0022-538X/94/$04.00+0
Copyright ©) 1994,American Society for Microbiology
Analysis of Integrated Ground Squirrel Hepatitis Virus and
Flanking Host DNA in
Two
Hepatocellular Carcinomas
CATHERINETRANSY,CLAIRE-ANGELIQUE RENARD,ANDMARIE-ANNICKBUENDIA* Departement des Retrovirus, Unitede Recombinaison etExpression Genetique (InstitutNational de la Santeetdela
RechercheMedicaleU163), Institut Pasteur, 75724Paris Cedex15, France
Received 28 January 1994/Accepted 17 May 1994
Wecloned theintegrated groundsquirrelhepatitisBvirus(GSHV) sequencesfromtwohepatomas showing
a single viral insertion. The GSHV inserts shared structural features with integrated DNAs of other
hepadnaviruses. Insertional activation ofa cellulargene appears unlikely: the integrated GSHV sequences
lacked the known viral enhancers andwerenot expressed in the tumors,and wefound noevidence forthe
presenceofa geneattheintegration site. Our results,togetherwith thosefrom earlier studies, suggestthat GSHV does not behave as an extensive insertional mutagen, in sharp contrast with the closely related woodchuck hepatitisvirus. GSHVmaythuscause carcinogenesis bymore indirectmechanisms, asdoes the
humanhepatitis B virus.
Whether viral integration playsamajor rolein hepatocarci-nogenesis associated with mammalian hepadnaviruses remains acontroversial issue (reviewed in references 2 and 15). That hepadnaviruses might act as insertional mutagens of proto-oncogenes, in a way similar to nonacute oncogenic retrovi-ruses, has been demonstrated in the case of the woodchuck hepatitis B virus (WHV). More than 50% of WHV-induced tumors showcis activation ofamyc family gene as a result of nearby insertion of viral enhancer regions (2, 8, 30). In contrast, few examples of insertional mutagenesis by the humanhepatitis Bvirus (HBV) are known, and these do not involve a unique gene or class of genes as targets. Further-more, inthe three examples well documented so far, namely, the retinoic acid receptor (5), cyclin A (29), and the meval-onatekinase (10) genes, mutagenesis does not proceed from an enhancer insertion mechanism as in the WHV model. Instead,aviral promoterreplaces the natural promoter of the affected gene and the gene product itself is also altered as a resultof the fusion with viralproteinsequences.Despite these
differences, WHV and HBVintegrated sequencesshow com-monstructuralfeatures.Forbothviruses,aspecific integration mechanism of thesortknown in retroviruses has been excluded
(9) and integration of a defective viral form encompassing
various subgenomic fragmentsseems to be the rule(2, 18). The ground squirrel hepatitis virus(GSHV) has been stud-ied in less detail than WHV and HBV with regard to the structure ofviral integrations and their potential mutagenic
effects. Wepreviously showed that the GSHV integrations in tumorsfromnaturally infectedgroundsquirrelsdonotinvolve myc loci(25), whereas c-mycamplification frequentlyoccurs. Hansen et al. (11) analyzed woodchuck tumors induced by
experimental infection with GSHV and reported similar re-sults. GSHV thus appearsmoresimilartoHBV thantoWHV both withregardtothe lateonsetof livertumorsfollowing viral infection (14) and with regard to the absence (or low
inci-dence) of insertional mutagenesis of myc genes. However, since GSHVintegrated sequences have notbeencloned, our
*Correspondingauthor.Mailingaddress:
Unite
deRecombinaisonet Expression Gen6tique (Institut National de la Sante et de la RechercheMedicaleU163),Institut Pasteur, Departement des
Retro-virus, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: 33-1-4568-8866. Fax: 33-1-4568-8943.
information is incomplete. In the present work, we cloned single integrated GSHV sequences from two squirrel liver tumors and analyzed the structure of viral DNA and host flanking sequences.
