JOURNAL OF VIROLOGY, Apr. 1980, p. 178-1860022-538X/80/04-0178/09$02.00/0

Vol. 34, No. 1

Viral DNA in Bursal Lymphomas Induced by Avian LeukosisViruses

PAUL NEIMAN,1 3* L. N. PAYNE,2 AND ROBIN A. WEISS'Imperial Cancer Research Fund Laboratories, Lincoln's Inn Fields, London WC2A 3PX, England';Houghton Poultry Research Station, Houghton, Huntingdon, Cambridgeshire, England2; and Fred

Hutchinson Cancer Research Center, Seattle, Washington 98144

Avian leukosis viruses (ALV) induce malignant lymphoma of the bursa ofFabricius. Viral DNA in tumors and normal tissues from infected birds wereanalyzed by using restriction endonucleases. Viral DNA fragments diagnostic ofthe exogenous ALV were easily detected in tumors, uninvolved bursal tissue,kidney, and erythrocyte nuclei. Exogenous viral DNA was more difficult to detectin liver. Using a restriction endonuclease (SacI) which cleaves linear unintegratedALV DNA in a single site to define integration sites in DNA from the varioustissues, we were able to detect ALV DNA only in tumor tissue. We concludedthat the proviral DNA detected in the various nontumor tissue must be integratedin multiple sites. The appearance of ALV integration sites uniquely in tumorssuggests that they are clonal growths. Furthermore, the data suggested thepresence of a single exogenous integration site for the ALV provirus in each of sixearly neoplastic bursal nodules. This provirus appeared to retain the organizationof EcoRI and BamHI recognition sequences present in the genome of virus usedto infect the birds. The ALV integration site appeared different in each of thetumors studied. In a widespread metastatic lymphoma, multiple ALV integrationsites were found as well as structural alterations in at least some copies of theALV provirus.

Malignant lymphomas of the bursa of Fabri-cius (lymphoid leukosis) are common neoplasmsin domestic chickens. The induction of thesetumors is closely associated with infection byavian leukosis viruses (ALV). The genome ofthis class of avian retroviruses encodes the genesknown to be required for viral replication, butno viral gene(s) mediating induction of bursallymphomas or the short list of other less com-mon neoplastic diseases (e.g., nephroblastomasand erythroleukemia) associated with theseagents has been identified. The mechanisms un-derlying tumor induction by ALV remain ob-scure. The genome of ALV is closely related(about 85% homology) to that of geneticallytransmitted endogenous retroviruses of chickensexemplified by Rous-associated virus type 0(RAV-0) (14). RAV-0, however, appears to lackthe oncogenic potential ofALV (10). The major-ity of sequence divergence that does exist be-tween ALV and RAV-0 is concentrated in the 3'end of the genomes of these agents (3, 13). Thesignificance, if any, of this region of divergencewith respect to the biological differences be-tween these viruses remains to be established.

In the field, ALV is often spread by congenitalinfection of embryos by viremic hens (2). Exper-imentally, newly hatched susceptible chicks can

be infected by intraperitoneal injection. Viralreplication has been observed in many organsafter infection, but the development of lympho-mas is absolutely dependent upon the presenceof the bursa of Fabricius (5, 15). The earliestrecognizable change in the bursa is the accu-mulation of lymphoblasts within individual fol-licles (5). We have observed such transformedfollicles in the majority of infected birds as earlyas 30 days after hatching and infection (P. E.Neiman, L. N. Payne, and R. A. Weiss, in Virusin Naturally Occurring Cancer, in press). In thestudy cited, we found that the vast majority ofthe 10 to 100 such transformed follicles per in-fected bursa did not appear capable of progres-sive growth. In fact, these structures disappearedwith the physiological regression of the bursa 5to 6 months after hatching. A much smallernumber (zero to two per bursa) of discrete butprogressively growing neoplastic nodules ap-peared beginning 14 to 15 weeks after infection.These tumors were composed of the same typeof pyroninophilic lymphoblasts which appearedin transformed follicles. That the progressivelygrowing nodules arise from the larger populationof transformed follicles seems a reasonable spec-ulation. These nodules grow rather slowly for aperiod of weeks. They presumably are the pre-

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ALV DNA IN BURSAL LYMPHOMAS 179

cursors of the rapidly growing metastatic bursallymphomas which kill most susceptible birdsbetween 20 and 40 weeks after hatching andinfection. Thus at least three distinct stages canbe observed in the development of bursal lym-phomas.

