JOURNAL OF VIROLOGY, Sept. 1992, p. 5265-52760022-538X/92/095265-12$02.00/0Copyright X 1992, American Society for Microbiology

Vol. 66, No. 9

Hepadnavirus Integration: Mechanisms of Activation of theN-myc2 Retrotransposon in Woodchuck Liver Tumors

YU WEI,' GENEVIEVE FOUREL,' ANTONIO PONZETTO,2 MARIA SILVESTRO,2PIERRE TIOLLAIS,' AND MARIE-ANNICK BUENDIAl*

Unite, de Recombinaison et Expression Genetique, Institut National de la Sante et de la Recherche MedicaleU. 163, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France, 1 and

Divisione di Gastroenterologia, Ospedale Molinette, Turn, Italy2

Received 21 February 1992/Accepted 27 May 1992

In persistent hepadnavirus infections, a distinctive feature of woodchuck hepatitis virus (WHV) is thecoupling of frequent viral integrations into myc family genes with the rapid onset of primary liver tumors. Wehave investigated the patterns ofWIHV DNA insertion into N-myc2, a newly identified retroposed oncogene, inwoodchuck hepatomas resulting from either natural or experimental infections. In both cases, integrated viralsequences were preferentially associated with the N-myc2 locus. In more than 40% of the woodchuck tumorsanalyzed, viral insertion sites were clustered in a 3-kb region upstream of N-myc2 or in the 3' noncoding region.Insertion of WHV sequences homologous to the human hepatitis B virus enhancers, either upstream ordownstream of the N-myc2 coding domain, was associated with the production of normal N-myc2 mRNA orhybrid N-myc2-WIHV transcripts, initiated at the normal N-myc2 transcriptional start site. Transient-transfection assays with different N-myc2-WHV constructs in HepG2 cells demonstrated that the viralenhancers could efficiently activate the N-myc2 promoter. These results, showing that cis activation of preferredcellular targets through enhancer insertion is a common strategy for tumor induction by WHV, emphasize thepreviously noted similarities between hepadnaviruses and nonacute oncogenic retroviruses.

Besides the well-known human hepatitis B virus (HBV),the hepadnavirus family includes several viruses whichinfect lower species (rodents and birds) and share many ofthe physical and biological properties of HBV (36). One ofthe members, the woodchuck hepatitis virus (WHV), and itsnatural host, the Eastern woodchuck, have been extensivelystudied as models for HBV infection, liver disease, andhepatocellular carcinoma (HCC) in humans (47). The closeassociation of chronic hepadnavirus infection with HCC,clearly shown in epidemiologic studies (3, 48), is evenstronger in woodchucks than in humans (35). Almost allwild-caught WEHV-infected and colony-born experimentallyinfected animals succumb to HCC within 2 to 4 years (17,35). A higher incidence and shorter latency of HCC inwoodchucks than in humans, after correction for the respec-tive life spans of the two species, indicate that WHVdisplays a greater oncogenic efficiency than its human ho-molog.The finding of viral DNA integration into the host chro-

mosomes in most cases of HCC in human and woodchuckcarriers (49) has suggested that viral integration may con-tribute to the oncogenic process. In human tumors, integra-tion of HBV DNA has been associated with chromosomaldeletions and translocations (28) and with the generation orderegulation of transcriptional trans activators encoded byrearranged viral sequences (23, 57). The hypothesis thathepadnaviruses, like nonacute oncogenic retroviruses, mayact as insertional mutagens has been supported only for rarecases of HBV-related HCC in humans. In two independenttumors, unique HBV insertion sites have been identifiedwith the retinoic acid receptor ,B gene (7, 11) and with thecyclin A gene (55). These genes play important roles in thecontrol of cell growth and differentiation, and their disrup-

* Corresponding author.

tion by virus insertion might be implicated in the genesis ofthe corresponding tumors. However, extensive studies ofHBV insertions in other human HCCs have failed to revealcommon integration sites and to detect coding regions inflanking cellular DNA (50).

In striking contrast, our recent studies of WHV-inducedwoodchuck HCC have demonstrated that insertional muta-genesis of myc-family oncogenes is commonly involved inthe tumorigenic process. Activation of c-myc, N-myc, and,predominantly, N-myc2, a woodchuck retrotransposedproto-oncogene, by nearby insertion ofWHVDNA has beenfound in about 30% of the woodchuck tumors analyzed (15,19).The family of myc proto-oncogenes, including c-myc,

N-myc, and L-myc, is well conserved during vertebrateevolution (9). These genes encode structurally related pro-teins, but they diverge in their regulatory elements anddisplay different expression patterns during normal develop-ment. Deregulated expression of myc genes has been asso-ciated with many human and animal neoplasms but onlyoccasionally with human HCC (52). Other myc-related,species-specific genes or pseudogenes have recently beendescribed (9), including the woodchuck N-myc2 gene, anintronless N-myc-related gene that presents all the hallmarksof a functional retrotransposon (15). N-myc2 has been ac-quired by rodents of the family Sciuridae, such as wood-chucks and squirrels (14, 51). It has been identified as a maintarget for hepadnavirus integration in woodchuck liver tu-mors (15). The sites for hepadnavirus insertion in myc genesappear to coincide with retroviral integration sites in murineT-cell lymphoma, suggesting that common mechanisms,leading to deregulated expression of the oncogenes, mayunderlie the tumorigenic pathways induced by both types ofviruses. In particular, WHV insertions in N-myc genes were

found to be clustered in a short sequence of the 3' untrans-lated region of N-myc, also depicted as a unique insertional

5265

5266 WEI ET AL.

hot spot in the murine N-myc gene in murine leukemiavirus-induced lymphomas (13, 43, 53).Woodchucks experimentally inoculated with WHV as

newborns afford the opportunity to conduct controlled stud-ies of the natural history of viral infection and liver disease(17, 25, 26, 35). We now report further studies of woodchuckHCCs, with the initial aim of comparing the patterns ofWHV DNA integration in tumors from experimentally andnaturally infected animals. Viral insertions in the N-myc2locus were observed with a similarly high frequency in thetwo groups of tumors. With the discovery of new viralinsertion sites upstream of the N-myc2 gene, we now de-scribe insertional activation of myc genes by WHV DNA inmore than 50% of the woodchuck HCCs analyzed so far.

