JOURNAL OF VIROLOGY, Sept. 1992,

p. 5265-5276

Vol. 66, No. 9

0022-538X/92/095265-12$02.00/0 Copyright X 1992, American Society for Microbiology

Hepadnavirus Integration: Mechanisms of Activation of the N-myc2 Retrotransposon in Woodchuck Liver Tumors YU WEI,' GENEVIEVE FOUREL,' ANTONIO PONZETTO,2 MARIA SILVESTRO,2 PIERRE TIOLLAIS,' AND MARIE-ANNICK BUENDIAl* Unite, de Recombinaison et Expression Genetique, Institut National de la Sante et de la Recherche Medicale U. 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 the coupling of frequent viral integrations into myc family genes with the rapid onset of primary liver tumors. We have investigated the patterns of WIHV DNA insertion into N-myc2, a newly identified retroposed oncogene, in woodchuck hepatomas resulting from either natural or experimental infections. In both cases, integrated viral sequences were preferentially associated with the N-myc2 locus. In more than 40% of the woodchuck tumors analyzed, 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 or downstream of the N-myc2 coding domain, was associated with the production of normal N-myc2 mRNA or hybrid N-myc2-WIHV transcripts, initiated at the normal N-myc2 transcriptional start site. Transienttransfection assays with different N-myc2-WHV constructs in HepG2 cells demonstrated that the viral enhancers could efficiently activate the N-myc2 promoter. These results, showing that cis activation of preferred cellular targets through enhancer insertion is a common strategy for tumor induction by WHV, emphasize the previously noted similarities between hepadnaviruses and nonacute oncogenic retroviruses.

Besides the well-known human hepatitis B virus (HBV), the hepadnavirus family includes several viruses which infect lower species (rodents and birds) and share many of the physical and biological properties of HBV (36). One of the members, the woodchuck hepatitis virus (WHV), and its natural host, the Eastern woodchuck, have been extensively studied as models for HBV infection, liver disease, and hepatocellular carcinoma (HCC) in humans (47). The close association of chronic hepadnavirus infection with HCC, clearly shown in epidemiologic studies (3, 48), is even stronger in woodchucks than in humans (35). Almost all wild-caught WEHV-infected and colony-born experimentally infected animals succumb to HCC within 2 to 4 years (17, 35). A higher incidence and shorter latency of HCC in woodchucks than in humans, after correction for the respective life spans of the two species, indicate that WHV displays a greater oncogenic efficiency than its human ho-

molog. The finding of viral DNA integration into the host chromosomes in most cases of HCC in human and woodchuck carriers (49) has suggested that viral integration may contribute to the oncogenic process. In human tumors, integration of HBV DNA has been associated with chromosomal deletions and translocations (28) and with the generation or deregulation of transcriptional trans activators encoded by rearranged viral sequences (23, 57). The hypothesis that hepadnaviruses, like nonacute oncogenic retroviruses, may act as insertional mutagens has been supported only for rare cases of HBV-related HCC in humans. In two independent tumors, unique HBV insertion sites have been identified with the retinoic acid receptor ,B gene (7, 11) and with the cyclin A gene (55). These genes play important roles in the control of cell growth and differentiation, and their disrup*

Corresponding author.

tion by virus insertion might be implicated in the genesis of the corresponding tumors. However, extensive studies of HBV insertions in other human HCCs have failed to reveal common integration sites and to detect coding regions in flanking cellular DNA (50). In striking contrast, our recent studies of WHV-induced woodchuck HCC have demonstrated that insertional mutagenesis of myc-family oncogenes is commonly involved in the tumorigenic process. Activation of c-myc, N-myc, and, predominantly, N-myc2, a woodchuck retrotransposed proto-oncogene, by nearby insertion of WHV DNA has been found 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 vertebrate evolution (9). These genes encode structurally related proteins, but they diverge in their regulatory elements and display different expression patterns during normal development. Deregulated expression of myc genes has been associated with many human and animal neoplasms but only occasionally with human HCC (52). Other myc-related, species-specific genes or pseudogenes have recently been described (9), including the woodchuck N-myc2 gene, an intronless N-myc-related gene that presents all the hallmarks of a functional retrotransposon (15). N-myc2 has been acquired by rodents of the family Sciuridae, such as woodchucks and squirrels (14, 51). It has been identified as a main target for hepadnavirus integration in woodchuck liver tumors (15). The sites for hepadnavirus insertion in myc genes appear to coincide with retroviral integration sites in murine T-cell lymphoma, suggesting that common mechanisms, leading to deregulated expression of the oncogenes, may underlie the tumorigenic pathways induced by both types of viruses. In particular, WHV insertions in N-myc genes were found to be clustered in a short sequence of the 3' untranslated region of N-myc, also depicted as a unique insertional 5265