In a previous study, hybridization reactionsbetween genomic viral RNA and cellular DNAin solution were used to detect ALV-specificproviral DNA sequences in bursas and bursaltumors from infected chickens (14). On the basisof the kinetics of hybridization, there are anaverage of one to two copies of ALV proviralDNA per haploid genome in bursal lymphomacells. In the present study, we have extractedDNA from early bursal nodules, from wide-spread bursal tumors, and from a series of othertissues from infected chickens. Viral DNA wasanalyzed, after digestion with a series of restric-tion endonucleases, by the Southern transfertechnique (17). The data provide further infor-mation on the distribution of ALV DNA ininfected birds, the clonal origin of bursal nod-ules, and the structure of the ALV provirus andadjacent host sequences in bursal lymphomas.

MATERIALS AND METHODSViruses and chickens. The virus used for this

experiment was ALV 5938, a field isolate which hasbeen characterized in detail in terms of both its onco-genicity and its extensive sequence homology withlaboratory strains of ALV such as RAV and transfor-mation-defective (td) deletion mutants of Rous sar-coma virus (RSV) (11, 14). We used 104 interferenceunits of ALV to infect by intraperitoneal injectionnewly hatched chicks from a mating between suscep-tible (to both infection and leukosis) inbred line 15Imales and outbred brown leghorn hens. Birds weresacrificed at intervals and examined for the appear-ance of bursal nodules, bursal lymphomas, and otherneoplasms such as nephroblastomas (which werefound in two instances). The course of development ofneoplastic change in this experiment, as monitored byhistological examination of serial sections of bursae, isdescribed elsewhere (Neiman et al., in press). Individ-ual neoplastic bursal nodules were dissected free ofsurrounding normal bursal tissue. Bursal nodules, lym-phomas, and nephroblastomas and normal bursa, kid-ney, erythrocytes (RBC), and liver were all collectedfor DNA extractions.DNA extraction. DNA was prepared from RBC

nuclei on the day of collection. RBC from about 2 mlof blood were washed in TNE (0.01 M Tris-hydrochlo-ride [pH 7.4]-0.1 M NaCl-0.001 M EDTA) and sus-pended in 0.01 M Tris-hydrochloride (pH 7.4)-0.01 MNaCl-0.005 M MgCl2-1% Nonidet P-40. After briefblending in a Vortex mixer, nuclei were pelleted andwashed two to four times in TNE until the hemoglobinwas removed. The nuclei were then suspended inabout 5 ml of 0.01 M Tris-hydrochloride (pH 7.4)-0.001 M EDTA (TE). Proteinase K and sodium do-decyl sulfate (SDS), were then added to 200 ug/ml and

0.5%, respectively, and the mixture was incubated at37°C for about 2 h, or until the solution was clear. Thesolution was then extracted with an equal volume ofchloroform-isoamyl alcohol (24:1), and the aqueousphase was obtained after centrifugation at 10,000 x gfor 10 min. After precipitation with 2 volumes ofethanol, the DNA was spooled on a glass rod, dissolvedin TE, and digested for 30 min with 20,g of RNase Aper ml (Worthington Biochemicals Corp.) at 37°C.This was followed by another 2-h digestion with pro-teinase K as before, extraction with an equal volumeof chloroform-phenol (1:1), extraction with an equalvolume of chloroform-isoamyl alcohol, and dialysiswith three changes of 2 liters of TE. If necessary, DNAwas concentrated to about 1 mg/ml by respooling anddissolution in TE.DNA from sources other than RBC was usually

extracted from tissues frozen and stored at -20°C.This was done by slicing the tissue while still frozeninto small slivers with a scalpel and dispersing the cellsin a small volume of cold saline-EDTA (0.1 M NaCl-0.1 M EDTA, pH 7.4), using a single stroke in a chilledDounce homogenizer. This suspension was added tosaline-EDTA containing 1% SDS preheated to 60°C(total volume, 5 ml/g of original tissue) and incubatedfor 10 min. The mixture was then cooled to roomtemperature, made 1 M in sodium perchlorate, andextracted with chloroform-isoamyl alcohol as for theRBC nuclear DNA preparations. The remainder ofthe procedure was also the same as that used for theRBC DNA.

Preparation of unintegrated ALV 5938 DNA.Twenty subconfluent plates of chicken embryo fibro-blasts were infected with ALV at a multiplicity ofinfection of about 1. At 40 h after infection, the cellslayers were rinsed with cold TNE, scraped into cen-trifuge tubes with a rubber policeman, and washedtwice more with cold TNE. Cells were lysed with SDSand fractioned by precipitation in the cold in thepresence of 1 M NaCl according to the proceduredescribed by Hirt (6). The supernatant fraction (Hirtsupernatant) containing unintegrated viral DNA wasdigested with RNase A and proteinase K, extractedwith chloroform-phenol, and precipitated with ethanolas previously described (13). When analyzed on 0.7%agarose gels by Southern transfer and hybridization to32P-labeled viral complementary DNA (cDNA) as de-scribed later, these preparations contained predomi-nantly a 5.1 x 106-dalton linear viral DNA moleculeand a small amount (roughtly 0.1 of the amount oflinear form) of a supercoiled viral DNA molecule (datanot shown). When portions representing 1 x 106 to 2x 10' fibroblasts in the original infected cultures wereused in subsequent experiments (e.g., Fig. 1A), nearlyall of the detected viral DNA was contributed by thelinear molecule.