MATERIALS AND METHODS

Animals and viruses. Animals were maintained in isolationin the laboratory animal facility at the Molinette Hospital,Turin, Italy. Woodchucks from two experimental sourceswere analyzed. Wild-caught woodchucks W157, W164,W188, W899, and W1332, trapped as WHV-infected year-lings, were purchased from Cocalico, Inc., Reamstown, Pa.,or North Eastern Wildlife, Inc., South Plymouth, N.Y. Theother animals were born in captivity in the woodchuckbreeding colony at the College of Veterinary Medicine,Cornell University, Ithaca, N.Y. Offsprings of dams provento be free of present or past WHV infection (W223, W231,W2238, W2249, W2260, W2284, and W2606) were inoculatedas newborns with a standardized virus infection pool, aspreviously described (35). All 12 woodchucks were produc-tively infected with WHV for their entire life span as shownby woodchuck hepatitis surface antigen and WHV DNA inthe serum. Most of them (with the exception of W223 andW1332) were inoculated at 2 to 3 years of age with differentdilutions of the serum of W1366 or W598, two woodchucksproducing high levels of human hepatitis D virus (HDV), asdescribed previously (33, 34). During follow-up, HDV RNAand delta antigen were detected in the serum of all inoculatedwoodchucks, except W157 and W2260. At autopsy, HDVRNA was revealed on Northern (RNA) blots (30) in W188and W2249 livers and was detected by polymerase chainreaction (PCR) methods (8) in the livers of the remainingHDV-positive animals. Short treatments with antiviral com-pounds such as arabinosyl-AMP were administered to someanimals, as part of an experiment to evaluate drug effects inhepadnavirus infection. The animals were sacrificed at 2.5 to4 years of age (mean age, 3.0 years), at an advanced stage ofHCC development. All animals had one or several tumormasses that were resected independently, quickly frozen inliquid nitrogen, and stored at -70°C.

Southern and Northern blot analysis. Total DNA or RNAwas extracted from frozen samples of woodchuck HCC,surrounding liver tissues, and normal woodchuck livers asdescribed previously (19), and 20 ,g of DNA or 40 ,g ofRNA was analyzed by Southern or Northern blotting (15)with alkaline transfer on Hybond-N+ (Amersham) (0.4 NNaOH for DNA and 0.05 N NaOH for RNA). The probesSplnt 1, specific for N-mycl, and TqEx3C, specific forN-myc exon 3, have been described previously (15). The165-bp Rsa-Hinfl (RsH) fragment covering N-myc2 exon 1,the 2.2-kb BglII-HindIII (BH) fragment covering exons 2 and3, and the 157-bp SspI-BglI (SsB) fragment of the 3' untrans-lated region of N-myc2 were subcloned into pBS+ (see Fig.1A). Cloned WHV DNA (32) and the subgenomic clones EnI(a 514-bp ApaI-PvuI fragment) and Enll (a 510-bp StuI-

HindIll fragment) were also used as hybridization probes.Hybridizations were carried out with 7% sodium dodecylsulfate (SDS)-0.5 M phosphate (pH 7)-i mM EDTA-50 p,gof salmon sperm DNA per ml at 65°C with 32P-labeledprobes. Southern blots were washed twice in 2x SSC (lxSSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDSfor 15 min at 65°C and twice in 0.lx SSC-0.1% SDS for 15min at 65°C. For Northern blots, filters were washed in 1%SDS-0.04 M phosphate (pH 7)-i mM EDTA at 65°C for 45min. Gels were exposed to Kodak X-Omat-AR 5 films at-80°C with intensifying screens.PCR amplification of genomic DNA. Genomic DNA (1 ,ug)

was amplified by PCR with Taq DNA polymerase (Northum-bria Biologicals Ltd.) and the following primers: SA2 (5'-GCGGTGAT[GAGATCTT-3'), complementary to the mi-nus strand ofWHV (positions 2533 to 2549) (16), and NMT18(5'-GTATCGATGCTCGAACCC-3'), complementary to thecoding strand of N-myc2 (positions -644 to -627). TheDNA samples were denatured at 94°C for 5 min and sub-jected to 35 cycles of PCR with 1 min of denaturation at94°C, 1 min of annealing at 55°C, and 1.5 min of elongation at72°C. The products were diluted 100-fold and amplified for asecond time by PCR with the same primers. The finalproducts were purified by electrophoresis and subcloned inpBS+. DNA was sequenced by the chain terminationmethod (39) with the Sequenase Version 2.0 Sequencing Kit(United States Biochemical Corp.).RNase protection. The 534-bp NheI-SmaI fragment of

N-myc2 (positions -238 to +296) was subcloned in pBS+and used as a template for mapping the transcription initia-tion sites in the tumors. The 2,627-bp RsaI-HindIII fragmentof N-myc2 (positions -310 to +2317) (15) was subcloned inpKS. The plasmid was linearized by SspI (position 1435),giving rise to a 882-bp SspI-HindIII template (positions 1435to 2317) for mapping of recombinational breakpoints intumors 2238T1 and 2260T1. Uniformly labeled complemen-tary RNAs were synthesized by using T3 and T7 polymer-ase, respectively. RNase mapping was performed with 20 ,gof total RNA from woodchuck liver tumors or adjacent livertissues (27).

Plasmid constructions, cells, and transient-expression as-says. Plasmid 1 was constructed by standard techniques (28)by subcloning a 3.2-kb HindIII fragment spanning wild-typeN-myc2 retroposon sequences from an EMBL3 recombinantphage (15) into the HindIII site of pBluescript BS+. TheWHV 1.65-kb ApaI-BglII fragment (see Fig. 9A) was iso-lated from a cloned WHV genome (32) and inserted atdifferent positions in plasmids 2, 3, and 4, as described in thelegend to Fig. 9.

Details of the construction of plasmid 5 will be describedelsewhere (14). Plasmid 5 contains N-myc2 promoter and5'-flanking sequences fused to the promoterless firefly lu-ciferase (LUC) reporter gene, at the unique HindIII site ofthe vector pSVOALD5' (12). Plasmid 9 was provided by H.de The (10). Plasmids 6, 7, 8, 10, 11, and 12 are described inthe legend to Fig. 10. pHBx2s, a plasmid carrying the HBVX gene controlled by the simian virus 40 early promoter, wasa kind gift from P. Hofschneider (57).The human hepatoma cell line HepG2 was grown in

Dulbecco modified Eagle medium with 10% fetal calf serumand antibiotics under 5% CO2. The plasmids bearing theN-myc2 gene or luciferase gene as a reporter (13 ,ug) and the13-galactosidase expression vector pCH110(3 ,ug), used as aninternal control for transfection efficiency, were introducedinto semiconfluent HepG2 cells (1.5 x 106 cells per 25 cm2)by calcium phosphate coprecipitation. In other experiments,

J. VIROL.

ACTIVATION OF N-myc2 BY WHV IN WOODCHUCK HCC 5267

TABLE 1. Frequency of WHV DNA integration in N-myc2

Animnal Modeof Tumor(s) WHV integrationinfection Tuo()in N-myc2W157 Natural 157T + (5')W164 Natural 164T + (5')W188 Natural 188T + (5')W899 Natural 899TaW1332 Natural 1332TW223 Experimental 223T2 + (5')