Genomic librarieswereconstructed in the A DASH IIvector
(Stratagene) from two hepatocellular carcinomas of ground squirrels, RV53 and RV50, that showed persistent and past GSHV infections, respectively. Both tumors carried a single GSHVintegration and showed c-myc gene amplification (25). Afterscreening withaGSHVprobe,twopositiveclones from each librarywere selected for further analysis. Phage inserts were excised by digestion at the flanking NotI sites, and restriction mapping was performed by the partial digestion method recommended by the vector supplier. Regions of overlap between phage inserts showed identical restriction maps, indicating thatno rearrangement had occurred during the cloning procedure. We next subcloned restriction frag-mentscontainingviral sequences anddetermined thestructure of the integrated viral DNA and virus-host junctions by sequencing analysis.
Structure ofintegrated fragments in RV53 and RV50 tu-mors. InRV53, the integratedviral DNA consists ofalinear
subgenomic fragmentabout 1kb inlength.Itextendsfrom 22 bp after thebeginningof thecoregene(C)tothelargesurface protein gene
(pre-Si),
134 bp downstream of the initiation codon(Fig.1A).InRV50, theintegratedsequencesconsist of twojuxtaposedGSHVfragments. Oneof thetwosegments is colineartothe GSHV genome andsimilar in structure tothe RV53viral insert.It startsinthe precoreregion (pre-C),spans the C gene, and ends within thepre-Si
region. The other segment, placed in the opposite transcriptional orientation,coversthe entirepre-Sl andpre-S2 regions,followed bya 3' truncated S geneshowing aninternal deletion of 280bp (Fig.
1B). Therefore, the two classes ofintegrations described for HBV, i.e., the simple and the complextypes (18), are repre-sented in RV53 andRV50,respectively.
Bothintegrated GSHV sequences have retained thepre-Sl
promoter region, and RV50 also contains the S promoter region. However, these promoters remained apparentlysilent in the tumors, since they did notgeneratetranscripts detect-able inNorthernhybridizationof
poly(A)+
RNAwithaGSHVgenomic probe(data not shown).
The left end of RV50 integrated DNA is located 4 bp
5291
on November 9, 2019 by guest
http://jvi.asm.org/
0 0 0
67% 64%
o a 0
o co t
64% 67%
pRbI2
H/
Retinoblastomagene
GSHV
0
'>
Fl
B 1kb
F3
ERXRRXXR
BaH H Ba
x
I r
200 bp
I
H Bai
esi1
I--- > -.r
1937 HOST 4-|-4 GSHV
tctttttaagaagaaaatctttCTTlTCACCTGTG
DRI
preS1 S S preS2 preS1 L
2851
GSHV HOST
AAAAAGTGTTCTtttcttc(t)10(ag)8ggg(ag) 4
FIG. 1. Structure of integrated GSHVsequencesinRV53(A)and RV50 (B) tumors. (Top) Restriction mapof the genomic insert: B, BamHI; Bg, BglII; R, EcoRI; H,HindIII; P, PvuII; S,Sall;X,XbaI. The shaded box represents the integrated viral fragment. Probes
derived fromflankinghostregionsareindicatedbythick lines above
themap.(Middle)Structureof the integratedviralsequences.Boxes
representGSHV integratedsequences(numberingsystemofWHV). Arrowsindicate thepositionsof initiationcodons andtranscriptional
orientation of the viralgenes.(Bottom) Sequenceof the viral-cellular
junctions.The tandem repeat present in the leftjunction of RV3 is underlined,and theDR1sequenceclosetothe viraljunctionofRV50 is boxed.
upstream ofDR1, one of the two 11-bp direct repeats that bracket the cohesive endregioninhepadnaviralgenomes.This site maps to the short region ofterminal redundancyin the minus-strand DNA that confersatriple-stranded structure to virion DNA (20). A strongpreference for integration within this region has been reported in studies of HBV integration (18, 23). Furthermore, thecorresponding region inthe WHV genome shows a cluster of topoisomerase I cleavage sites thought to mediate illegitimate recombination between viral and cellular DNAs (28).
