H~l~ne Mabit, 1 Sylvie Dubanchet, 1 Francis Capel, 1 Charlie Dauguet 2 and Marie-Anne Petit 1.

Transcription

1 Journal of General Vrology (1994), 75, Prnted n Great Brtan 2681 n vtro nfecton of human hepatoma cells (HepG2) wth hepatts B vrus (HBV): spontaneous selecton of a stable HBV surface antgen-producng HepG2 cell lne contanng ntegrated HBV DNA sequences H~l~ne Mabt, 1 Sylve Dubanchet, 1 Francs Capel, 1 Charle Dauguet 2 and Mare-Anne Pett 1. 11NSERM Untd 131, 32 rue des Carnets, Clamart and 2 Untd d'oncologe Vrale, nsttut Pasteur, 28 rue du Dr Roux, Pars Cedex 15, France The degree of susceptblty of human hepatoma (HepG2) cells to drect hepatts B vrus (HBV) nfecton remans unknown. We prevously observed a low level of Dane partcle producton and vral DNA replcaton after n vtro nfecton of HepG2 cells wth serum-derved HBV. However, ths culture system appeared to be affected by varatons as human hepatocyte cultures. n the present study, HBV nfecton of HepG2 cells led to a sgnfcant ncrease n the secreton of three envelope antgens (HBsAg, pres2ag and pres1ag) at 4 days post-nfecton, and Northern blot analyss revealed the presence of both pres1 (2.6 kb) and pres2/s (2.2 kb) transcrpts. Expresson of pres1ag and the correspondng vral RNA became undetectable on 21 days post-nfecton whereas the 2"2 kb RNA speces perssted and was assocated wth secreton of subvral HBs partcles expressng pres2-eptopes and bandng between 30 and 35% sucrose. At 35 days post-nfecton (ffth passage), a sudden hgh level producton of HBsAg and pres1ag was observed, followed by a massve cell death (90 %). A stable HBsAg-producng HepG2 cell lne, desgnated HepG2-BV3, grew out of the survvng cells. HepG2- BV3 cells could ntegrate HBV DNA sequences and produce the three HBV surface antgens. Treatment wth dexamethasone ncreased the HBsAg and pres1ag secreton. Such a HBsAg-producng HepG2 cell lne obtaned by n vtro HBV nfecton seems to mmck events that occur n the naturally occurrng persstent chronc nfecton, and therefore may be an effcent n vtro model for studyng the contrbuton of vral ntegraton n the dysregulaton of HBV and lverspecfc genes expresson. ntroducton Attempts have been made to establsh an n vtro model of hepatts B vrus (HBV) that allows nfecton and replcaton of the vrus to be examned. ndeed, prevous studes have shown that HBV enters and replcates n prmary fetal human hepatocytes (Ochya et al., 1989) as well as n normal adult human hepatocytes n the presence of DMSO (Grpon et al., 1988). However, the dfferentated state of the hepatocytes n these cultures can be mantaned only for a lmted amount of tme. Propagaton of HBV n cell culture has been acheved by transfecton of closed crcular HBV DNA n human hepatoma cell lnes (HepG2 or HUH7) (Sureau et al., 1986; Sells et al., 1987; Tsurmoto et al., 1987). However, the latter system dd not mmc the n vvo process of vral nfecton, ncludng vrus penetraton and early bochemcal events. Therefore, we have focussed on the problem of n vtro nfecton of HepG2 cells (Aden et al., 1979; Knowles et al., 1984) wth HBV. The mechansm of HBV entry s not yet understood, however, we have prevously demonstrated that several factors are essental for successful n vtro nfecton of HepG2 cells wth HBV (Bchn et al., 1990). n partcular () morphologcal ntegrty of vron partcles and () hgh level expresson of pres1 sequences (at postons 21 to 47) of the large HBs envelope proten (LHBs) are determnant for the bndng of HBV to hepatocytes (HepG2) (Pett et al., 1991 a and c). n addton, we have dentfed presl-specfc bndng protens at the HepG2 cell membrane (Pett et al., 1992) suggestng that HepG2 cells dsplay a specfc receptor for HBV attachment and penetraton. Usng ths model, we have succeeded n nfectng HepG2 cells wth HBV (Bchn et al., 1990), and an HepG2 cell lne (HepG2-BV) that produces complete vrons wth the vral DNA replcaton beng measurable was obtaned. However, smlar to normal human hepatocyte prmary cultures (Grpon et al., 1988), ths latter n vtro nfecton model also appeared subject to mportant varatons (Pett et al., 1991b). n the present report, we nvestgated the course of producton of HBV-assocated partcles, and of the relatve expresson of all three HBV envelope protens followng n vtro nfecton of HepG2 cells wth serum SGM