223T3 + (5')223T5223T6 + (5')

W231 Experimental 23117 + (5')W2238 Experimental 2238T1 + (3')

2238T2 + (5')W2249 Experimental 2249T1 + (5')

2249T2W2260 Experimental 2260T1

22601T2W2284 Experimental 2284T + (5')W2606 Experimental 2606T1 + (3')

2606T2 + (5')

a WHV DNA was integrated in the c-myc gene in 899T.b An additional DNA rearrangement unlinked to viral integration was also

observed in the 3' noncoding domain of N-myc2 in 231T.

various amounts (0.6 to 15 ,ug) of pHBx2s or pWB15 (aplasmid carrying WHV fragment AB) were mixed with 3 p,gof plasmid 5 and 3 ,g of pCH110, made up to 20 ,g of totalDNA with pBS+ DNA, and applied to the culture medium.Luciferase activity was determined from cell extracts pre-pared 30 to 48 h after transfection as reported previously (10,12t. For RNA analysis, the transfections were scaled up to10 cells and total cellular RNA was prepared by thehot-phenol procedure followed by two rounds of acidicextraction (22) to remove contaminating DNA.

RESULTSWHV DNA integration in the N-myc2 gene in woodchuck

HCC. We analyzed woodchuck HCCs associated with nat-ural or experimental infection with WHV. As listed in Table1, five wild-caught animals had acquired persistent WHVinfection through natural transmission, whereas seven colo-ny-born animals had been inoculated as newborns with aviral infectious pool. Most woodchucks carried one or twotumor masses, and one (W223) bore six distinct tumorousnodules, only four of which were available for the presentanalysis. A total of 19 independent tumors were recoveredfrom the panel of 12 WHV-infected animals.DNA from the woodchuck HCCs was analyzed for viral

insertion into the N-myc loci. Figure 1A shows a map of thenormal N-myc (N-mycl) and N-myc2 regions with the posi-tions of the probes and relevant restriction endonucleasesites indicated. In Southern blot hybridizations of HCCDNA digested with HindIII, the N-myc probe BH recog-nized the germ line 7.4-kb N-mycl and 3.5-kb N-myc2fragments and detected additional bands of various sizes ineight tumors originating from both wild-caught and experi-mentally infected animals (Fig. 1B). The N-myc loci retainedwild-type configuration in the nontumorous parts of thecorresponding 4ivers (data not shown). Hybridization of theblots with aWHVDNA probe showed discrete bands of highmolecular weight corresponding to viral insertion events inall tumor DNAs (Fig. 1C). Distinct viral integration patternsindicated independent clonal origins for the tumorous nod-

ules isolated from the same liver (compare lanes 223T2,223T3, and 223T6 and lanes 2238T1 and 2238T2 in Fig. 1C).In six of the eight tumors harboring a rearranged N-mycallele, the tumor-specific N-myc fragment comigrated withone of the virus-specific bands and could represent a junc-tion fragment linking WHV DNA and N-myc sequences. Wealso observed the remnants of viral replication in mosttumors (Fig. 1C), and all surrounding liver tissues containedabundant viral replicative forms corresponding to intensesmears beneath the linearized 3.3-kb genome (not shown).

Further hybridization experiments were carried out toconfirm viral insertions and characterize the recombinationsites in the eight HCCs. The observed rearrangements inN-myc loci could be assigned to the N-myc2 gene, sincehybridization of the same Southern blot with a specificN-mycl probe revealed only the unaltered 7.4-kb N-myclfragment (Fig. 1D). In previous studies, viral insertions intoN-myc genes had been invariably mapped to the 3' untrans-lated region of N-myc (15). We therefore hybridized TaqI-digested DNAs with a probe specific for the third N-mycexon (TqEx3C [Fig. 1A]). As shown in Fig. 2, a tumor-specific band was detected only in three tumors (231T,2238T1, and 2606T1). Hybridization of the same blot with aviral probe revealed viral integration into N-myc2 in 2238T1and 2606T1 but not in 231T (data not shown). To map theviral integration sites more precisely and to determine theorientation of WHV DNA relative to N-myc2, we performeda restriction analysis of 2238T1 and 2606T1 DNAs by usinga probe of the N-myc2 3' untranslated region (SsB [Fig. 1A]).Digestions with DraI or SspI or with a combination of ClaIand XbaI or ClaI andApaI generated novel bands that werealso detected with a virus-specific probe, indicating viralinsertion in the N-myc2 3' untranslated region, as illustratedin Fig. 3. Fine mapping of the viral sequences adjacent toN-myc2 and comparison with the restriction map of theWHV genome showed that, in both tumors, WHV andN-myc2 sequences were placed in the same transcriptionalorientation (Fig. 3). The recombinational breakpoints weretentatively localized 220 and 430 bp downstream of theN-myc2 termination codon in 2238T1 and 2606T1, by usingRNase mapping with total cellular RNA and a probe span-ning the N-myc2 3' untranslated sequences (data not shown).

Frequent viral integrations upstream of the N-myc2 retro-poson. The absence of junction fragments linking the 3'domain of N-myc2 and WHV DNA in five tumors harboringa rearranged N-myc allele prompted us to explore the entireN-myc2 gene and flanking sequences for viral insertionevents in all tumors. Genomic DNA was digested withPvuII, an enzyme with no recognition site within the WHVgenome, and hybridized with various probes specific fordifferent regions of N-myc and WHV. Strikingly, the RsHprobe specific for N-myc exon 1 (Fig. 1A) detected novelbands not only in the five remaining tumors harboring arearranged HindIll fragment but also in five other tumorsand revealed an additional rearrangement in 231T (Fig. 4A).Rearrangements in the upstream sequences of N-myc2 wereconfirmed in all cases by hybridizing the PvuII blots with anN-mycl-specific probe, which did not detect additional frag-ments (data not shown). Hybridization of the blots with aviral probe revealed WHV-specific bands comigrating witheach of the additional N-myc2 bands (Fig. 4B), suggestingviral-N-myc junction fragments. Figure 4B also shows thatmultiple independent viral insertions occurred in all HCCDNAs (average of three insertions per tumor). Furtherhybridizations with probes specific for various viral subge-nomic fragments detected viral sequences homologous to the

VOL. 66, 1992

J. VIROL.5268 WEI ET AL.

A 0.5 kbp

N-mycl

Bg R HA P Ba P A Ba

1-p -

Splntl--I

BaT T T Ba H Bg

TqEx3C3rqx1------

N-myc2

A HBg, 11

P R Ba H T BgTP CIT H T

RSH SsB -

C\ C' (C)

-

c0 Cn C' C',

B t' C C C\. C\.

