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J. Biol. Chem., Vol. 277, Issue 10, 7645-7647, March 8, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/10/7645    most recent C100729200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rang, A. Articles by Will, H. Search for Related Content PubMed PubMed Citation Articles by Rang, A. Articles by Will, H.

ACCELERATED PUBLICATION
Antiviral Activity of Interferon- against Hepatitis B Virus Can Be Studied in Non-hepatic Cells and Is Independent of MxA^*

Andreas Rang, Michael Bruns, Tilman Heise, and Hans Will

From the Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistraße 52, 20251 Hamburg, Germany

Received for publication, December 14, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

It is well established that interferon- can induce non-cytotoxic intracellular suppression of hepatitis B virus replication, but the mechanisms involved are unclear. Cell culture studies to characterize these mechanisms are restricted, in part because hepatitis B virus replicates almost exclusively in liver-derived cells. To overcome this limitation we used a cytomegalovirus promoter-controlled hepatitis B virus expression system, which leads to intracellular viral replication even in non-hepatic cell lines. In this experimental system interferon- treatment specifically suppressed viral replication demonstrating that antiviral activities against hepatitis B virus are not restricted to hepatic cells. Furthermore, the interferon-inducible MxA protein was recently reported to play a key role in the antiviral action of interferon- against hepatitis B virus. Our data demonstrate that interferon- also suppresses hepatitis B virus replication in MxA-deficient HEp2 cells, indicating that MxA is not essential for these activities. Taken together, our data imply that the experimental approach presented can also be adapted to established cell lines which are deficient in parts of the signal transduction pathway or other elements located further downstream, providing important insights into mechanisms specifically suppressing hepatitis B virus.

	INTRODUCTION

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Hepatitis B virus (HBV)¹ is a small (3.2 kb) enveloped DNA virus, the replication of which involves reverse transcription of a pregenomic RNA within nucleocapsids in the cytoplasm, resulting in production of replicative DNA intermediates (1). Thereafter, nucleocapsids containing replicative intermediates are converted into mature virions and shuttled out of the cell. Transcripts essential for HBV replication in vitro are unspliced and share an identical 3'-region, which contains a posttranscriptional response element (PRE) (2-5). The PRE is supposed to participate in nucleocytoplasmic transport of the viral RNA (6, 7), essential for initiation of the cytoplasmic steps of viral replication. Worldwide, more than 300 million people are chronically infected with HBV (6). Treatment with interferon- (IFN) is one of the few effective therapies for chronic HBV carriers. However, it results in efficient reduction of the viral load only in 10-30% of treated patients and rarely, if at all, in complete elimination of the virus. So far, the mechanisms responsible for the IFN-mediated reduction of the viremia in responding patients are elusive. IFN treatment of cells is known to induce an antiviral state, which prevents productive infection by several viruses (8-11), and it is well established that this state also inhibits replication of HBV in liver-derived cell lines (12-20). The antiviral state is established by induction of the expression of a wide pattern of different genes and the functional activation of a variety of cellular proteins. Among others, the IFN-inducible human MxA protein is per se known to confer antiviral activity against several RNA viruses (10, 21-23) and was also recently reported to block the replication of HBV in the human hepatoma cell line Huh7 after transient transfection of HBV-DNA (14). Compared with control cells, in cells stably expressing ectopic MxA the levels of cytoplasmic HBV-RNA and replicative DNA intermediates were reduced. Furthermore, the reduced levels of the cytoplasmic RNA were associated with down-regulation of the PRE activity. From these and other published data, the authors conclude that it is likely that MxA protein plays a key role in the antiviral action of IFN against HBV, since IFN and MxA have comparable inhibitory effects on HBV (14).