2 2682 H. Mabt and others e~ (a) "7 l A HBV Tme post-nfecton (d) nfecton 3000 ~.~ (c) (b) 28s- :!: : ~ 18s kb kb derved vral partcles. At 35 days post-nfecton, sudden ncreased producton of HBs partcles and pres1ag was spontaneously observed, followed by c.p.e. A stable HepG2 cell lne that produced hgh levels of pres2- postve HBs partcles and contanng ntegrated HBV DNA sequences was therefore dentfed through selecton and desgnated the HepG2-BV3 cell lne. Our results ndcate that () n vtro HBV nfecton of HepG2 cells s subject to wde varatons not only from one experment to another (Bchn et al., 1990; Pett et al., 1991b) but also durng the long-term follow-up of nfecton n the same experment as observed n HBV-nfected patents and also that () dysregulaton n surface gene expresson probably resultng from ntegraton of HBV contrbutes to HBV persstence. Methods HepG2 cell culture. HepG2 cells were grown n Dulbecco's MEM supplemented wth 10 % fetal bovne serum, 1% non-essental amno acds, 1% sodum pyruvate, 2 mm-l-glutamne and antbotcs (penclln 100 unts/ml, and streptomycn 100 gg/ml) (n complete medum). Cells were seeded at a concentraton of 2 x 105 cells per ml n T-75 flasks. The medum was changed every 2 days. Cultures were observed daly usng a phase-contrast mcroscope. At confluency, cell monolayers were trypsnzed, and passaged weekly thereafter. HBV solate and nfecton of HepG2 cells. The HBV solate (HBsAg stran We: subtype adw2, genogroup A; Norder et al., 1993) was obtaned from a patent wth envelope antgen HBeAg-postve chronc hepatts B, who had elevated alanne amnotransferase levels (> 100 U/ml). HBV-assocated partcles were concentrated (150-fold) from serum by means of ultracentrfugaton (230000g for 18h) through a sucrose-densty gradent (30 %, w/w). The fnal vral partcleenrched pellet was resuspended n TNE buffer (10 mm-trs-hcl, ph 7.2, 140 mm-nac1 and 10 mm-edta) at a concentraton of 1 mg of proten per ml and stored at --80 C. HBV DNA content was of 6 ng/ml usng the Abbott 125-labelled HBV DNA hybrdzaton assay (Abbott). Four days after platng, HepG2 cells (about 5 x 105 per ml) were ncubated for 16h at 37 C wth serum-derved vral preparaton dluted n complete medum to a fnal concentraton of 30 gg of proten per ml (correspondng to 200 pg of HBV DNA per ml). The m.o.. corresponded to approxmately 100 vral genomes equvalent (v.g.e.) per cell. After removal of the noculum, cells were washed three tmes n PBS (threefold), and then cultured n complete medum Fracton no. Fg. 1. Knetc analyss of HBsAg (0), pres2ag (&) and pres1ag (11) secreton by HepG2 cells followng HBV nfecton (day 0). HepG2 cells (10 ~ cells/ml) were exposed for 16 h at 37 C to serum-derved HBV partcles at 10 s v.g.e, per ml and per 106 cells. Passages (ndcated as'~/) were performed every week. Results were expressed as c.p.m, measured n the culture supernatants (P). Negatve controls (N) corresponded to the mean value of 150 c.p.m. (for many determnatons) and the results were consdered postve when values were hgher than 315 c.p.m. (P/N = 2.1). (b) Northern blot analyss of total RNA from HBVnfected HepG2 cells at 1, 2, 4 and 21 days after nfecton (lanes 1 to 4, respectvely). Hybrdzaton was performed wth a2p-labelled whole HBV DNA used as probe. (c) Sedmentaton profle obtaned after sopycnc centrfugaton n sucrose gradent of HBV partcles present n the supernatant of nfected-hepg2 cells at day 21. The bulk of HBsAg actvty sedmented between 30 and 35 % sucrose.