Incontrast, theviraljunctions in RV53 integration do not
fall intherecombination-proficient regions identified alongthe HBV genome, namely, the cohesive overlap (including the
aforementioned site upstream of DR1) and the transcription initiation sites ofthepre-S and Sgenes (18). Itis of interest
that the cellularsequenceadjacenttotheleft end of viralDNA shows a perfect direct duplication of 23 bp (Fig. 1A). One
repeat is located close to and the other is located at the cellular-viraljunction,the viralsequencecontributing the four
[image:2.612.62.302.70.358.2]lastnucleotidesof the motif. Since the unoccupied sitein the host genome was not accessible for molecular cloning (see below),wedonotknowwhether theduplication resulted from the integration event or preexisted. In the latter case, patch
FIG. 2. Homology betweenthe cellularregion flankingtheRV53 viral insert and known human sequences. The ground squirrel
se-quence is represented by a thin line, and the integrated GSHV
sequence is represented by a hatched box. Stippled boxes indicate
sequenceshomologoustohuman Rbgeneregions depicted byarrows
in the top part. Open boxes indicate sequences homologous tothe human transposon L1.2 sequences represented by arrows in the bottompart.Thedirection ofarrowsshowsthetranscriptional orien-tationof humansequences,and the numbersplaced verticallyreferto
the actual numbering in the database entry. The percentages of
identity between theground squirrel and the human sequences are indicated. Theground squirrel Fland humanpRbI2 probesareshown by thick solidbars.
homology betweenviral and hostsequences might have
con-tributedtotherecombination process,aspreviouslysuggested
inseveral casesofHBVintegration (12, 13, 23).
Highlyandmoderatelyrepetitivesequencesinhostflanking
regions. The cellular sequence adjacent to the right viral junction in RV50 and host regions surrounding the viral segment in RV53 strongly hybridized with labelled total ground squirrel genomic DNA (not shown), indicating the
presenceofhighly repetitive DNA.Furthermore, exploration ofmore distalcellular sequencesdidnotrevealany fragment
behavingas auniqueprobe,whichprecludedfurthercloningof
theunoccupied site.
Inspection of the cellular DNA sequence in the 400-bp
EcoRI fragment encompassingtherightviral-cellularjunction in RV50 revealed the presence of a microsatellite sequence
consistingofanAG dinucleotiderepeatfollowingastretchof
10 T's butnootherknownrepetitiveelement(Fig. 1Band data
notshown).InRV53,theintegratedviral DNAwasembedded
in a cellular sequence showing strong homology with the 3' region of human and rodent insertion elements of the long interspersedrepeatedDNAfamily,referredtoasLINEorLi sequences (Fig. 2) (24). Li sequences have been found inall mammaliangenomes examined,inwhichtheymakeup about 5% of total genomic DNA. Most LI copies are truncatedor
rearrangedand arethought tobe defectiveretrotransposition products from a few full-length competent Li elements (6). Thegroundsquirrelsequenceadjacenttothe left viraljunction islikelytobe ofthe defectivetypesinceit lacked the3' endof Li sequences that typically includes a poly(A) stretch (27).
However,thesequenceslocatedoneachside of the viral insert
could be aligned with the full-length human L1.2 sequence without introduction ofagap(Fig. 2), indicatingthatnomajor
A
Fl
1kb
F2
xDAS
0) CD
o f
a)
65%
Transposon Li.2 0
-4-.
67%
3247841
R, r-issP1Fv471-1 -1
11 ts
on November 9, 2019 by guest
http://jvi.asm.org/
[image:2.612.316.556.73.255.2]o? 0) xCl m3 v o 4) ell}v o oz el) v 4)Om 0) o co CUV to X ) co° cQ to xl t o co N
.:Z -:1 2z N C- N N N N C-) N >.U
-c t zir cL()cJ ir ir rQ. c) a: z cc Z4C c)
Fl
pRbI2
kb
23.0-9.6_w_9 t!.
6.6 - -.a
4.4- V
*1
2.2
-2.0
-Fl
X-_is--l --w_-,
....