So far, in vitro studies of drug-induced intracellular antiviral mechanisms against HBV are restricted to liver-derived cell lines, since activity of the pregenomic promoter depends on liver-specific transcription factors (24). To determine whether MxA is required for IFN-induced suppression of HBV, we established a HBV expression system, which allows expression of replicative DNA intermediates even in non-hepatic cell lines and used a non-hepatic cell line, which does not express endogenous MxA (HEp2) (22). Using this experimental system, we demonstrate that IFN-induced suppression of HBV replication is not restricted to hepatoma cells and that MxA is dispensable for this activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Plasmids-- Plasmid pHBV-dimer contains the HBV-DNA of subtype ayw (25) as head to tail linked dimer cloned via the EcoRI site (17). Plasmid pCMV-HBV is a CMV promoter-controlled HBV expression construct (kindly provided by Heinz Schaller, containing a 1.3-fold overlength HBV genome, the HBV pregenomic RNA synthesized from this construct is driven by the immediate early promoter of CMV). Plasmid pCMV-SEAP contains the secreted alkaline phosphatase (SEAP) gene under control of the same CMV promoter. Expression of SEAP was used to standardize efficiency of transfection and to monitor potential IFN-mediated cytotoxic effects.

Cell Culture and Transfections-- The human hepatoma (Huh7) and the human cervix carcinoma (HEp2) cell lines were grown as monolayer in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Plasmid DNAs used for transfection were purified by ion exchange chromatography and transfected using FuGENE 6 transfection reagent (Roche Deutschland Holding GmbH, Grenzach-Wyhlen, Germany) according to the protocol supplied. Cells (1.3 × 10⁶ cells per 5-cm plate) were transfected with 2 µg of the corresponding HBV expression plasmids and 0.25 µg of pCMV/SEAP. Sixteen hours after transfection cells were treated with human IFN-2b (1,000 IU/ml, Intron A, Essex Pharma, Munich, Germany) for 48 h and harvested for analysis afterward. The amount of SEAP activity secreted into the medium of transfected cells was determined by quantification of the SEAP activity as increase of the optical density nm × 10³ at 405 (mOD) per min (mOD/min) as described previously (17).

Purification of HBV-DNA from Intracellular Core Particles and Southern Blot Analysis-- Isolation of HBV-DNA from cytoplasmic core particles was performed as described recently (18). DNA was separated on a 1.2% agarose gel, blotted onto Hybond N nylon membranes (Amersham Biosciences, Inc., Buckinghamshire, United Kingdom) and hybridized with a ³²P-labeled full-length HBV-DNA fragment. Blots were exposed to Fuji imaging screens and signals were quantified by a Fujix BAS 2000 bio-imaging analyzer (Fuji, Tokyo, Japan) and by TINA software (Raytest, Straubenhardt, Germany).

Western Blot Analysis-- Protein extracts were prepared as described previously (17). Extracts were separated by use of a 5-20% gradient SDS-PAGE and blotted onto polyvinylidene difluoride membrane (Bio-Rad). After incubation of the lower part of the membrane with core-specific (17) and the upper part with an MxA-specific rabbit antiserum (kindly provided by Peter Stäheli) and a rabbit-specific horseradish peroxidase-coupled secondary antiserum (Dianova, Hamburg, Germany, 1:50,000), the signals were detected by enhanced chemiluminescence (Pierce) visualized by exposure to x-ray films.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

We have established a transient HBV expression system in Huh7 cells that allows determination of IFN-induced antiviral activities against HBV (17, 18). After transient transfection with the plasmid pHBV dimer into Huh7 cells, addition of IFN induces specific posttranscriptional suppression of the viral transcripts, proteins, and encapsidated DNA intermediates (17). To determine whether MxA is necessary for IFN-mediated suppression of HBV we adapted the established test system for HEp2 cells, which do not express MxA and are not liver-derived cells (22). Because transcription of the HBV promoter that controls expression of the pregenomic RNA requires liver-specific factors (24) and is therefore not supported in the HeLa-derived HEp2 cells (data not shown), a CMV-controlled HBV expression construct (pCMV-HBV) was used for the study. HEp2 or Huh7 cells were transfected with pCMV-HBV or pHBV-dimer, respectively, and pCMV-SEAP. Afterward, cells were treated with 1,000 IU/ml IFN and harvested for analysis 48 h later. As shown in Fig. 1, transfection of pCMV-HBV into HEp2 cells resulted in production of intracellular replicative DNA intermediates, indicating that the pregenomic RNA expressed under control of the CMV promoter is identical to the one expressed from the authentic pregenomic promoter. In samples from cells treated with IFN the amount of replicative DNA intermediates was reduced to 36% in HEp2 and to 47% in Huh7 cells, compared with untreated controls. These values reflect quantified signal intensities standardized with the expression levels of the control gene SEAP (Fig. 1). The expression levels of the cotransfected control gene SEAP were not affected by the IFN-treatment (Fig. 1), indicating that the activities induced specifically suppress HBV in HEp2 and Huh7 cells. Furthermore, these data suggest that IFN induces antiviral activities against HBV that act on a posttranscriptional level, since expression of the control gene SEAP and of the pregenomic RNA are controlled by the same CMV promoter.