3 Stable HBsAg producng HepG2 cell lne 2683 Sucrose-densty gradent fractonaton. Vral partcles n culture meda were spun down at g for 2 h, and the pellet was then resuspended n TNE buffer (concentrated 10-fold) and centrfuged at g for 18 h n a sucrose densty gradent that ranged from 20 to 60 % (w/w) concentraton Fractons (1.2 ml) were collected, tested for sucrose concentraton usng refractometry, and then dluted 1 : 10 for the detecton of HBsAg and pres antgens by radommunoassays as descrbed below. Polyelonal-monoelonal antbody radommunoassays (PAb-MAb RAs). Quantfcaton of three antgenc specfctes of the HBV envelope eptopes (HBs, pres2 and pres 1) was performed usng an nhouse PAb-MAb RA system utlzng rabbt polyclonal ant-hbs ggs on the sold phase and HBs(F39.20)-, pres2(f124)- or presl(f35.25)- specfc monoclonal antbodes n the revelaton phase, as descrbed n detals elsewhere (Pett et al., 1990). Bndng of specfc monoclonal antbodes was revealed by ncubaton wth the 125-labelled (F(ab') 2 fragment of ant-mouse mmunoglobulns (gs) (Amersham). Western blot analyss. HepG2 cell culture supernatants (over 3 days at subconfluency) were mmunoprecptated wth HBs(F39.20)-specfc monoclonal antbody (Bchn et al., 1990). The mmune complex was made soluble n SDS-PAGE sample buffer, run on 12.5% polyacrylamde gels and assayed by mmunoblottng usng rabbt polyclonal ant-hbs ggs that were detected by ncubaton wth the 12~-labelled F(ab')~ fragment of ant-rabbt gs (Amersham). mmunocytochemcal stanng. Subconfluent HepG2 cells were trypsnzed, harvested, counted, resuspended n cold PBS and placed onto sldes (2 10 ~ per slde) wth a Cytospn 3 nstrument (Shandon). mmedately after preparaton, the sldes were ar-dred for 2 h, fxed n acetone for 10 mn, n chloroform for 30 mn and washed n PBS for 5 to 10 mn. The sldes were then stored at -80 C untl used. A threestep mmunoperoxdase technque was performed (Mason & Sammons, 1979) usng HBs(F39.20)-, pres2(f376)- and presl(f35.25)-specfc monoclonal antbodes. Non-specfc proten stanng was reduced by dlutng monoclonal mmunoglobulns n PBS contanng 50% of normal human serum. Analyss of lb V DNA sequences by Southern blot. After trypsnzaton and washngs n PBS, subconfluent HepG2 cells were lysed n 0.5 % SDS (50 mm-trs-hc1, ph 8, 100 mm-nac and 1 mra-edta), and ncubated wth 50 ~tg of protenase K per ml for 12 h at 37 C. The DNA was frst extracted wth phenol/chloroform and then wth chloroform. The DNA soluton was adjusted to a concentraton of 0.3 M wth NaC1, precptated wth 2 vol of cold ethanol, and then chlled for 12 h at -20 C. The precptate was resuspended n TNE buffer. Hgh M r DNA was dgested for 5 h at 37 C wth restrcton endonucleases, EcoR and Hndl (Boehrnger Mannhem) (45 U of enzyme for 15 lag of DNA). DNA was then analysed by electrophoress on a 0.8 % agarose gel and transferred to a Hybond-N membrane (Amersham; Southern, 1975). Flters were hybrdzed wth a azp_ labelled HBV DNA probe (HBsAg: subtype ayw3, genegroup D; Charnay et al., 1979). The probe was labelled by nck-translaton (sp. act. 1 x 109 c.p.m, per ~tg). Hybrdzaton was performed at 65 C n the presence of 10 % dextran sulphate. Flters were washed, dred and then exposed for 12 h to 1 week at -70 C to Cronex flm (Dupont) wth an ntensfyng screen. RNA analyss. Total RNA was extracted from subconfluent HepG2 cells usng RNAfl soluton (Boprobe systems). Twenty lag of RNA were dluted n 1 x MOPS buffer contanng formaldehyde and deonzed formamde, ncubated for 15 mn at 65 C and chlled on ce. RNA samples were electrophoresed through 1% agarose gels contanng 1.1 M-formaldehyde, and