N rnlycl1.4ko

_ 4y4i.tw:. _im

('NF-

C\') C') (01-

C"!Cl C"J

_4

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'3.5kb~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

A_ 4 =~~~~

*^-i- *".C

D

N- rmnycl

.!.4kb

- _ _ _ _. _ _ _

I31I

FIG. 1. Integration ofWHV DNA in N-myc2 in woodchuck liver

tumors. (A) Structural organization of the woodchuck N-myc gene(N-mycl) and N-myc-related retroposon (N-myc2). The N-myc loci

are represented as lines, with exons as boxes, coding regionsshaded, and untranslated regions open. The probes used in hybrid-izations are shown under the corresponding diagrams. SpIntl is an

N-mycl-specific probe. TqEx3C, RsH, SsB, and BH recognize both

N-myc genes. Abbreviations: ex, exon; A, ApaI; Ba, BamHI; Bg,BglII; Cl, ClaI; H, HindIll; P, PvuII; R, EcoRI; T, TaqI (not all

TaqI sites are shown). (B through D). Southern blot analysis of

HindIll-digested DNA from nine woodchuck tumors and a normal

liver (NL), hybridized sequentially with the N-myc probe BH (panelB), WHV DNA (panel C), and the N-mycl probe SpIntl (panel D).WHV-specific fragments and tumor-specific N-myc2 fragments that

migrated at the same position are shown by arrowheads. Opentriangles indicate rearranged N-myc2 bands unlinked to viral se-

quences. No rearrangement of N-myc genes could be detected in

HindIII digests of 2249T1 DNA. The positions and sizes of the germline N-myc alleles are marked on the left. Exposure to X-ray filmwas continued for 5 days.

probe and different WHV subgenomic probes established theviral localizations within the N-myc2 upstream region. Asshown in Fig. 5B, the structural organization of the viralinserts, partially deduced from the restriction map andcomparison with the WHV genome (illustrated in Fig. 3),

HBV enhancers EnI, EnII, or both in all viral inserts lyingnear N-myc2, except in 2238T2 (Fig. 4C). In most cases,

both WHV enhancer sequences were present. However,WHV EnI was absent in 164T and 2284T and EnII was

absent in 157T (data not shown).The sites of viral integration were investigated by South-

ern blot analysis of HCC DNAs digested with EcoRI,BamHI, BglII, and TaqI (data not shown). The virus-hostjunction near the 5' end of N-myc2 was mapped precisely in2238T2 by a PCR procedure and nucleotide sequencing ofthe cloned reaction product. As shown in Fig. 5A, a 93-bpfragment was amplified by using two primers complementaryto the N-myc2 sequence at positions -644 to -627 withrespect to the N-myc2 initiation codon and to WHV se-

quences at positions 2533 to 2549 (16). Sequencing of theamplified fragment indicated that viral sequences of the Cgene were juxtaposed to cellular sequences located 688 bpupstream of the N-myc2 coding domain, in the oppositetranscriptional orientation. In four other tumors, the sizes ofthe additional fragments detected with the N-myc exon 1

\- C\ut C M F CtZ F- Z H-

OD Oa) a) coZ CD 0C)

coC') CM CN CN cD QD ()SC"! N CU C\M CM CM CN IN

N rryc2 L .

4Kb~~~~~

FIG. 2. Rearrangements in the 3' part of N-myc2 in three livertumors. DNA from five woodchuck HCCs was digested with TaqI,an enzyme which cuts inside N-mycl and N-myc2 genes, and probedwith the N-myc exon 3-specific fragment TqEx3C. Black arrow-

heads and the open triangle denote novel bands generated by viralintegration or DNA rearrangement unlinked to WfIV. The germ lineN-myc2 fragment is indicated on the left. The probe also detects twoN-mycl fragments of 250 and 300 bp (not shown). The additionalcommon band originates from an N-myc-related pseudogene (ourunpublished data).

_8k-

3.8kb

ACTIVATION OF N-myc2 BY WHV IN WOODCHUCK HCC 5269

WHV

N N Ss H Bg SsII - I-5

151)1 x' '20b050

---- P

200:BaRT T X

I I I330611Pre S1/S2

Dr A N

S

500bp

CIT DrSsTx DrA Ss Ss

2238T1 |I 1 1 1 __1___1_B

CIT DrSs Ba RTX DrA H

2606Ti -r-

FIG. 3. Viral integrations in the 3' untranslated region of N-myc2. (A) Physical and genetic map of the WHV genome. The restriction mapand numbering of the nucleotides correspond to WHV1 (16). The viral genes are represented as arrows. Abbreviations: A, ApaI; Ba, BamHI;Bg, BglII; Dr, DraI; H, HindIII; N, NcoI; R, EcoRI; Ss, SspI; T, TaqI; X, XbaI (not all TaqI and DraI sites are shown). (B) Schematicrepresentation of the viral integrations in 2238T1 and 2606T1. N-myc2 sequences are shown as boxes (coding regions are shaded), and WHVsequences are shown as thick black bars. Identified viral subgenomic fragments are represented by continuous bars, and their transcriptionalorientation is indicated by an arrow. Dashed bars denote viral sequences that could not be mapped on the viral genome or orientated relativeto N-myc2.

was strikingly heterogeneous. We observed that no completeviral genome but only various, frequently rearranged subge-nomic fragments were juxtaposed to N-myc2. The transcrip-tional orientation of viral DNA adjacent to N-myc2 could bedetermined only in three of these tumors (188T, 223T2, and

CMi N--0CDcC0

c to C"} OI C\J n co IN 0* CDzCI C\ C" C\ N

N\ -N

\t

0

N-myc23.8kb

B

WHV_i3.3kb

.3R

MAW..,- _;-4v*X!s

C

. w. 'k_

w w~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.el*

VVHV3.3kb

:,.m.._ _

FIG. 4. Viral integrations in the 5' cellular sequences flankingN-myc2. Genomic DNA from woodchuck HCCs and normal liver

(NL) was digested with PvuII, and Southern blots were hybridizedsequentially with the N-myc exon 1 probe RsH (A), total WHVDNA (B), and the subgenomic WHV probes EnI and EnII (C). Thepositions of the germ line N-myc2 fragment and the linear WHVgenome are indicated on the left.