5266

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WEI ET AL.

hot spot in the murine N-myc gene in murine leukemia virus-induced lymphomas (13, 43, 53). Woodchucks experimentally inoculated with WHV as newborns afford the opportunity to conduct controlled studies of the natural history of viral infection and liver disease (17, 25, 26, 35). We now report further studies of woodchuck HCCs, with the initial aim of comparing the patterns of WHV DNA integration in tumors from experimentally and naturally infected animals. Viral insertions in the N-myc2 locus were observed with a similarly high frequency in the two groups of tumors. With the discovery of new viral insertion sites upstream of the N-myc2 gene, we now describe insertional activation of myc genes by WHV DNA in more than 50% of the woodchuck HCCs analyzed so far.

MATERIALS AND METHODS Animals and viruses. Animals were maintained in isolation in the laboratory animal facility at the Molinette Hospital, Turin, Italy. Woodchucks from two experimental sources were analyzed. Wild-caught woodchucks W157, W164, W188, W899, and W1332, trapped as WHV-infected yearlings, were purchased from Cocalico, Inc., Reamstown, Pa., or North Eastern Wildlife, Inc., South Plymouth, N.Y. The other animals were born in captivity in the woodchuck breeding colony at the College of Veterinary Medicine, Cornell University, Ithaca, N.Y. Offsprings of dams proven to be free of present or past WHV infection (W223, W231, W2238, W2249, W2260, W2284, and W2606) were inoculated as newborns with a standardized virus infection pool, as previously described (35). All 12 woodchucks were productively infected with WHV for their entire life span as shown by woodchuck hepatitis surface antigen and WHV DNA in the serum. Most of them (with the exception of W223 and W1332) were inoculated at 2 to 3 years of age with different dilutions of the serum of W1366 or W598, two woodchucks producing high levels of human hepatitis D virus (HDV), as described previously (33, 34). During follow-up, HDV RNA and delta antigen were detected in the serum of all inoculated woodchucks, except W157 and W2260. At autopsy, HDV RNA was revealed on Northern (RNA) blots (30) in W188 and W2249 livers and was detected by polymerase chain reaction (PCR) methods (8) in the livers of the remaining HDV-positive animals. Short treatments with antiviral compounds such as arabinosyl-AMP were administered to some animals, as part of an experiment to evaluate drug effects in hepadnavirus infection. The animals were sacrificed at 2.5 to 4 years of age (mean age, 3.0 years), at an advanced stage of HCC development. All animals had one or several tumor masses that were resected independently, quickly frozen in liquid nitrogen, and stored at -70°C. Southern and Northern blot analysis. Total DNA or RNA was extracted from frozen samples of woodchuck HCC, surrounding liver tissues, and normal woodchuck livers as described previously (19), and 20 ,g of DNA or 40 ,g of RNA was analyzed by Southern or Northern blotting (15) with alkaline transfer on Hybond-N+ (Amersham) (0.4 N NaOH for DNA and 0.05 N NaOH for RNA). The probes Splnt 1, specific for N-mycl, and TqEx3C, specific for N-myc exon 3, have been described previously (15). The 165-bp Rsa-Hinfl (RsH) fragment covering N-myc2 exon 1, the 2.2-kb BglII-HindIII (BH) fragment covering exons 2 and 3, and the 157-bp SspI-BglI (SsB) fragment of the 3' untranslated 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-