transferred to a nylon membrane (Hybond-N, Amersham). Flters were prehybrdzed for 2 h at 65 C n 6 SSC, 5 Denhardt's soluton, 0.1% SDS, and 100~g/ml of denatured herrng sperm DNA, and then were allowed to hybrdze wth the nck-translated full-length HBV DNA as descrbed above. Dexamethasone treatment of cell cultures. HBV-nfected HepG2 cells (106/ml) were cultured for 3 days n complete medum contanng 10-6 M-dexamethasone. Culture medum was removed and assayed for HBsAg, pres2ag and pres1ag by PAb-MAb RAs. dentfcaton of HBsAg parteles by electron mcroscopc examnaton. HepG2-BV3 cell culture supernatant (about 40 ml) was centrfuged at g for 2 h. The pellet was dssolved n TNE buffer (0 1 ml), mxed wth 0-1 ml of HBs(F39.20)-specfc monoclonal antbody (100 lag of gg per ml) and ncubated at 37 C for 1 h, then at 4 C for 16 h. The mxture was then dluted n 10 ml of TNE buffer and centrfuged at g for 1 h. The pellet was resuspended n 0.1 ml of TNE buffer, dropped on a carbon-coated grd, and observed after negatve stanng usng a JEOL 100 CX electron mcroscope. Results Expresson of vral protens and RNAs durng 3 weeks followng HBV nfecton HepG2 cells were nfected wth the vral preparaton (approx. 108 v.g.e, per ml and per 106 cells) for 16 h at 37 C. After removal of the noculum, vrus weakly assocated wth the cells was eluted by extensve washngs. No HBV surface antgens were detected n the thrd washng (Fg. 1 a, day 1 post-nfecton). Culture medum was collected at days 2, 4, 7, and 21 after nfecton, ntally clarfed and then centrfuged at g for 2 h. The resultng supernatants and pellets were tested for HBsAg, pres2ag and presag. All HBV surface antgens were recovered n the partcle-assocated fracton, ndcatng that nfected HepG2 cells secreted ,,, ~ ' ' 8000 ~ Tme post-nfecton (d) Fg. 2. Knetc analyss of HBsAg (t), pres2ag (&) and preslag () secreted by HBV-nfected HepG2 cells from day 35 to day 75 after nfecton. A massve cell death (90 %) was observed from day 55 to day 75.

4 2684 H. Mabt and others (a) Recprocal supernatant dluton (b) (c) 1-36K - 33K - 24K (a) Fg. 3. For legend see opposte

5 Stable HBsAg producng HepG2 cell lne 2685 vral antgens as partculate forms. Fg. 1 (a) shows the knetc analyss of HBsAg, pres2ag and pres1ag produced by HepG2 cells durng the frst 3 weeks followng nfecton (ncludng three passages of cells after treatment wth trypsn). Maxmal secreton of three surface antgens was observed at day 4 post-nfecton. HBsAg and pres2ag producton then slghtly decreased but remaned sgnfcantly postve after three successve subcultures (at day 21 post-nfecton). n contrast, pres1ag rapdly dropped at day 7 post-nfecton and then became negatve at 21 days (Fg. 1 a). The presence of HBV-specfc transcrpts n nfected- HepG2 cells at days 1, 2, 4 and 21 after nfecton was analysed by Northern blot. As shown n Fg. 1 (b) HBV RNAs at 2.2 kb and 2"6 kb rose from nearly undetectable levels at day 1 to hgh levels at 2 and 4 days postnfecton. The former could be the transcrpt for the major (SHBs, p24/gp27) and the mddle (MHBs, gp33/gp36) surface protens, whereas the latter could be the transcrpt for the large (LHBs, p39/gp42) surface protens. The 2"6 kb/lhbs RNA could not be seen at day 21 whereas the 2"2 kb/shbs-mhbs RNA perssted. These results are n agreement wth prevous mmunoassay fndngs on the secreton of HBV surface antgens (Fg. l a). Of note, the 3"6 kb RNA correspondng to pregenomc RNA that codes for core protens (HBe/cAgs) was not detected (Fg. 1 b). HBV-nfected HepG2 cell cultures were tested for HBeAg and for the presence of HBV DNA by RA and hybrdzaton assay (Abbott Laboratores). HBV replcaton markers were not detected (results not shown). The latter results ndcate early actve transcrpton of HBV DNA that leads to synthess of envelope protens alone, and absence of vrus replcaton. Sedmentaton analyss of secreted vral partcles To characterze the vral partcles released from HBVnfected HepG2 cells, culture meda collected at day 21 post-nfecton were subjected to sopycnc centrfugaton n a sucrose densty gradent (Fg. 1 c). By usng PAb- MAb RAs, only one populaton of vral partcles postve for HBsAg and pres2ag was found n fractons 8 to 11 between 30 and 35 % sucrose. Nether HBV DNA nor the major core proten (determned by Southern or Western blottng, respectvely) could be detected n fracton 10, confrmng the secreton of vral partcles only composed of HBs protens, but not of mature complete vrons. Spontaneous selecton of a stable HBsAg-producng HepG2 cell lne At day 35 after nfecton (ffth passage), a fourfold ncrease n c.p.m, measured for HBsAg and a strong postve sgnal (P/N > 100) for pres1ag were observed (Fg. 2). Even after concentraton, nether HBeAg nor HBV DNA could be detected n supernatants of HBVnfected HepG2 cells (results not shown). From day 40 to day 75 post-nfecton, the amounts of HBsAg and pres1ag that were released nto the culture medum progressvely decreased. Cell death began at day 50 postnfecton and rapdly ncreased thereafter (about 90 % of cells ded at day 75 post-nfecton). The remanng vable HepG2 cells were grown frst n culture plate wells, and then propagated n flasks as descrbed n Methods. A stable HBsAg-producng HepG2 cell lne, desgnated HepG2-BV3, was thus spontaneously selected. The HepG2-BV3 cells dd not morphologcally dffer from the parental HepG2 cells as observed by phase-contrast mcroscopy. However, HepG2-BV3 grew one and a half tmes faster than the parental HepG2 cells dd. HBV protens and DNA analyss n nfected HepG2- BV3 cells The producton of HBV envelope antgens (HBs, pres2 and pres1) was followed usng PAb-MAb RAs after every passage durng 16 weeks (results not shown). Nne months after nfecton (36th passage), the levels of HBsAg secreton by HepG2-BV3 cells were stablzed to reach a concentraton between 50 and 100 ng of HBsAg per ml of supernatant over a 3-day culture perod (determned usng our n-house PAb-MAb RA test as Fg. 3. (a) HBsAg (left), pres2ag (mddle) and preslag (rght) ttres n untreated (O) and Dex-treated (11) HepG2-BV3 cell cultures at 9 months after nfecton. HBV surface antgens were determned usng our Own PAb-MAb RA system. Results were expressed as descrbed n Fg. 1. (b) Western blot analyss of the surface protens secreted by the HepG2-BV3 cells nto the culture medum. HBV protens were mmunoprecptated wth monoclonal antbody rased aganst HBsAg (F39.20) and then subjected to mmunoblottng. Transfer blots were ncubated wth rabbt ggs rased aganst HBsAg. Antbody bndng to the transferred protens was detected wth a~s-labelled ant-rabbt mmunoglobuln F(ab')2. The numbers on the rght ndcate Mrs. Lane 1, Parental HepG2 cell lne; lane 2, untreated HepG2-BV3 cells; lane 3, HepG2-BV3 cells after treatment wth dexamethasone. (e) Electron mcroscope examnaton of 200- fold concentrated supernatant from HepG2-BV3 cells n the presence of monoclonal antbody rased aganst HBsAg (F39.20). Bar marker represents 100 nm. (d) mmunoperoxdase stanng of HepG2-BV3 cells reacted wth () rrelevant monoclonal antbody (MAb), () MAb rased aganst HBsAg (F39.20), () MAb rased aganst pnes2ag (F376), and (v) MAb rased aganst pres lag (F35.25) at a fnal concentraton of 10 ~tg/ml, 9 months after nfecton.