223T3), showing the opposite orientation relative to N-myc2,whereas in 2249T1 the viral sequences were highly rear-

ranged near the virus-host junction (Fig. SB). The detectionof a WHV-N-myc2 junction fragment in RsaI-digested223T3 DNA, by using the N-myc2 RsH probe and a viralprobe, indicated that the 5'-most N-myc2 RsaI site was lostin the tumorous allele and suggested viral integration withinthe 5' end of N-myc2 exon 1 region. In the six remainingtumors, the viral integration sites were localized in a PvuII-BamHI fragment, 2 to 3 kb upstream of N-myc2 (Fig. 6). Theabsence of informative restriction sites or the presence of thecomplex restriction patterns probably resulting from recom-bination of different viral subgenomic fragments in theinserted sequences did not allow us to further characterizethe structure of the WHV inserts. Evidence for rearrange-ments in cellular DNA at the integration sites was obtainedfor two HCCs. In 223T6, a rearrangement of N-myc2 was

observed in both HindIII and PvuII digests (Fig. 1B and 4A),but only with PvuII could a viral junction fragment bedetected (Fig. 1C and 4B), suggesting that viral integrationwas accompanied by a large rearrangement of cellular DNAat the 5' side of N-myc2. In 223T3, a deletion of cellularDNA in the same region was deduced from the restrictionmap of the viral integration site (Fig. 5B).

In our earlier study of 30 other woodchuck tumors (15), noDNA alterations had been observed in the 2-kb upstreamN-myc2 region. The finding of frequent viral insertionsfurther upstream of this region (Fig. 6, between the 5' PvuIIand BamHI sites) prompted us to reexamine HCC DNAsfrom the earlier study by using ApaI or PvuII digestion andthe N-myc2 RsH probe. In three tumors, viral integrationswere mapped to the -3 to -2 kb region, and in one case a

DNA rearrangement without apparent involvement of thevirus was found in the same region (results not shown).To search for potential DNA rearrangements and viral

insertions further upstream or downstream of the previouslyexplored region, HCC DNAs were digested with ApaI or

A

- b~~~~~~~~~~

zo

VOL. 66, 1992

5270 WEI ET AL.

A 0.5kb

H RI BgT BgT P T H T

2238 T2 J - - - -

WHV .4|.< N-myc 2CGCCACTAACTCTAGAAGAGGACGCTCCTCTgggagattttggtgtagatttttg2549 WHV primer 2519-688

N188T

RBa Bg H T-_ _ -

.....gggttcgagcatcgatacN-myc 2 prmer -627

Bg TP

H Bg A TRBa Bg P

223T2 -_ _ I I I-x--- 4-

223T3Pv R T N RsA BgT P

I L_. I''e-L~Mm4-*-

M x

A X BaRBgH T B TP2249Ti

FIG. 5. Schematic representation of viral integrations 5' to N-myc2. (A) Illustration of the mutated N-myc2 allele and nucleotide sequenceof the virus-host junction in the woodchuck HCC 2238T2. Symbols are as in Fig. 1A and 3; WHV DNA is shown as a thick bar, and viralregions which could not be characterized are represented as a dashed bar. The amplification product obtained after PCR from 2238T2 genomicDNA, by using N-myc2 and WHV primers, was entirely sequenced. Nucleotides of the viral sequence are shown in capital letters, and thoseof N-myc2 are shown in lowercase letters. (B) Restriction map of viral integrated sequences in 188T, 223T2, 223T3, and 2249T1. The RsaIsite (Rs) shown in 223T3 (nucleotide 1211 on the viral genome) could be mapped because of the deletion of the RsaI site at the 5' end ofN-myc2 retroposed sequences. In this tumor, the location of the cellular PvuII and EcoRI sites upstream of viral integrated sequences isconsistent with a 2.5-kb deletion in N-myc2 5'-flanking sequences.

doubly digested with ApaI and ClaI and hybridized withN-myc2 probes or with a specific cellular probe derived fromunique sequences located 12 kb upstream of N-myc2 (14).No additional alterations or integrations were found in the28-kb region surrounding N-myc2 (-15 to +13 kb) in thewoodchuck HCCs analyzed (results not shown).As summarized in Table 1, 13 of 19 HCCs from naturally

and experimentally infected animals showed integratedWHV DNA in the vicinity of the N-myc2 coding domain,either within the 3' untranslated region of the gene (in 2

cases) or in the 3-kb cellular region flanking the 5' side ofN-myc2 (in 11 cases). Figure 6 presents an overview of theintegration sites and, when determined, the transcriptionalorientation of the viral inserts. No viral insertion could bedetected in the woodchuck N-mycl gene in the presentstudy. Insertional activation of c-myc by WHV DNA, dem-onstrated in one additional tumor, has been described else-where (56).

Activated expression of N-myc2 in woodchuck HCC. Wehave previously described abundant 2.3-kb N-myc2 tran-

0.5 kb

N-myc 2 RNAP Ba H T Bg PTI I I I

2606T2 2249T11 88T1 223T3

2284T 2238T2223T2

H Till ex3

2238T1 2606T1

223T6

231T1 64T157T

FIG. 6. Position and orientation of integrated WHV DNA in the woodchuck N-myc2 locus in 13 liver tumors. Localizations of viralinsertions are shown by vertical arrows when mapped by PCR or RNase protection and by horizontal blocks when deduced from restrictionmapping. HCC numbers correspond to the listing in Table 1. The arrows above HCC numbers show the orientation of viral sequences.

B

HBg11

J. VIROL.

ACTIVATION OF N-myc2 BY WHV IN WOODCHUCK HCC 5271

HFZ HFZF Z cm Cm co cO ac cccc 0c cO co CY CY co 0o ocC03 cecm c\ c\ co

_ CC\J CM

z F- F- FHZW H HZo o -r- ; zr

(0 Co O ) CDcc CY) CyCJC\j c cmc\ z

S F a F F (ii co <

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Zftc)Cj U) c ooCC4 CCN _N C

)CD D C\ ).- '- ,-C IC\ C\

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2.3kb _18S5 .

FIG. 7. Enhanced expression of N-myc2 in woodchuck HCCs.Total cellular RNA from liver tumors (lanes T), adjacent livers(lanes NT), and normal liver (lane NL) was analyzed by Northernhybridization with the N-myc2 probe BH (Fig. 1A). The normal sizeof N-myc2 transcripts and the positions of rRNAs are indicated. Thedifferent sizes of abnormal N-myc2 RNAs produced from mutatedN-myc2 alleles are given in the text.

scripts in many woodchuck HCCs and longer chimericN-myc-WHV RNAs in tumors harboring viral integration inthe 3' untranslated region of N-myc (15). To assess the effectof upstream viral insertions on N-myc2 transcription, weisolated total RNA from frozen samples of tumorous andnontumorous woodchuck livers and hybridized it with theN-myc-specific probe BH (Fig. 7). In agreement with ourearlier report (15), N-myc2 was silent in most normal livertissues from uninfected and tumor-bearing animals. Highlevels of the 2.3-kb N-myc2 RNA were observed in mosttumor samples, except in 899 HCC, in which the c-myc genewas activated by viral insertion (data not shown) (56). In2238T1 and 2606T1, integration of viral DNA in the 3'noncoding region of N-myc2 resulted in the production ofabnormal N-myc transcripts, 4.0 and 4.3 kb in size, respec-tively (Fig. 7). These transcripts hybridized with a viralprobe (data not shown), indicating the presence of N-myc2-WHV fusion transcripts. In 231T, the expression of a 2.65-kbN-myc2 RNA might result from two different genetic alter-ations that occurred on the same allele: viral integrationupstream of the gene, leading to activated N-myc2 transcrip-tion, and DNA rearrangement in the 3' noncoding region,providing a novel transcription termination signal.