were also used as hybridization probes. Hybridizations were carried out with 7% sodium dodecyl sulfate (SDS)-0.5 M phosphate (pH 7)-i mM EDTA-50 p,g of salmon sperm DNA per ml at 65°C with 32P-labeled probes. Southern blots were washed twice in 2x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS for 15 min at 65°C and twice in 0.lx SSC-0.1% SDS for 15 min 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 45 min. 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 (Northumbria Biologicals Ltd.) and the following primers: SA2 (5'GCGGTGAT[GAGATCTT-3'), complementary to the minus strand of WHV (positions 2533 to 2549) (16), and NMT18 (5'-GTATCGATGCTCGAACCC-3'), complementary to the coding strand of N-myc2 (positions -644 to -627). The DNA samples were denatured at 94°C for 5 min and sub-

HindIll fragment)

jected to 35 cycles of PCR with 1 min of denaturation at 94°C, 1 min of annealing at 55°C, and 1.5 min of elongation at 72°C. The products were diluted 100-fold and amplified for a second time by PCR with the same primers. The final products were purified by electrophoresis and subcloned in pBS+. DNA was sequenced by the chain termination method (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 initiation sites in the tumors. The 2,627-bp RsaI-HindIII fragment of N-myc2 (positions -310 to +2317) (15) was subcloned in pKS. The plasmid was linearized by SspI (position 1435), giving rise to a 882-bp SspI-HindIII template (positions 1435 to 2317) for mapping of recombinational breakpoints in tumors 2238T1 and 2260T1. Uniformly labeled complementary RNAs were synthesized by using T3 and T7 polymerase, respectively. RNase mapping was performed with 20 ,g

of total RNA from woodchuck liver tumors or adjacent liver tissues (27). Plasmid constructions, cells, and transient-expression assays. Plasmid 1 was constructed by standard techniques (28) by subcloning a 3.2-kb HindIII fragment spanning wild-type N-myc2 retroposon sequences from an EMBL3 recombinant phage (15) into the HindIII site of pBluescript BS+. The WHV 1.65-kb ApaI-BglII fragment (see Fig. 9A) was isolated from a cloned WHV genome (32) and inserted at different positions in plasmids 2, 3, and 4, as described in the legend to Fig. 9. Details of the construction of plasmid 5 will be described elsewhere (14). Plasmid 5 contains N-myc2 promoter and 5'-flanking sequences fused to the promoterless firefly luciferase (LUC) reporter gene, at the unique HindIII site of the vector pSVOALD5' (12). Plasmid 9 was provided by H. de The (10). Plasmids 6, 7, 8, 10, 11, and 12 are described in the legend to Fig. 10. pHBx2s, a plasmid carrying the HBV X gene controlled by the simian virus 40 early promoter, was a kind gift from P. Hofschneider (57). The human hepatoma cell line HepG2 was grown in Dulbecco modified Eagle medium with 10% fetal calf serum and antibiotics under 5% CO2. The plasmids bearing the N-myc2 gene or luciferase gene as a reporter (13 ,ug) and the 13-galactosidase expression vector pCH110 (3 ,ug), used as an internal control for transfection efficiency, were introduced into semiconfluent HepG2 cells (1.5 x 106 cells per 25 cm2) by calcium phosphate coprecipitation. In other experiments,

ACTIVATION OF

VOL. 66, 1992

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

Animnal

Modeof infection

W157 W164 W188 W899 W1332 W223

Natural Natural Natural Natural Natural Experimental

integration WHV N-myc2 Tumor(s) Tuo()in 157T 164T 188T

899Ta 1332T 223T2

223T3 223T5 223T6

W231 W2238

Experimental Experimental

+ (5') + (5') + (5')

23117 2238T1

2238T2 W2249

Experimental

2249T1

W2260

Experimental

2249T2 2260T1

W2284 W2606

Experimental Experimental

+ (5') + (5')