6 2686 H. Mabt and others HMW , ~ ::~!: f Fg. 4. Southern blot analyss of DNA extracted from HepG2-BV3 cells. Lane 1, Control (parental HepG2 cell lne); lane 2, undgested DNA; lane 3, EcoR-dgested DNA; lane 4, Hnd-dgested DNA. The mass of DNA loaded was 15 gg. Sze markers (kb) for Hnddgested bacterophage 2 DNA are ndcated on the rght. well as the Abbott Ausra kt). Fg. 3 (a) shows HBsAg, pres2ag and pres1ag ttres n untreated (-Dex) and Dex-treated (+Dex) HepG2-BV3 cell cultures. Sgnfcant ncrease n HBsAg and pres1ag secreton was observed after treatment wth dexamethasone (Fg. 3 a). HBsAg producton by HepG2-BV3 cells was much hgher than that of the cell lne (Sells et al., 1987) as judged by usng RA (postve-to-negatve-sample ratos of 50 and 10, respectvely, for unconcentrated medum). The rate of HBsAg producton by the HepG2- BV3 cell lne dd not change over a 2-year perod even after several freezng and thawng cycles. Partcles that were mmunoprecptated from untreated and Dex-treated HepG2-BV3 cells culture supernatants by HBs(F39.20)-specfc monoclonal antbody were tested for the presence of HBsAg protens usng Western blottng. As depcted n Fg. 3(b) lanes 2 and 3, respectvely, partcles secreted by HepG3-BV3 cells contaned the major HBs surface proten (SHBs, 24K) and the pres2-contanng HBs mddle surface protens (MHBs, 33K and 36K). Furthermore, electron mcroscopc examnaton revealed the presence of sphercal HBsAg partcles wth an average dameter of 25 nm, whch were aggregated after ncubaton wth monoclonal antbody to HBsAg (Fg. 3c). The mmunoperoxdase stanng (Fg. 3 d) ndcate that HBsAg () and pres2ag () are present n the majorty of HepG2-BV3 cells whereas only few of them express pres1ag (v). The ntracellular SHBs, MHBs and LHBs syntheszed by HepG2-BV3 cells show cytoplasmc localzaton. Together, these results demonstrate that the HepG2-BV3 cell lne produces all forms of the surface proten and secreted HBsAg partcles n large quanttes. To examne the physcal state of the HBV sequences n HepG2-BV3 cells, Southern blot analyss was performed (Fg. 4). Non-dgested DNA and restrcton fragments usng EcoR (one restrcton ste n the HBV genome) and Hndl (no restrcton ste) were subjected to agarose gel electrophoress. Cloned P-labelled full-length HBV DNA was used as the probe to detect vral sequences. No HBV-related sequences could be detected n the parental HepG2 cell lne (Fg. 4 lane 1). Undgested total DNA from the HepG2-BV3 cell lne mgrated n the hgh M r (HMW) regon (Fg. 4 lane 2). After dgeston wth EcoR, a very strong sgnal was observed at 2.6 kb and three addtonal fant bands at 6"6 kb, 3"3 kb and 1.95 kb (Fg. 4 lane 3). After dgeston wth Hndl, a major band at 22 kb was obtaned and two others larger than HBV DNA (3.2 kb) at 11 and 5.2 kb (Fg. 4 lane 4). Dscusson HBV studes have been consderably lmted because of the vrus' restrcted host and tssue range and the lack of an n vtro culture system. Prevously, we successfully nfected well-dfferentated HepG2 human hepatoma cells wth serum-derved HBV partcles (Bchn et al., 1990). Furthermore, we dentfed HepG2 cell pres1- bndng protens as receptor canddates that were requred for ntaton of nfecton through the pres1 doman of vrons (Pett et al., 1992). Therefore, we beleve that hepatoma-derved HepG2 cells are permssve and also susceptble to drect HBV nfecton. However, ths n vtro nfecton model has ts lmtatons : () the reproducblty of the nfecton process, as well as that of models based on prmary cultures of human hepatocytes (Grpon et al., 1988; Ochya et al., 1989), and () hepatoma cells seem partcularly susceptble to mportant dysregulaton of HBV gene expresson whch often results n a loss of ther ablty to produce progeny vrus partcles, leadng n some cases to a non-productve nfecton (Pett et al., 1991b). n the present report, we descrbe spontaneous development and selecton of a stable HBsAg producng HepG2 cell lne, named HepG2-BV3, whch was obtaned by n vtro HBV nfecton. We examned the levels of surface gene transcrpts and surface protens n nfected hepatoma cells. As well as the reference PLC/PRF/5 cell