Transcriptional initiation of the 2.3-kb N-myc2 RNAs andN-myc-WHV cotranscripts in woodchuck tumors and adja-cent livers was analyzed by RNase protection assays, usingtotal RNA and an antisense RNA probe specific for the 5'end of the N-myc2 retroposon, covering 296 bases of theN-myc2 coding region and 238 bases of upstream sequences.A doublet of protected fragments of 322 to 325 (+5) baseswas detected with various intensities in all tumorous samplesexcept 899T (Fig. 8), indicating identical transcriptional startsites in HCCs carrying viral insertions upstream and down-stream of the N-myc2 coding region, as well as in cases whenno viral insert could be detected in the 28-kb region sur-rounding the N-myc2 retroposon. 98T is a woodchuck HCC,from the previously analyzed panel (15), in which a cellularDNA alteration unlinked to WHV has been mapped 2 to 3 kbupstream of N-myc2. Transcriptional initiation has beenobserved at the same positions, 24 to 27 bp upstream of thefirst initiation codon of N-myc2 in normal adult woodchuckbrain tissues, and N-myc2 promoter sequences have beencharacterized in the 5'-terminal, noncoding region of N-mycexon 2 (14). Therefore, in tumors harboring WHV insertions5' to the N-myc2 gene, enhanced N-myc2 expression prob-ably results from activation of the normal N-myc2 promoterrather than from transcriptional initiation from a nearbyinserted viral promoter.

Activation of the N-myc2 promoter by WHIV enhancers in

R58ntRNA probe

-_________ 322-325nt protected fragments

FIG. 8. Identical transcriptional start sites of N-myc2 RNA indifferent tumors. Total HCC RNA was analyzed by RNase protec-tion with a 32P-labeled riboprobe spanning the 5' end of N-myc2coding sequences and upstream region, as illustrated at the bottomof the figure. The same reaction was carried out with 20 j±g of yeasttRNA. The input probe and the molecular size markers (pBR322DNA digested with HpaII) are shown on the left. Sizes are innucleotides (nt).

vitro. The WHV ApaI-BglII fragment (AB), spanning se-quences homologous to the HBV enhancers EnI and EnII(predicted positions: 1200 to 1360 and 1765 to 1870), as wellas the entire X open reading frame (Fig. 9A), was introducedat different positions and in different orientations in a plas-mid bearing the N-myc2 gene (Fig. 9B). The effect of thisviral sequence on the expression of N-myc2 was analyzed intransient-expression assays after transfection in subconflu-ent HepG2 cells. A low level of expression was observedwith the basic N-myc2 construct 1 (Fig. 9C). The variousWHV-containing recombinants yielded high steady-statelevels of N-myc2 RNAs of different sizes. Construct 2produced a 3.1-kb N-myc2-WHV cotranscript. The 2.3-kbRNA expressed in cells transfected with constructs 3 and 4is identical in size to the normal N-myc2 RNA previouslyobserved in vivo, and it correctly initiated at the N-myc2promoter (data not shown), indicating transcriptional activa-tion of the N-myc2 promoter by WHV sequences. In addi-tion, construct 4 produced a 2.7-kb RNA initiated in the5'-flanking region of N-myc2 retrotransposed sequences, butno transcription was detected from the viral C promoter.This suggests that introduction of the viral AB fragment atthe 5' side of N-myc2 in the same transcriptional orientationmay activate a cryptic cellular promoter, a situation notobserved so far in vivo in woodchuck HCC.To better delineate the contribution of transcriptional

mechanisms to the observed up regulation of N-myc2 mRNAand characterize the role of WHV enhancers in the activa-tion of the N-myc2 promoter, we constructed a chimericplasmid containing a segment of the 5' N-myc2 noncodingregion, known to include N-myc2 promoter sequences (14),ligated to the luciferase gene (LUC). As shown in Fig. 10,transfection of this plasmid in subconfluent HepG2 cellsdemonstrated a weak basal promoter activity. Introductionof the viral AB fragment in the N-myc2-LUC plasmid atthree different locations, either upstream or downstream ofthe LUC transcriptional unit, resulted in a 4- to 11-foldactivation of LUC expression. In parallel experiments, the

285>-

404.-9.

309-

Nhel

N-myc2 ex I

I ..

- Smal

ex2

VOL. 66, 1992

5272 WEI ET AL.

A

P gene

I XgeneDPF lCge ne

492 887 100015`C

Apal2535

O-ggiI

B

3X>

_

ex3c

3.1 kb _2.7 kb -

2.3 kb-

1 2 3 4

,, ....

FIG. 9. Activation of N-myc2 expression by cis-acting viral

sequences in HepG2 cells. (A) WHV genome structure between

nucleotides 492 and 2535 (16). Large open arrows represent the viral

genes. Location of the sequences homologous to HBV enhancers

EnI and EnlI is indicated by shaded ellipses, and the polyadenyla-

tion signal is indicated by a star. (B) Schematic structure of the

N-myc2-WHV constructs. Regions of homology with N-myc exons

are indicated by large boxes, transcription start sites are indicated

by arrows, and the coding region is shaded. The N-myc2 3' poly(A)

stretch is represented as a thick vertical bar, and the flanking direct

repeats are shown as two black arrowheads. The hatched arrow

represents viral sequences indicating the viral positive-strand orien-

tation and the Enl and En2 sequences homologous to HBV enhanc-

ers EnI and EnIl. In plasmid 2, theWHVApaI-BglII fragment was

inserted downstream of the N-myc2 coding region, in the same

transcriptional orientation, in a plasmid carrying the rearranged

N-myc2 gene from a woodchuck HCC (15). Plasmids 3 and 4 were

constructed by inserting theWHVApaI-BglII fragment upstream of

N-myc2 in plasmid 1, either in the opposite (plasmid 3) or in the

same (plasmid 4) transcriptional orientation with respect to N-myc2.