+ + + + +

(5') (5') (3') (5') (5')

22601T2 2284T 2606T1

2606T2

+ (5') + (3') + (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 (a plasmid carrying WHV fragment AB) were mixed with 3 p,g of plasmid 5 and 3 ,g of pCH110, made up to 20 ,g of total DNA with pBS+ DNA, and applied to the culture medium. Luciferase activity was determined from cell extracts prepared 30 to 48 h after transfection as reported previously (10, 12t. For RNA analysis, the transfections were scaled up to 10 cells and total cellular RNA was prepared by the hot-phenol procedure followed by two rounds of acidic extraction (22) to remove contaminating DNA. RESULTS WHV DNA integration in the N-myc2 gene in woodchuck HCC. We analyzed woodchuck HCCs associated with natural or experimental infection with WHV. As listed in Table 1, five wild-caught animals had acquired persistent WHV infection through natural transmission, whereas seven colony-born animals had been inoculated as newborns with a viral infectious pool. Most woodchucks carried one or two tumor masses, and one (W223) bore six distinct tumorous nodules, only four of which were available for the present analysis. A total of 19 independent tumors were recovered from 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 the normal N-myc (N-mycl) and N-myc2 regions with the positions of the probes and relevant restriction endonuclease sites indicated. In Southern blot hybridizations of HCC DNA digested with HindIII, the N-myc probe BH recognized the germ line 7.4-kb N-mycl and 3.5-kb N-myc2 fragments and detected additional bands of various sizes in eight tumors originating from both wild-caught and experimentally infected animals (Fig. 1B). The N-myc loci retained wild-type configuration in the nontumorous parts of the corresponding 4ivers (data not shown). Hybridization of the blots with a WHV DNA probe showed discrete bands of high molecular weight corresponding to viral insertion events in all tumor DNAs (Fig. 1C). Distinct viral integration patterns indicated independent clonal origins for the tumorous nod-

N-myc2

BY WHV IN WOODCHUCK HCC

5267

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-myc allele, the tumor-specific N-myc fragment comigrated with one of the virus-specific bands and could represent a junction fragment linking WHV DNA and N-myc sequences. We also observed the remnants of viral replication in most tumors (Fig. 1C), and all surrounding liver tissues contained abundant viral replicative forms corresponding to intense smears beneath the linearized 3.3-kb genome (not shown). Further hybridization experiments were carried out to confirm viral insertions and characterize the recombination sites in the eight HCCs. The observed rearrangements in N-myc loci could be assigned to the N-myc2 gene, since hybridization of the same Southern blot with a specific N-mycl probe revealed only the unaltered 7.4-kb N-mycl fragment (Fig. 1D). In previous studies, viral insertions into N-myc genes had been invariably mapped to the 3' untranslated region of N-myc (15). We therefore hybridized TaqIdigested DNAs with a probe specific for the third N-myc exon (TqEx3C [Fig. 1A]). As shown in Fig. 2, a tumorspecific band was detected only in three tumors (231T, 2238T1, and 2606T1). Hybridization of the same blot with a viral probe revealed viral integration into N-myc2 in 2238T1 and 2606T1 but not in 231T (data not shown). To map the viral integration sites more precisely and to determine the orientation of WHV DNA relative to N-myc2, we performed a restriction analysis of 2238T1 and 2606T1 DNAs by using a probe of the N-myc2 3' untranslated region (SsB [Fig. 1A]). Digestions with DraI or SspI or with a combination of ClaI and XbaI or ClaI and ApaI generated novel bands that were also detected with a virus-specific probe, indicating viral insertion in the N-myc2 3' untranslated region, as illustrated in Fig. 3. Fine mapping of the viral sequences adjacent to N-myc2 and comparison with the restriction map of the WHV genome showed that, in both tumors, WHV and N-myc2 sequences were placed in the same transcriptional orientation (Fig. 3). The recombinational breakpoints were tentatively localized 220 and 430 bp downstream of the N-myc2 termination codon in 2238T1 and 2606T1, by using RNase mapping with total cellular RNA and a probe spanning the N-myc2 3' untranslated sequences (data not shown). Frequent viral integrations upstream of the N-myc2 retroposon. The absence of junction fragments linking the 3' domain of N-myc2 and WHV DNA in five tumors harboring a rearranged N-myc allele prompted us to explore the entire N-myc2 gene and flanking sequences for viral insertion events in all tumors. Genomic DNA was digested with PvuII, an enzyme with no recognition site within the WHV genome, and hybridized with various probes specific for different regions of N-myc and WHV. Strikingly, the RsH probe specific for N-myc exon 1 (Fig. 1A) detected novel bands not only in the five remaining tumors harboring a rearranged HindIll fragment but also in five other tumors and revealed an additional rearrangement in 231T (Fig. 4A). Rearrangements in the upstream sequences of N-myc2 were confirmed in all cases by hybridizing the PvuII blots with an N-mycl-specific probe, which did not detect additional fragments (data not shown). Hybridization of the blots with a viral probe revealed WHV-specific bands comigrating with each of the additional N-myc2 bands (Fig. 4B), suggesting viral-N-myc junction fragments. Figure 4B also shows that multiple independent viral insertions occurred in all HCC DNAs (average of three insertions per tumor). Further hybridizations with probes specific for various viral subgenomic fragments detected viral sequences homologous to the