1-....
W.-.... "
W
S
H
kb 23
-I
9.6
-6.6
-W S H '-W S H
'U.-4ON.
4.4
[image:3.612.363.509.78.257.2]-F2 F3
FIG. 3. Moderately repeated sequences in the cellular regions flanking integrated GSHVsequences. Genomic DNAs (15 ,iLg)from the ground squirrel liver tumors indicated above each lane were digested withBglII. After Southern blotting, hybridizationwas per-formedwith theprobesindicated beloweach panel. ProbesFland F2 arederived from RV53, and probe F3 is derived from RV50 (Fig. 1).
rearrangement of host sequences has occurred upon viral integration.
Hepadnaviral integration into repetitive DNA is not without precedent, since HBV integration into satellite and Alu se-quences hasbeenreported for several tumors (16, 18, 21). This may simply be the consequence of the relative abundance of repetitive sequences in the genome. Alternatively, since Alu or
Li
sequencesexpand via a retrotransposition mechanism that requires integration of reverse-transcribed RNA at a newgenomic site, one might speculate that the genomic regions harboring such repetitive elements are especially prone to recombination events.
In both RV5O and RV53 cloned genomic inserts, several restriction fragments were not revealed by labelled total ground squirrel genomic DNA. However, each fragment re-vealed a distinct series of -20 discrete bands in Southern
analysis of groundsquirrelgenomicDNAasillustrated inFig. 3. One of these fragments (probe Fl, located -2.5 kb away from the left viraljunction in RV53) (Fig. 1A) detected related sequencesin human aswell as inwoodchuck genomes, when usedat arelativelyhighstringency(Fig. 4B).Thispromptedus todetermine the sequenceof the -3.9-kbregion flanking the left viral junction. The resulting nucleotide sequence, deter-minedonbothstrands by thedideoxynucleotide method,was submitted to the EMBL data bank. FASTA and BLAST programs detected a significant homologyscore between an -2-kb region encompassing the Fl probe and only one sequence of the nucleotide data banks, namely, the hu-man retinoblastoma gene (Rb) sequence (accession number
L11910).Homologywaslocatedin intron 2 of Rb, which spans
position5551 toposition 33894, andwassplitintwoblocksof
1,200 and 650nucleotideswith respectto the gene
transcrip-tional orientation (Fig. 2). Ahigh degree of conservation of intron sequences betweenspecies is not expected; however, Rb is a tumor suppressor gene, first identified because of its
systematic inactivation in human retinoblastomas and later found inactivated in various human tumors (reviewed in reference 1), including hepatocellular carcinomas (17). We therefore examined the possibility that GSHV haddisrupted
one of theground squirrel Rb alleles. However,we obtained noevidencesupportingthishypothesis.Rbexon2andexon3 probes generated byPCR with ahuman Rb cDNA clone did
2.2
-A
B
C
FIG. 4. DetectionofFl-relatedsequences inwoodchuck and hu-mangenomes.GenomicDNAs(15
p.g)
fromwoodchucks(W), ground squiffels (S), andhumans(H)weredigested withBamHIand trans-ferred to a nylon membrane. (A) Hybridization with the ground squirrelFlprobe(15-hexposure); (B)sameprobe as for panel A(72-h exposure); (C) the blot stripped and rehybridized with the human pRbI2 probe depicted in Fig. 2. The hybridization and washing solutionsdescribedinreference4wereusedat60°C.The human DNA fragments revealedby both probesareindicatedbydots. Theposition of the band generated by the human Rb gene is shown by an arrowhead.nothybridizewith the RV53 X clone insertscontaining GSHV sequences;furthermore, noalterationof thesquirrelRb gene structure could be detected in RV53 tumor by Southern
analysis, and Rb transcripts of normal size wererevealed by Northernblotting of tumor RNA(data not shown). Finally, by
using recombinant X phage DNAs spanning the human Rb intron 2(31) (generouslyprovided by T. Dryja, Boston, Mass.), we synthesized a 400-bp PCR fragment (pRbI2) from the region of homology with the ground squirrel Fl probe (Fig. 2). This probe revealed several bands in human genomic DNA (Fig. 4C); therefore, thecorrespondingsequenceis moderately
repeated inthe human genome and isnotspecificfor the Rb locus. In addition, thefragment generated by the human Rb locus doesnotcorrespondtothemostintense bandrevealed in human genomicDNAby the groundsquirrelFl probe (com-parelanesHofpanelsBandC).This suggests that thesquirrel Fl and the human Rb intron 2 probes do not map to homologous loci but rather represent membersofapreviously noncharacterized family of moderately repeated DNA con-served in several mammalianspecies.