View larger version (72K): [in this window] [in a new window]   Fig. 1.   IFN induces suppression of HBV DNA replication in Huh7 and HEp2 cells. Southern blot analysis of intracellular replicative DNA intermediates (upper panel) and expression levels of the control gene SEAP (lower panel) derived from Huh7 or HEp2 cells transfected with HBV-DNA and pCMV-SEAP and treated with or without IFN for 48 h.

The effect of IFN on the levels of viral core protein in MxA-negative HEp2 and in MxA-positive Huh7 cells was analyzed by Western blotting. The top panel of Fig. 2 demonstrates IFN-induced MxA expression in Huh7 but not in HEp2 cells. Irrespective of the presence of MxA, the IFN treatment reduced the amount of core protein to a similar extent in both cell types compared with untreated controls. After Western blot analysis the membrane was stained with Amido Black (Fig. 2), demonstrating that equal amounts of proteins were loaded onto the gel.

View larger version (68K): [in this window] [in a new window]   Fig. 2.   IFN induces suppression of HBV core protein in Huh7 and HEp2 cells, irrespective of the presence of MxA. Western blot analysis of intracellular MxA and core levels were analyzed from cells treated as described in the legend to Fig. 1. After Western analysis the membrane was stained with Amido Black to control consistent loading of the gel.

Taken together, our data clearly demonstrate that IFN induces suppression of HBV replication in vitro in the absence of MxA, indicating that MxA is dispensable for the antiviral activity in HEp2 cells. Whether these mechanisms differ from that exerted by MxA reported previously (14) remains to be studied.

Gordien et al. (14) reported that IFN-induced depletion of cytoplasmic HBV-RNA is associated with suppression of the PRE-activity as determined by reporter gene analysis and suggested that this inhibition is, at least in part, responsible for IFN-induced HBV suppression. However, data published previously indicate that the activity of the PRE is not required for IFN-induced suppression of the viral RNA, because RNA synthesized from a subgenomic region (nucleotide position 1-1,306, pRK-HBs/Eco) that lacks a functional PRE is also efficiently degraded in response to IFN (17). Thus, it is unlikely that the PRE is a major target for the antiviral activity mediating IFN-induced degradation of the viral RNA.

At least as important, the results demonstrate for the first time that IFN-induced posttranscriptional suppression of HBV replication can be studied not only in liver-derived cell lines but also in different non-hepatic cell lines (Vero, data not shown), and probably in all cell lines that support the function of the CMV promoter or similar promoters that can replace that of the pregenomic HBV RNA. Thus, the experimental approach presented can be adapted to established cell lines, which are deficient in parts of the IFN-signal transduction pathway or that lack certain genes induced downstream of this pathway (11, 26-28). These studies are likely to provide important insights into mechanisms that specifically suppress HBV.

	ACKNOWLEDGEMENT

We appreciate critical reading of the manuscript by David Zuckerman.

	FOOTNOTES

^* This work was supported by grants from the Bundesministerium für Bildung und Forschung and the Deutsche Forschungsgemeinschaft. The Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie is supported by the Bundesministerium für Gesundheit und the Freie and Hansestadt Hamburg.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.

To whom correspondence should be addressed: Bundesinstitut für Gesundheitlichen Verbraucherschutz und Veterinärmedizin, Thielallee 88-92, 14195 Berlin, Germany. Tel.: 49-188-8412-3716; Fax: 49-188-8412-3635; E-mail: a.rang@bgvv.de.

Published, JBC Papers in Press, January 7, 2002, DOI 10.1074/jbc.C100729200

	ABBREVIATIONS

The abbreviations used are: HBV, hepatitis B virus; PRE, posttranscriptional response element; IFN, interferon-; SEAP, secreted alkaline phosphatase; CMV, cytomegalovirus.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/10/7645    most recent C100729200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rang, A. Articles by Will, H. Search for Related Content PubMed PubMed Citation Articles by Rang, A. Articles by Will, H.