(C) Northern blot of total RNA from HepG2 cells, transfected with

the corresponding constructs as indicated by lane numbering. The

probe was the 32P-labeled N-myc2 BH fragment (Fig.1A). The sizes

of the major RNA species are indicated.

positive TK-LUC controls, which contain the basal pro-

moter of the thymidine kinase (TK) gene of herpes simplex

virus type 1 cloned upstream of the luciferase gene, showed

similar behavior to that of the N-myc2-LUC constructs (Fig.

lOB). A 10- to 16-fold activation of the TK promoter was

induced by adjacent WHV enhancer sequences from differ-

ent positions. Transient-transfection assays with HepG2cells cultured at low density revealed a reduced ability of theWHV sequences to activate the N-myc2 promoter (data notshown). It seems probable that the known density-depen-dent modulation of liver phenotype in HepG2 cell cultures(24) affects the efficiency of the WHV enhancers, as alsoshown for the HBV enhancers (46).The viral AB fragment contains the X open reading frame

and its regulatory sequences; the observed up regulation ofthe N-myc2 promoter by the juxtaposed AB fragment mighttherefore be attributed to a trans-acting effect of the viraltranscriptional transactivator. To address this possibility, wecotransfected N-myc2-LUC construct 5 with a plasmidcarrying WHV fragment AB or with an HBx expressionvector (pHBx2s) in HepG2 cells under the same experimen-tal conditions as above. The LUC expression levels wereincreased 1.5- to 2-fold compared with basal LUC expres-sion driven by the N-myc2 promoter (data not shown). Theseresults indicate that the X trans-acting activity might con-tribute to a minor extent to N-myc2 promoter activation byadjacent viral sequences in constructs 6, 7, and 8. However,transcriptional activation of N-myc2 by the cis-acting effectof the viral enhancers appears to be mainly responsible forthe accumulation of N-myc2 mRNA observed both in vivoand in vitro.

DISCUSSION

Previous studies have demonstrated that woodchucksnaturally or experimentally infected with WHV are at ex-ceptionally high risk for HCC (17, 35). Our initial observa-tions of frequent viral integrations into woodchuck myc locihave suggested that the rapid onset of liver tumors in thismodel might be correlated with a direct, cis-acting effect ofintegrated viral sequences in activating myc family onco-genes (15, 19).

This report describes a detailed analysis of WHV DNAinsertions into myc genes in a new panel of 19 woodchuckHCCs from wild-caught and colony-born animals that ac-quired persistent WHV infection from natural transmissionor inoculation with a selected viral stock at birth. Confirmingand extending our previous data, we found that integratedviral sequences were preferentially associated with N-myc2,a recently identified retrotransposed oncogene peculiar towoodchucks: 13 of 19 tumors showed insertional mutagene-sis of the N-myc2 locus, whereas none harbored viralintegration in the classical N-myc gene. In a parallel analy-sis, WHV DNA integration in c-myc was observed in one ofthese tumors, as described elsewhere (56). Experimentalinfections yielded integration events at the same frequencyas did natural infections. In agreement with this, recentinvestigations of the evolution of experimental laboratoryinfections have shown kinetics of HCC development andvirological and histological patterns that were essentiallyidentical to those observed in feral animals (17, 25, 26, 35).More striking was the detection of viral integration sites in

a 3-kb region flanking the 5' side of the N-myc2 retrotrans-poson. In our earlier study of 30 different HCCs (15), wedetected viral insertions exclusively in the N-myc2 3' non-coding region; however, the cellular domain extending fur-ther than 2 kb upstream of N-myc2 had not been explored atthat time. Reexamination of the previously analyzed tumorsrevealed viral integration in a region between -3 and -2 kbin the N-myc2 map in three additional HCCs, showing thatthe two panels of woodchuck HCCs differ only in thefrequency of viral insertions near N-myc2. These variations,

......e x 3

J. VIROL.

1

ACTIVATION OF N-myc2 BY WHV IN WOODCHUCK HCC 5273

A5'- 3'T+ 3-NT+

/ / I

yc5Z1oo Ncmyc2:50LUC LV4

Relative luciferase activity (arbitrary units)2 3 4 5 6 7 8 9 10

l1 12 13 14 15 16

N-myc2 promoter basal activity

3'T3NT+5,-

-5- 3'T(- NT_

%% I, & % 11 %%/ 0 3F1-1 9TK-55 i-3.

TK promoter basal activity

3T3'NT+5'-

Relative luciferase activity (arbitrary units)

6 9 12 15 18 21 24 27 30

l l

6

678

33 36 39 42 45 48

9

101112

FIG. 10. Activation of N-myc2 and TK promoters by cis-acting viral sequences in HepG2 cells. A schematic diagram of plasmids used isshown on the left. Plasmid 5 contains N-myc2 promoter sequences (open box with coding region shaded) fused to the luciferase (LUC) gene

(wavy box). The transcription initiation site is indicated by an arrow, and the simian virus 40 (SV40) small t intron and polyadenylation signal

are indicated by a large, lightly stippled arrow. The WHV ApaI-BglII fragment (Fig. 9A) is shown as a black arrow. It was placed in threedifferent positions: either downstream of the luciferase gene, in transcribed (3'T+, plasmid 6) or untranscribed (3'NT+, plasmid 7)configurations, or upstream of the N-myc2 promoter (5'-, plasmid 8). In plasmids 6 and 7, WHV sequences were placed in the same

transcriptional orientation as N-myc2, whereas in plasmid 8 they were in the opposite transcriptional orientation. Constructs 9 to 12 are similarto constructs 5 to 8 but contain the TK basal promoter in place of N-myc2 promoter sequences. The plasmids were transfected in subconfluentHepG2 cells, and luciferase activities were determined after 30 to 48 h and normalized for 3-galactosidase activities. The results shown are

the average of three independent transfection experiments.