GSHVintegrationandhepatocarcinogenesis.Thepossibility
ofadirect role of viral integrationinhepatocarcinogenesisis best addressed withtumors showing asingle viralintegration
event. The two GSHV-associated tumors analyzed here met thiscriterion.Theyalso shared c-myc gene
amplification,
which might have reflectedapossible cooperationbetween c-mycand another oncogene activated by viral insertion. Thus, the so-called provirus tagging strategy (26) has identified several oncogenes cooperating with c-myc in the development of mouse B-cell lymphomas. However, in the two particulartumors examined here, a mechanism of oncogene activation viaenhancer insertion isunlikelybecause bothGSHV
integra-tions lacked theregionextendingfrom the end of the S geneto the beginning of the C gene, which carries the known viral enhancers (22, 32). This feature should be
underlined,
sinceon November 9, 2019 by guest
http://jvi.asm.org/
[image:3.612.80.273.78.234.2]the relevant activated oncogene need not necessarily reside close to the viral integrationsite;thus, we have recently found that in several woodchuck liver tumors, oncogene activation results from WHV insertions at a distance of over 150 kb (7). A promoter insertion mechanism appears unlikely as well, given the absence of detectable transcripts driven by the integrated promoters in the corresponding tumors.
As pointed out earlier (19), viral integration might also inactivate a tumor suppressor gene, which theoretically re-quires only a physical disruption of regulatory signals (e.g., promoter-enhancer regions, splicing signals) or the coding capacity of the gene. We obtained no evidence for such a mechanism; however, we keep in mind the possible existence of very large introns and/or tiny exons at the site of viral integration which might be difficult to recognize.
Finally, there is no reason to anticipate an alteration in trans of host gene expression by the integrated viral sequences: the latter did not include the viral X transactivator gene, and the truncated pre-S2/S viral transactivator (3) potentially encoded by theRV50 viral insert was not expressed at a detectable level. In conclusion, our analysis of integrated GSHV DNA has revealed features frequently described in HBV and WHV integration events: (i) integration of a defective genome con-sisting of either a linear or a rearranged sequence, (ii) a viral recombination breakpoint close tothedirectrepeatDR1,and (iii) the presence of highly repetitive DNA at the hostsite of integration. This suggests that mechanisms ofintegration are common to all mammalian hepadnaviruses, in keeping with their genetic similarity. As in thevastmajority ofcharacterized HBV integration sites, we obtained no evidence for direct alteration of a hostgene byGSHV DNA.We of course do not wish to imply from our study that insertional mutagenesis by GSHV neveroccurs; however, the picture nowemerging from thecomparison of the threemammalianhepadnaviruses is that WHV may be unique in its extensive ability to give rise to insertional activation ofoncogenes.
Nucleotide sequenceaccessionnumber.The sequenceof the -3.9-kb region flanking the leftviraljunctionwas given EMBL accessionnumberX77801.
We thank P. Tiollais for his constant interest in our work. We are indebted to P. Marion for the gift of ground squirrel tumor samples and toT. Dryja for providing the human Rb cDNA and Rb gene X phages. We thank members of our team and especially Y. Wei for support andsuggestions and P. Legrain for many helpful discussions. We are grateful to A.Tartakoff for help in writing the manuscript.
