m1-Luc); the CArG box 2 at base pair -283 to -266 was converted from CCATAAAAGGTTTTTCCC to CCATAAAAAATTTTTCCC (SM22alpha m2-Luc). Combinations of mutations were generated by performing mutagenesis reactions on a template already containing a mutation (SM22alpha m1m2-Luc). As for the HF456, the CArG box at base pair -314 to -305 was converted from CCATATTAGG 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 that 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, Japan). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO2.

Transfection and Luciferase Assays-- Approximately 4 × 105 HeLa, HuH7, or HepG2 cells were plated into a 6-well tissue culture plate (Iwaki Glass, Chiba, Japan) 24 h 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 of reporter plasmids, 0.05 µg of pFA-GALElk or pSG5-GALSRF, and 0.2 µg of pCXN2, pCXN2-DL, or pCXN2-DS. For evaluation of the transcriptional ability of LHDAg, 0.2 µg of reporter plasmids and 0.2 µg of pM vector or pM-GAL4DL were used. For the assay using HF456 containing the c-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 sea pansy luciferase activity. The luciferase activity of the cells that were transfected with the reporter plasmid plus pCXN2 was set arbitrarily at 1.0, and then the relative luciferase activity was compared with 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-2Kk 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 h, cells were treated with 0.05% trypsin and dispersed by being transferred by pipet into single-cell suspensions after the addition of 100 µl of 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-2Kk and incubated for 15 min at room temperature. Then magnetically labeled cells were recovered by the magnetic separation column and used for an EMSA.

EMSA-- Annealed oligonucleotides for the CArG box (5'-GGATGTCCATATTAGGACATC-3') were end-labeled with [alpha -32P]ATP using T4 polynucleotide kinase. Nuclear extracts were obtained from HeLa cells transfected with pCXN2, pCXN2-DL, or pFC-PKA as described previously (30). The protein concentration of nuclear extracts was measured by a micro-BCA protein assay reagent kit (Pierce) and was 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) was 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 min 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 × Tris borate-EDTA at 4 °C. 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 Pharmacia Biotech) using the soluble protein extracts of HeLa cells that were transfected with pCXN2, pCXN2-DL, or pCXN2-DS (31). HDAg expression was examined using serum obtained from a patient with 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of SRF- and SRE-dependent Signal Transduction Pathways by LHDAg-- Each of the five reporter plasmids (pSRF-Luc, pSRE-Luc, pNF-kappa B-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 ± S.D.)-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 mass of 27 kDa and SHDAg at a molecular mass of 24 kDa (5).


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  		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-kappa B-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 sea pansy 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 (1st lane) and SHDAg (2nd lane). 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 versus control.

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 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).


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  		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 µg of pCXN2-DL and with 0.033, 0.1, and 0.2 µg of pCXN2-DS (2nd through 4th lanes), respectively. pCXN2 was added to each transfection to keep the total amount of DNA constant (0.4 µg).

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 SM22alpha 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 (), 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.


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  		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 was used as a reporter plasmid. pFR-Luc, pRL-TK, and either GAL4-SRF-expressing plasmid or empty plasmid were cotransfected into HeLa cells with pCXN2 or pCXN2-DL. The results of the luciferase assay are expressed as the fold of luciferase activity above that induced from pFR-Luc plus pCXN2 plasmid without GAL4-SRF-expressing plasmid. pRL-TK was used as an internal control for transfection efficiency. Data shown are the average ± S.D.*, 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), 0.3 (lanes 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 having the firefly luciferase gene controlled by the yeast GAL4 upstream activation sequence was used as a reporter plasmid. pFR-Luc, pRL-TK, and Gal4-Elk1-expressing plasmid or empty 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 pFR-Luc plus pCXN2 without GAL4-Elk1-expressing plasmid. pRL-TK was used as an internal control for transfection efficiency. Data shown are the average ± S.D. 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 Western blotting analysis. Elk1 and phosphorylated Elk1 were detected by anti-Elk1 antibody and anti-phospho-specific Elk1 (Ser-383) antibody.

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 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, Inc., 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 SM22alpha Promoter-- We examined whether LHDAg activated a rat promoter containing the CArG box. By use of SM22alpha -Luc, the reporter plasmid with a SM22alpha promoter containing two CArG boxes, luciferase activity increased by a value 3.3 ± 0.5-fold higher than the control with LHDAg (Fig. 4B), whereas 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 was examined in transient transfection assays. As shown in Fig. 4B, the effect of LHDAg on the SM22alpha promoter were decreased when the CArG boxes were mutated, indicating that activation of the SM22alpha promoter by LHDAg is dependent on enhancement of the transcriptional ability of SRF.


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  		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 SM22alpha -luc and HF456 which have a luciferase gene under the human c-fos promoter. B, the effect of LHDAg on the SM22alpha 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 SM22alpha -luc plus pCXN2 plasmid. *, p < 0.05 versus 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 a 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 versus control.

LHDAg Activates the SRE-dependent Pathway through SRF, Not Elk1-- LHDAg activated not only the SRF-dependent pathway but also the SRE-dependent pathway. SRE is composed of the CArG box bound SRF and nearby Ets motif bound ternary complex factors such as Elk1. Elk1, regulated by mitogen-activated protein kinase (32-34), is phosphorylated by mitogen-activated protein kinase 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), although 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 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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 SM22alpha 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 cannot 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 mitogen-activated protein kinase 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 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, whereas 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 cannot 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 delta interacting protein A, nucleolin, and karyopherin alpha 2 (52-54). Overexpression of nucleolin (nucleolin-expressing plasmid pCGN-Nu was kindly provided by Dr. S. C. Lee, National Taiwan University, Taiwan (55)) 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 delta interacting protein A or karyopherin alpha 2 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 and Ganem (57) showed that LHDAg, but not SHDAg, could activate transcription from several promoters, including the AP-1 binding site. Their findings are consistent with our results that only LHDAg can activate transcription, although Wei and Ganem did not investigate the mechanism of how LHDAg activates the pathway. Although transfection of 0.2 µg of pCXN2-DL did not activate the AP-1-dependent pathway, transfection of 0.4 µg of 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. (58) demonstrated that both LHDAg and SHDAg inhibited SP1-activated and basal RNA polymerase II transcription. 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 SM22alpha 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 SM22alpha (28), alpha -smooth muscle actin (alpha -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, alpha -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 up-regulates transcription of alpha -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.

    ACKNOWLEDGEMENTS

We thank Drs. J. Taylor, J. Miyazaki, R. Treisman, M. E. Lee, S. C. Lee, T. Ishikawa, and M. Fujii for plasmids.

    FOOTNOTES

* This study was supported in part by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-3-3815-5411 (ext. 33070); Fax: 81-3-3814-0021; E-mail: kato-2im@h.u-tokyo.ac.jp.

Published, JBC Papers in Press, August 28, 2000, DOI 10.1074/jbc.M002947200

    ABBREVIATIONS

The abbreviations used are: HDAg, hepatitis delta antigen; HBV, hepatitis B virus; HDV, hepatitis delta virus; SHDAg, small hepatitis delta antigen; aa, amino acids; LHDAg, large hepatitis delta antigen; SRF, serum response factor; SRE, serum response element; NF-kappa B, nuclear factor kappa B; 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's medium; FBS, fetal bovine serum; alpha -SMA, alpha -smooth muscle actin; HBx, hepatitis B virus X protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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