J. VIROL.

WEI ET AL.

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FIG. 1. Integration of WHV DNA in N-myc2 in woodchuck liver (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 regions shaded, and untranslated regions open. The probes used in hybridizations 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 (panel B), 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. Open triangles indicate rearranged N-myc2 bands unlinked to viral sequences. No rearrangement of N-myc genes could be detected in HindIII digests of 2249T1 DNA. The positions and sizes of the germ line N-myc alleles are marked on the left. Exposure to X-ray film was continued for 5 days.

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probe and different WHV subgenomic probes established the viral localizations within the N-myc2 upstream region. As shown in Fig. 5B, the structural organization of the viral inserts, partially deduced from the restriction map and comparison with the WHV genome (illustrated in Fig. 3),

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HBV enhancers EnI, EnII, or both in all viral inserts lying near 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 Southern blot analysis of HCC DNAs digested with EcoRI, BamHI, BglII, and TaqI (data not shown). The virus-host junction near the 5' end of N-myc2 was mapped precisely in 2238T2 by a PCR procedure and nucleotide sequencing of the cloned reaction product. As shown in Fig. 5A, a 93-bp fragment was amplified by using two primers complementary to the N-myc2 sequence at positions -644 to -627 with respect to the N-myc2 initiation codon and to WHV sequences at positions 2533 to 2549 (16). Sequencing of the amplified fragment indicated that viral sequences of the C gene were juxtaposed to cellular sequences located 688 bp upstream of the N-myc2 coding domain, in the opposite transcriptional orientation. In four other tumors, the sizes of the additional fragments detected with the N-myc exon 1

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VOL. 66, 1992

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ACTIVATION OF N-myc2 BY WHV IN WOODCHUCK HCC

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indicated

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223T3), showing the opposite orientation relative to N-myc2, whereas in 2249T1 the viral sequences were highly rearranged near the virus-host junction (Fig. SB). The detection of a WHV-N-myc2 junction fragment in RsaI-digested 223T3 DNA, by using the N-myc2 RsH probe and a viral probe, indicated that the 5'-most N-myc2 RsaI site was lost in the tumorous allele and suggested viral integration within the 5' end of N-myc2 exon 1 region. In the six remaining tumors, the viral integration sites were localized in a PvuIIBamHI fragment, 2 to 3 kb upstream of N-myc2 (Fig. 6). The absence of informative restriction sites or the presence of the complex restriction patterns probably resulting from recombination of different viral subgenomic fragments in the inserted sequences did not allow us to further characterize the structure of the WHV inserts. Evidence for rearrangements in cellular DNA at the integration sites was obtained for 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 be detected (Fig. 1C and 4B), suggesting that viral integration was accompanied by a large rearrangement of cellular DNA at the 5' side of N-myc2. In 223T3, a deletion of cellular DNA in the same region was deduced from the restriction map of the viral integration site (Fig. 5B). In our earlier study of 30 other woodchuck tumors (15), no DNA alterations had been observed in the 2-kb upstream N-myc2 region. The finding of frequent viral insertions further upstream of this region (Fig. 6, between the 5' PvuII and BamHI sites) prompted us to reexamine HCC DNAs from the earlier study by using ApaI or PvuII digestion and the N-myc2 RsH probe. In three tumors, viral integrations were mapped to the -3 to -2 kb region, and in one case a DNA rearrangement without apparent involvement of the virus was found in the same region (results not shown). To search for potential DNA rearrangements and viral insertions further upstream or downstream of the previously explored region, HCC DNAs were digested with ApaI or