This work was supported in part by grant ARC 6550 from the Association pour la Recherche contre le Cancer.
REFERENCES
1. Bookstein, R., and W. H. Lee. 1991. Molecular genetics of the retinoblastoma suppressor gene. Crit. Rev. Oncog. 2:211-227. 2. Buendia, M. A. 1992. Hepatitis B viruses and hepatocellular
carcinoma. Adv. Cancer Res. 59:167-226.
3. Caselmann, W. H., M. Meyer, A. S. Kekule, U. Lauer, P. H. Hofschneider, and R. Koshy. 1990. A trans-activator function is generated by integration of hepatitis B virus pre-S/S sequences in human hepatocellular carcinoma DNA. Proc. Natl. Acad. Sci. USA87:2970-2974.
4. Church, G., and W. Gilbert. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA81:1991-1995.
5. Dejean, A., L. Bougueleret, K. H.Grzeschik, and P. Tiollais. 1986. Hepatitis B virus DNAintegration in a sequence homologous to v-erbA and steroid receptor genes in ahepatocellular carcinoma. Nature (London) 322:70-72.
6. Dombrosky, B. A., A. F. Scott, and J. Kazazian. 1993. Two additional potential retrotransposons isolated from a human LI subfamily that contains an activeretrotransposable element. Proc.
Natl.Acad. Sci. USA 90:6513-6517.
7. Fourel, G., J. Couturier, Y. Wei, F.Apiou, P.Tiollais, and M. A. Buendia. Evidence for long range activation by hepadnavirus insertion. EMBO J.,in press.
8. Fourel, G., C.Trepo, L. Bougueleret, B.Henglein, A.Ponzetto,P. Tiollais, and M. A.Buendia. 1990. Frequentactivation ofN-myc genes by hepadnavirus insertion in woodchuck liver tumours. Nature (London)347:294-298.
9. Ganem, D., and H. E. Varmus. 1987. The molecular biology of the hepatitis B viruses. Annu. Rev.Biochem.56:651-693.
10. Graef, E., W. H. Caselman, and R. Koshy. 1993. Differential splicing ofoverexpressedviral-cellular fusiontranscriptsin human hepatoma celllinePLC/PRF/5, p. 72. Program Abstr. Mol. Biol. Hepatitis B Virus 1993 Meet.
11. Hansen, L. J., B. C.Tennant, C. Seeger, and D. Ganem. 1993. Differential activation of myc gene family members in hepatic carcinogenesis by closely related hepatitis B viruses. Mol. Cell. Biol. 13:659-667.
12. Koch, S., A. Freytag von Loringhoven, R. Kahmann, P. H. Hofschneider, and R. Koshy. 1984. The genetic organizationof integrated hepatitis B virus DNAin the humanhepatomacell line PLC/PRF/5. Nucleic Acids Res.12:6871-6876.
13. Koshy, R., S. Koch, A. Freytag vonLoringhoven,R.Kahmann,K. Murray, and P. H.Hofschneider. 1983. IntegrationofhepatitisB virus DNA: evidence for integration in the single-stranded gap. Cell34:215-223.
14. Marion, P. L. 1991. Groundsquirrelhepatitisvirus,p. 39-51. In A. McLachlan (ed.), Molecularbiology of thehepatitisBvirus. CRC Press, Inc.,Boca Raton, Fla.
15. Matsubara, K. 1991. Chromosomal changesassociated with hep-atitis B virus DNA integration and hepatocarcinogenesis, p. 245-256. In A.McLachlan(ed.), Molecular biologyof thehepatitis Bvirus. CRC Press, Inc., Boca Raton, Fla.
16. Meyer, M., K. H. Wiedorn, P. H. Hofschneider, R. Koshy, and W. H. Caselman. 1992. A chromosome 17:7 translocation is associated with a hepatitis B virus DNA integration in human hepatocellularcarcinoma DNA. Hepatology 15:665-671. 17. Murakami, Y., K. Hayashi, S. Hirohashi, and T. Sekiya. 1991.