7 Stable HBsAg producng HepG2 cell lne 2687 lne derved from human hepatocellular carcnoma (Alexander et al., 1976), the HepG2-BV3 cell lne contans HBV genomes n ntegrated forms. The cascade of events leadng to a clonal growth of hepatocytes that have ntegrated vrus are unknown. Therefore, we focussed our nvestgatons on the pathway leadng to changes n the HBV gene expresson durng chronc nfecton, n a model that bypassed the cellular mmune response. At an early stage of HepG2 cell nfecton wth HBV (durng week 1 post-nfecton), all three envelope antgenc specfctes (HBsAg, pres2ag and pres1ag) were syntheszed and secreted nto the medum by nfected hepatoma cells. Both the pres1 and S promoter transcrpts were ncreased wthn the cells, whereas nether the 3.5 kb pregenome RNA nor HBV DNA replcatve ntermedate forms were detected n ths nfecton assay. Expresson of the large HBsAg transcrpt n HepG2 cells wthout extensve vrus replcaton at an early stage of nfecton s n agreement wth the report of Yuasa et al. (1991). Ths fndng suggests that the pres1 promoter could be regulated by unknown cellular factors that probably enhanced ts expresson. The amount of pres 1 transcrpts n the cells and pres 1 antgen n the medum rapdly dropped below undetectable levels (3-weeks postnfecton). HBV-nfected HepG2 cells contnued, however, to express the S promoter transcrpt and to secrete HBsAg partcles expressng few pres2-specfc eptopes. At a late stage of HepG2 cell nfecton wth HBV (2-months post-nfecton), producton of HBsAg and pres1ag spontaneously greatly ncreased and ths was followed by an hepatocytotoxc effect. Ths dysregulaton of surface gene expresson suggests dsrupton of the HBV genome, as observed durng chronc nfecton by HBV n humans when vral core proten expresson and vraema s at low or undetectable levels (Denes et al., 1990). HepG2-BV3 cell lne stably producng hgh levels of HBsAg (6 to 9 months postnfecton) was thus selected, and the state of the HBV DNA further analysed by the Southern blot technque. The lack of replcatve ntermedates and crcular forms of the HBV genome, and the presence of HMW HBV DNA n the undgested DNA pattern suggested that HBV envelope antgens were produced from ntegrated copes. Dgeston wth Hndl revealed the presence of three DNA fragments wth a major band at 22 kb, reflectng the exstence of a lmted number of ntegraton stes n the cellular DNA. The uncut cellular DNA pattern and the Hndl restrcton pattern dffered demonstratng that HBV DNA ntegraton occurred, rather than free olgomers were present. Addtonal nformaton was provded by the use of EcoR restrcton endonuclease. The presence of a major band at the 2.6 kb poston suggested that the majorty of HepG2-BV3 cells had ntegrated several ncomplete HBV DNA sequences (2"6 kb) n tandem wth a head-to-tal orentaton. Ths would correlate well wth only the expresson of HBV envelope protens and the loss of an ablty to express core protens and to produce complete vrons by the HepG2-BV3 cell lne. However, clonng of the ntegrated vral sequences s necessary to establsh ths conclusvely. Further nvestgaton s now n progress. t cannot be excluded that undetectable mnute amounts of complete vrons may be produced by a few cells. n support of ths hypothess, we demonstrated the presence of HBV DNA n untreated and Dex-treated HepG2-BV3 culture supernatants usng a hghly senstve PCR amplfcaton technque (results not shown), ndcatng that HBV replcaton occurred at very low levels n the HepG2-BV3 cell lne. Sudden ncreased producton of both HBsAg and pres1ag could be explaned by HBV genomc fragmentaton and rearrangement resultng from ntegraton nto the host cell genome. Such a dysregulated surface gene expresson was followed by a massve cell death. Ths phenomenon s not well documented, but abnormal transcrpton of the LHBs probably resulted n ntracellular retenton of all forms of the surface proten (Ou & Rutter, 1987), and ths has been shown to be cytotoxc (Chsar et al., 1987). Ths supports the dea that lver cell njury durng HBV