5270

J. VIROL.

WEI ET AL.

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FIG. 5. Schematic representation of viral integrations 5' to N-myc2. (A) Illustration of the mutated N-myc2 allele and nucleotide sequence of 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 viral regions which could not be characterized are represented as a dashed bar. The amplification product obtained after PCR from 2238T2 genomic DNA, by using N-myc2 and WHV primers, was entirely sequenced. Nucleotides of the viral sequence are shown in capital letters, and those of N-myc2 are shown in lowercase letters. (B) Restriction map of viral integrated sequences in 188T, 223T2, 223T3, and 2249T1. The RsaI site (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 of N-myc2 retroposed sequences. In this tumor, the location of the cellular PvuII and EcoRI sites upstream of viral integrated sequences is consistent with a 2.5-kb deletion in N-myc2 5'-flanking sequences.

doubly digested with ApaI and ClaI and hybridized with N-myc2 probes or with a specific cellular probe derived from unique sequences located 12 kb upstream of N-myc2 (14). No additional alterations or integrations were found in the 28-kb region surrounding N-myc2 (-15 to +13 kb) in the woodchuck HCCs analyzed (results not shown). As summarized in Table 1, 13 of 19 HCCs from naturally and experimentally infected animals showed integrated WHV 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 of N-myc2 (in 11 cases). Figure 6 presents an overview of the integration sites and, when determined, the transcriptional orientation of the viral inserts. No viral insertion could be detected in the woodchuck N-mycl gene in the present study. Insertional activation of c-myc by WHV DNA, demonstrated in one additional tumor, has been described elsewhere (56). Activated expression of N-myc2 in woodchuck HCC. We have previously described abundant 2.3-kb N-myc2 tran-

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ACTIVATION OF N-myc2 BY WHV IN WOODCHUCK HCC

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viral sequences indicating the viral positive-strand orientation and the Enl and En2 sequences homologous to HBV enhancers 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 were constructed by inserting theWHVApaI-BglII fragment upstream of N-myc2 in plasmid 1, either in the opposite (plasmid 3) or 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. represents

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DISCUSSION Previous studies have demonstrated that woodchucks naturally or experimentally infected with WHV are at exceptionally high risk for HCC (17, 35). Our initial observations of frequent viral integrations into woodchuck myc loci have suggested that the rapid onset of liver tumors in this model might be correlated with a direct, cis-acting effect of integrated viral sequences in activating myc family oncogenes (15, 19). This report describes a detailed analysis of WHV DNA insertions into myc genes in a new panel of 19 woodchuck HCCs from wild-caught and colony-born animals that acquired persistent WHV infection from natural transmission or inoculation with a selected viral stock at birth. Confirming and extending our previous data, we found that integrated viral sequences were preferentially associated with N-myc2, a recently identified retrotransposed oncogene peculiar to woodchucks: 13 of 19 tumors showed insertional mutagenesis of the N-myc2 locus, whereas none harbored viral integration in the classical N-myc gene. In a parallel analysis, WHV DNA integration in c-myc was observed in one of these tumors, as described elsewhere (56). Experimental infections yielded integration events at the same frequency as did natural infections. In agreement with this, recent investigations of the evolution of experimental laboratory infections have shown kinetics of HCC development and virological and histological patterns that were essentially identical 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 retrotransposon. In our earlier study of 30 different HCCs (15), we detected viral insertions exclusively in the N-myc2 3' noncoding region; however, the cellular domain extending further than 2 kb upstream of N-myc2 had not been explored at that time. Reexamination of the previously analyzed tumors revealed viral integration in a region between -3 and -2 kb in the N-myc2 map in three additional HCCs, showing that the two panels of woodchuck HCCs differ only in the frequency of viral insertions near N-myc2. These variations,