Aberrations of the tumor suppressor p53 and retinoblastoma genesin human hepatocellularcarcinomas. Cancer Res. 51:5520-5525.
18. Nagaya, T., T. Nakamura, T. Tokino, T. Tsurimoto,M. Imai,T. Mayumi, K. Kamino, K. Yamamura, and K. Matsubara. 1987. Themode ofhepatitis B virus DNA integration in chromosomes ofhumanhepatocellular carcinoma. Genes Dev. 1:773-782. 19. Ogston, C. W., G. J. Jonak, C. E. Rogler, S. M. Astrin, and J.
Summers. 1982. Cloning and structural analysis of integrated woodchuck hepatitis virus sequencesfromhepatocellular carcino-masofwoodchucks. Cell 29:385-394.
20. Seeger, C., D. Ganem, and H. E. Varmus. 1986. Biochemical and genetic evidence for the hepatitis B virus replication strategy. Science 232:477-484.
21. Shaul, Y., P. D. Garcia, S. Schonberg, and W. J. Rutter. 1986. Integration of hepatitis B virus DNA in chromosome-specific satellite sequences. J. Virol. 59:731-734.
22. Shaul, Y., W.J. Rutter, and 0.Laub. 1985. Ahuman hepatitis B viralenhancerelement. EMBOJ. 4:427-430.
23. Shih,C., K. Burke, M.-J. Chou, J. B. Zeldis, C.-S. Yang, C.-S.Lee, K.J.Isselbacher, J. R. Wands, and H. M. Goodman. 1987. Tight clustering of human hepatitisBvirusintegration sites in hepato-mas near atriple-strandedregion. J. Virol. 61:3491-3498. 24. Singer, M. F., and J. Skowronski. 1985. Making sense out of
LINES: long interspersed repeat sequences in mammalian ge-nomes. Trends Biochem. Sci. 10:5739-5745.
25. Transy, C., G. Fourel, W. S. Robinson, P. Tiollais, P. L. Marion, and M. A. Buendia. 1992. Frequent amplification of c-myc in ground squirrel liver tumors associated with past or ongoing infection with a hepadnavirus. Proc. Natl. Acad. Sci. USA 89: 3874-3878.
26. van Lohuizen, M., and A. Berns. 1990. Tumorigenesis by slow-transforming retroviruses-an update. Biochim. Biophys. Acta 1032:213-235.
27. Voliva, C. F., C. L. Jahn, M. B. Comer, M. H. Edgell, and C. A.
on November 9, 2019 by guest
http://jvi.asm.org/
Hutchisson III. 1983. The LlMd long interspersedrepeatfamily in
themouse:almost all examplesaretruncatedatoneend. Nucleic
Acids Res. 11:8847-8859.
28. Wang, H.-P., andC. E. Rogler. 1991. Topoisomerase I-mediated integration of hepadnavirus DNA in vitro. J. Virol. 65:2381-2392. 29. Wang, J., X. Chenivesse, B. Henglein, and C. Br6chot. 1990. Hepatitis B virus integration in a cyclin A gene in a human hepatocellular carcinoma. Nature (London) 343:555-557. 30. Wei, Y., G. Fourel, A. Ponzetto, M. Silvestro, P. Tiollais, and M. A.
Buendia. 1992. Hepadnavirus integration: mechanisms of
activa-tion of the N-myc2retrotransposoninwoodchuck livertumors.J. Virol. 66:5265-5276.
31. Wiggs, J., M. Nordenskjold, D. Yandell, J. Rapaport, V. Grondin, M. Janson, B. Welerius, R. Petersen, A. Craft, K. Riedel, R. Lieberfarb, D. Walton, W. Wilson, and T. P.Dryja.1988. Predic-tion of the risk of hereditary retinoblastoma, using DNA polymor-phisms within the retinoblastomagene.N.Engl. J. Med. 318:151-157.
32. Yee, J. K. 1989. A liver-specific enhancer in thecore promoter region of human hepatitis B virus. Science 246:658-661.