if statistically significant in a limited number of samples,possibly reflect different stages of tumor progression atsacrifice; no clear correlation with other parameters, such as

superinfection with HDV or treatment with antiviral drugs,could be established. Investigations of a 28-kb cellulardomain surrounding the N-myc2 retroposon did not provideevidence for additional viral integration sites located furtherupstream or downstream of N-myc2 in the tumors from bothseries. Together, these results now demonstrate integrationof WHV DNA next to the N-myc2 proto-oncogene in morethan 40% of the 49 woodchuck HCCs analyzed. Further-more, it can be estimated that about 25% of the totalintegration events detected in these tumors occur in theN-myc2 locus.The reasons for the strong clustering of viral insertions

adjacent to N-myc2 are not yet clear. Common integrationsites, including myc genes and different proto-oncogenes,have been found in cells infected by many nonacute retro-viruses (6, 18, 29, 31, 42). This apparent specificity mightreflect molecular requirements to produce oncogenic effectsand might be selected for by clonal outgrowth of transformedcells. However, the rate of viral targeting to the woodchuckN-myc2 retrotransposon in HCC is much higher than that toc-myc and N-myc genes, despite comparable oncogenicefficiencies of the three genes in rat embryo fibroblastcotransformation assays (14, 15). Previous studies havedemonstrated the use of highly preferred target sites forintegration of different RNA and DNA viruses in the absence

of selective pressure (4, 38, 45). Integration of HBV DNA inhuman HCC has also been observed at a rate higher thanaverage in chromosomes 11 and 17, although no evidencehas been provided so far for a target gene at the integrationsites (50). Preferred retrovirus integration sites have beenmapped near DNase I-hypersensitive sites and CpG-richislands located in the vicinity of many eucaryotic genes (37,40, 54). N-myc2 sequences, which are extremely rich in CpGdoublets, appear to be a related example (15) and might beconsidered an HCC susceptibility gene peculiar to wood-chucks. In this regard, it is interesting that a locus homolo-gous to the woodchuck N-myc2 retrotransposon is alsopresent in the squirrel genome, although information is stilllacking on its coding and transforming potential. Insertionalmutagenesis of the squirrel N-myc2 locus by hepadnavirusDNA has not been observed in studies of liver tumorsassociated with ground squirrel hepatitis virus infection (51).Differences in intrinsic oncogenic properties have beennoted between the two rodent hepadnaviruses (41), and therelatively long latent period ofHCC (5 to 9 years) in squirrelsmight be correlated with a reduced ability of ground squirrelhepatitis virus to integrate into host genomic DNA, therebylowering the overall chances of insertional mutagenesis (51).In human HCCs as well, insertional activation of the mycproto-oncogenes has never been observed, despite frequentviral integration events. The absence of a human N-myc2retroposed oncogene might be a major host factor possiblyimplicated in these discrepancies. However, it has recently

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5274 WEI ET AL.

been shown that the human c-myc and N-myc genes canprovide occasional targets for integration of another DNAtumor virus, human papillomavirus (5), and the reasons fordifferent oncogenic steps in the development of HBV- andWHV-associated HCCs represent an intriguing problem.A common feature of woodchuck HCC is enhanced accu-

mulation of N-myc2 mRNAs compared with adjacent livertissues, whereas N-myc2 is apparently silent in normal adulthepatocytes (15). In many tumors, viral integration inter-rupted different regions of the N-myc2 locus and juxtaposedWHV sequences homologous to the well-characterized HBVenhancers (44, 59) to the N-myc2 coding domain. Our invitro studies clearly indicate that the WHV genome containsenhancer elements capable of activating heterologous pro-moters in a position- and orientation-independent manner ina differentiated hepatoma cell line. In woodchuck HCCs,integration of viral DNA in the 5'-flanking N-myc2 regionoccurred frequently in the opposite transcriptional orienta-tion and was associated with the production of abundantN-myc2 transcripts of normal size, initiated in the 5' non-coding part of N-myc exon 2. A cryptic N-myc promoter thatcontrols N-myc2 expression in adult brain tissues has beenrecently identified in the corresponding N-myc2 region (14).No chimeric RNA starting at a viral promoter could bedetected, excluding a mechanism of promoter insertion, asdescribed for avian lymphomas (18). It seems likely, there-fore, that nearby integration of WHV DNA activates theN-myc2 promoter through enhancer insertion, as in manyretrovirus models (6, 42, 53). A similar mechanism mightalso prevail in other woodchuck HCCs, in which the viralinserts interrupted the N-myc2 3' noncoding domain, leadingto N-myc2-WHV cotranscripts. It has been suggested thatthe removal of control elements which might interfere withefficient translation or RNA stability may potentiate partic-ular proto-oncogenes. The 3' region of N-myc2, like those ofc-myc and N-myc (1, 21), might contain negative regulatorysequences, and their deletion might contribute to enhancedaccumulation of N-myc2 mRNA. In addition, variations inthe relative N-myc2 RNA levels among different HCCscould not be strictly correlated with a position-dependenteffect of virus integrated sequences containing the enhanc-ers. It is possible that the modulations in liver-specificphenotype, which occur during hepatocyte transformation,influence the efficiency of the WHV enhancers to stimulateN-myc2 expression, as also suggested by recent studies ofthe activity of the HBV enhancers on viral promoters indifferent cell lines (46).The production of abundant N-myc2-specific transcripts in

a significant number of tumors which do not harbor viralintegration in N-myc2 indicates that N-myc2 may be acti-vated through alternative mechanisms. An attractive hy-pothesis suggests that viral integration in a cellular locusgenetically related to the N-myc2 locus, but located aconsiderable distance away, might cause its aberrant expres-sion. Such a mechanism has been demonstrated in myeloidleukemias by the finding of retroviral insertions 90 kbproximal to the Evi-1 myeloid transforming gene (2). Asimilar question arises from recent studies of avian nephro-blastomas induced by myeloblastosis-associated virus,showing that overexpression of the transforming nov gene isassociated only occasionally with viral insertion at this locus(20). Alternatively, deregulated N-myc2 expression mightresult from trans-acting mechanisms associated with persist-ent expression of viral genes or with programmed resurgenceof a fetal phenotype in transformed liver cells. Our in vitrostudies, showing a weak but reproducible transactivation of

the N-myc2 promoter by the viral X protein, suggest thatexpression of the X transactivator might participate in transin the transcriptional activation of N-myc2. More detailedstudies with various WHV-N-myc2 constructions and differ-ent cell lines are necessary to establish the relative contri-butions of cis and trans mechanisms in the observed upregulation of N-myc2 RNA in woodchuck HCCs. The detec-tion of high levels of N-myc2 transcripts in precancerousnodules from chronically infected livers has suggestedthat N-myc2 activation might occur at an early step ofwoodchuck hepatocarcinogenesis (58). Whether enhancedN-myc2 expression in preneoplastic cells is due to an earlyviral integration event or to a trans-acting mechanism re-mains to be determined to further delineate the role ofWHVintegration in the genesis of woodchuck HCC.

ACKNOWLEDGMENTS

We thank L. Johnson, J. Gerin, and B. Tennant for providingwoodchucks experimentally infected with WHV from the NationalInstitute of Allergy and Infectious Diseases colony at CornellUniversity. We are grateful to C. Transy and M. Robertson forhelpful discussions and critical reading of the manuscript, to C. A.Renard for excellent technical assistance, and to L. M. Da forsecretarial assistance.

This work was supported by the Commission of the EuropeanCommunity and the Association pour la Recherche contre le Can-cer. Y.W. is supported by the Fondation Merieux.

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