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ACTIVATION OF N-myc2 BY WHV IN WOODCHUCK HCC

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if statistically significant in a limited number of samples, possibly reflect different stages of tumor progression at sacrifice; no clear correlation with other parameters, such as superinfection with HDV or treatment with antiviral drugs, could be established. Investigations of a 28-kb cellular domain surrounding the N-myc2 retroposon did not provide evidence for additional viral integration sites located further upstream or downstream of N-myc2 in the tumors from both series. Together, these results now demonstrate integration of WHV DNA next to the N-myc2 proto-oncogene in more than 40% of the 49 woodchuck HCCs analyzed. Furthermore, it can be estimated that about 25% of the total integration events detected in these tumors occur in the N-myc2 locus. The reasons for the strong clustering of viral insertions adjacent to N-myc2 are not yet clear. Common integration sites, including myc genes and different proto-oncogenes, have been found in cells infected by many nonacute retroviruses (6, 18, 29, 31, 42). This apparent specificity might reflect molecular requirements to produce oncogenic effects and might be selected for by clonal outgrowth of transformed cells. However, the rate of viral targeting to the woodchuck N-myc2 retrotransposon in HCC is much higher than that to c-myc and N-myc genes, despite comparable oncogenic efficiencies of the three genes in rat embryo fibroblast cotransformation assays (14, 15). Previous studies have demonstrated the use of highly preferred target sites for integration of different RNA and DNA viruses in the absence

of selective pressure (4, 38, 45). Integration of HBV DNA in human HCC has also been observed at a rate higher than average in chromosomes 11 and 17, although no evidence has been provided so far for a target gene at the integration sites (50). Preferred retrovirus integration sites have been mapped near DNase I-hypersensitive sites and CpG-rich islands located in the vicinity of many eucaryotic genes (37, 40, 54). N-myc2 sequences, which are extremely rich in CpG doublets, appear to be a related example (15) and might be considered an HCC susceptibility gene peculiar to woodchucks. In this regard, it is interesting that a locus homologous to the woodchuck N-myc2 retrotransposon is also present in the squirrel genome, although information is still lacking on its coding and transforming potential. Insertional mutagenesis of the squirrel N-myc2 locus by hepadnavirus DNA has not been observed in studies of liver tumors associated with ground squirrel hepatitis virus infection (51). Differences in intrinsic oncogenic properties have been noted between the two rodent hepadnaviruses (41), and the relatively long latent period of HCC (5 to 9 years) in squirrels might be correlated with a reduced ability of ground squirrel hepatitis virus to integrate into host genomic DNA, thereby lowering the overall chances of insertional mutagenesis (51). In human HCCs as well, insertional activation of the myc proto-oncogenes has never been observed, despite frequent viral integration events. The absence of a human N-myc2 retroposed oncogene might be a major host factor possibly implicated in these discrepancies. However, it has recently

5274

WEI ET AL.