nfecton may be due to dysregulated surface gene expresson (Huang & Yen, 1993), and that human lver-specfc factors are lkely to be crtcal for vral pathogeness. n addton, the pres regon of HBV could nduce drect suppresson of cellular gene expresson through ts bndng capacty for hepatocyte nuclear factor-1 (Courtos et al., 1988). Thus, the vrus tself may exert c.p.e.s durng chronc n vvo nfecton along wth the HLA class restrcted cytotoxc T lymphocyte response to HBV-encoded antgens, as well-documented by the Chsar's group (Penna et al., 1991; Nayersna et al., 1993). Dysregulaton of surface gene expresson due to vral DNA ntegraton seems to be a predomnant phenomenon occurrng durng n vtro nfecton of hepatoma cells (HepG2 or HUH7) wth HBV. Ths model appears therefore adequate not only for the study of the mechansms mplcated n chronc hepatts B but also for the locaton of ntegrated vral sequences and ther contrbuton n hepatocarcnogeness. Therefore, we are nvestgatng the expresson by HepG2-BV3 cell lne of the X gene and 3' end-truncated mddle surface genes (pres2/s t) whch have been found to have a transcrptonal transactvator functon (K~kul6 et al., 1993; Lauer et al., 1994). Dexamethasone s known to exert a postve regulatory nfluence on HBV replcaton va glucocortcod responsve elements wthn the HBV genome (Tur-Kaspa

8 2688 H. Mabt and others et al., 1986; Farza et al., 1987). ncreased secreton of HBsAg partcles by Dex-treated HepG2-BV3 cells as prevously descrbed for HBV-transfected HepG2 cells (Tur-Kaspa & Laub, 1990) ndcates that the glucocortcod-dependent enhancer element n the HBV genome s present and functonally ntact n the ntegrated DNA fragment. Therefore, the HepG2-BV3 cell lne may be of use n nvestgatons of the response to varous drugs, bologcal stmul and nfectons wth other vruses n modfyng HBV gene expresson durng chronc nfecton. n concluson, the HepG2-BV3 cell lne obtaned by n vtro HBV nfecton of HepG2 cells mmcks events whch occur n naturally nfected hepatocytes of patents at a late stage of chronc HBV nfecton (HBeAgnegatve), when the DNA of HBV s present n an ntegrated form wth vraema occurrng at low or undetectable levels. Ths system may serve as an effcent model for studyng the sequence of events leadng to hepatocellular carcnoma and assst us to delneate the contrbuton of vral ntegraton n the dysregulaton of HBV and lver-specfc genes expresson. We gratefully thank D. Kremsdorf for helpful dscussons, and Y. Thomas who revewed the manuscrpt. Ths work was supported by grants from the Assocaton pour la Recherche sur le Cancer (ARC No. 6838) and from NSERM. References ADEN, D. P., FOGEL, A., PLOTKN, S., DAMJANOV, 1. & KNOWLES, B. B. (1979). Controlled synthess of HBsAg n a dfferentated human lver carcnoma-derved cell-lne. Nature, London 282, ALEXANDER, J. J., BEY, E. M., GEDDES, E. W. & LECATSAS, G. (1976). Establshment of a contnuously growng cell lne from prmary carcnoma of the lver. South Afrcan Medcal Journal 50, BCHN, R., CAPEL, F., DAUGUET, C., DUBANCHET, S. & PETT, M. A. (1990). n vtro nfecton of human hepatoma (HepG2) cells wth hepatts B vrus. 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9 Stable HBsAg producng HepG2 cell lne 2689 TSURMOTO, T., GRPON, P., FOUREL,., HANTZ, O., TREPO, C. & GUGUEN-GULLOUZO, C. (1987). Stable expresson and replcaton of hepatts B vrus genome n an ntegrated state n a human hepatoma cell lne transfected wth the cloned vral DNA. Proceedngs of the Natonal Academy of Scences, U.S.A. 84, TuR-KASPA, R. & LAUB, O. (1990). Cortcods stmulate hepatts B vrus DNA, mrna and proten producton n a stable expresson system. Journal of Hepatology 11, TUR-KASPA, R., BURK, R. D., SHAUL, Y. & SHAFRTZ, D. A. (1986). Hepatts B vrus contans a glucocortcod-responsve element. Proceedngs of the Natonal Academy of Scences, U.S.A. 83, YUASA, T., KAJNO, K., SATO,. MYAMURA, T. (1991). Preferental expresson of the large hepatts B vrus surface antgen gene by an adenovrus~epatts B vrus recombnant. Journal of General Vrology 72, (Receved 16 December 1993; Accepted 12 Aprl 1994)