been shown that the human c-myc and N-myc genes can provide occasional targets for integration of another DNA tumor virus, human papillomavirus (5), and the reasons for different oncogenic steps in the development of HBV- and WHV-associated HCCs represent an intriguing problem. A common feature of woodchuck HCC is enhanced accumulation of N-myc2 mRNAs compared with adjacent liver tissues, whereas N-myc2 is apparently silent in normal adult hepatocytes (15). In many tumors, viral integration interrupted different regions of the N-myc2 locus and juxtaposed WHV sequences homologous to the well-characterized HBV enhancers (44, 59) to the N-myc2 coding domain. Our in vitro studies clearly indicate that the WHV genome contains enhancer elements capable of activating heterologous promoters in a position- and orientation-independent manner in a differentiated hepatoma cell line. In woodchuck HCCs, integration of viral DNA in the 5'-flanking N-myc2 region occurred frequently in the opposite transcriptional orientation and was associated with the production of abundant N-myc2 transcripts of normal size, initiated in the 5' noncoding part of N-myc exon 2. A cryptic N-myc promoter that controls N-myc2 expression in adult brain tissues has been recently identified in the corresponding N-myc2 region (14). No chimeric RNA starting at a viral promoter could be detected, excluding a mechanism of promoter insertion, as described for avian lymphomas (18). It seems likely, therefore, that nearby integration of WHV DNA activates the N-myc2 promoter through enhancer insertion, as in many retrovirus models (6, 42, 53). A similar mechanism might also prevail in other woodchuck HCCs, in which the viral inserts interrupted the N-myc2 3' noncoding domain, leading to N-myc2-WHV cotranscripts. It has been suggested that the removal of control elements which might interfere with efficient translation or RNA stability may potentiate particular proto-oncogenes. The 3' region of N-myc2, like those of c-myc and N-myc (1, 21), might contain negative regulatory sequences, and their deletion might contribute to enhanced accumulation of N-myc2 mRNA. In addition, variations in the relative N-myc2 RNA levels among different HCCs could not be strictly correlated with a position-dependent effect of virus integrated sequences containing the enhancers. It is possible that the modulations in liver-specific phenotype, which occur during hepatocyte transformation, influence the efficiency of the WHV enhancers to stimulate N-myc2 expression, as also suggested by recent studies of the activity of the HBV enhancers on viral promoters in different cell lines (46). The production of abundant N-myc2-specific transcripts in a significant number of tumors which do not harbor viral integration in N-myc2 indicates that N-myc2 may be activated through alternative mechanisms. An attractive hypothesis suggests that viral integration in a cellular locus genetically related to the N-myc2 locus, but located a considerable distance away, might cause its aberrant expression. Such a mechanism has been demonstrated in myeloid leukemias by the finding of retroviral insertions 90 kb proximal to the Evi-1 myeloid transforming gene (2). A similar question arises from recent studies of avian nephro-

blastomas induced by myeloblastosis-associated virus, showing that overexpression of the transforming nov gene is associated only occasionally with viral insertion at this locus

(20). Alternatively, deregulated N-myc2 expression might

result from trans-acting mechanisms associated with persistent expression of viral genes or with programmed resurgence of a fetal phenotype in transformed liver cells. Our in vitro studies, showing a weak but reproducible transactivation of

J. VIROL.

the N-myc2 promoter by the viral X protein, suggest that expression of the X transactivator might participate in trans in the transcriptional activation of N-myc2. More detailed studies with various WHV-N-myc2 constructions and different cell lines are necessary to establish the relative contributions of cis and trans mechanisms in the observed up regulation of N-myc2 RNA in woodchuck HCCs. The detection of high levels of N-myc2 transcripts in precancerous nodules from chronically infected livers has suggested that N-myc2 activation might occur at an early step of woodchuck hepatocarcinogenesis (58). Whether enhanced N-myc2 expression in preneoplastic cells is due to an early viral integration event or to a trans-acting mechanism remains to be determined to further delineate the role of WHV integration in the genesis of woodchuck HCC. ACKNOWLEDGMENTS

We thank L. Johnson, J. Gerin, and B. Tennant for providing woodchucks experimentally infected with WHV from the National Institute of Allergy and Infectious Diseases colony at Cornell University. We are grateful to C. Transy and M. Robertson for helpful discussions and critical reading of the manuscript, to C. A. Renard for excellent technical assistance, and to L. M. Da for secretarial assistance. This work was supported by the Commission of the European Community and the Association pour la Recherche contre le Cancer. Y.W. is supported by the Fondation Merieux. REFERENCES

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