Restriction endonuclease digestion and elec-trophoresis of viral DNA. Restriction endonucle-ases EcoRI, SacI, BamHI, and HpaI were obtainedfrom New England Biolabs. Digestions, carried out in50 pl of the recommended buffer solution, containedeither 10 Mig of cellular DNA preparation or a portionof the Hirt supernatant DNA preparation representingabout 2 x 106 infected fibroblasts along with 1 to 5 Uof the appropriate restriction endonuclease. One mi-

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180 NEIMAN, PAYNE, AND WEISS

crogram of lambda phage DNA was dissolved in 5-p(0.1 volume) portions of the complete reaction mix-ture, and both mixtures were incubated for 2 to 4 h at37°C. The small reaction containing the lambda DNAwas analyzed by agarose gel electrophoresis (as de-scribed below) to confirm that the reaction had goneto completion. Only limit digests were analyzed fur-ther. The main reaction mixture was reduced in vol-ume by evaporation in a vacuum, redissolved in 50 pdof 0.01 M Tris-hydrochloride (pH 7.4)-0.001 MEDTA-0.1% SDS-50% glycerol containing bromophe-nol blue, and loaded on a 0.4-cm-thick 1.1% horizontalagarose (Seakem) gel. Electrophoresis was at 15 V/cmfor about 24 h.

Transfer to nitrocellulose membranes and hy-bridization. DNA was transferred to nitrocellulosemembranes (0.1 jam; Sartorius) by the blotting tech-nique described by Southern (17). Membranes weredried in a vacuum oven at 80°C for 2 h and prehy-bridized for 16 h at 420C in 5x SSC (SSC = 0.15 NaClplus 0.015 sodium citrate), 50% formamide containingDenhardt solution (0.02% albumin, Ficoll, and polyvi-nylpyrrolidone), 20 ,ug of single-stranded calf thymusDNA per ml, and 50 ,ug of yeast RNA per ml. Themembrane was then transferred to a second plasticenvelope containing the same buffer plus 106 cpm of a32P-labeled viral cDNA probe. The latter was preparedby in vitro reverse transcriptase reactions using puri-fied ALV 3958 35S genomic RNA as template preparedas described previously (12) and limit DNase I-di-gested calf thymus DNA oligomers as primer (19).Reaction conditions and isolation of cDNA were es-sentially as described previously (1978). Hybridizationwas carried out for 48 to 72 h. The membrane wasthen washed in 200 ml of2x SCC in Denhardt solutionfor 20 min at room temperature followed by 200 ml of0.lx SSC-0.1% SDS at 500C with shaking for 1 h.Final washing was with four to six rapid changes of0.lx SSC followed by air drying. Viral DNA bandswere then visualized by radioautography in the pres-ence of calcium tungstate intensifying screens.

RESULTSDigestion of viral and cellular DNA with

EcoRI. Shank et al. (16) have recently reportedthat EcoRI cleaves the linear forms of bothparental and td RSV unintegrated DNA veryclose to both the 3' and 5' ends of the moleculeowing to the presence of a recognition site withina 300-nucleotide repeated sequence at each ter-minus. Two additional internal sites are recog-nized, giving a characteristic three-band patternof internal fragments released by digestion ofRSV and td RSV DNA with EcoRI. Given theextensive sequence homology between ALV5938 and td RSV (11), it was not surprising thatdigestion of the linear form of unintegrated ALVDNA yielded three fragments: 2.5 x 106, 1.65 x106, and 0.9 x 106 daltons (Fig. 1A, lane HS).These sizes are reasonably close to predictedvalues from the EcoRI map of td RSV (16). Weassumed, therefore, that these three fragments

represented three internal fragments of ALV,the terminal fragments being too small to detectby this method.

Figure 1 also shows the results of digestion ofa series of tissues from tumor-bearing birds withEcoRI. In Fig. 1A, digests of DNA from twoindividual bursal nodules (T6 and Tb), from non-tumorous portions of the bursa, and from RBCand the liver of one bird (2989) are shown. ViralDNA fragments of 2.5 x 106, 1.65 x 106, and 0.9x 106 daltons were easily detected in all tissuesexcept the liver, where only the largest fragmentwas easily observed. This fragment is commonlyobserved in EcoRI digests of DNA from unin-fected chicken cells (e.g., Fig. 1, bird 2407), andit, along with the other higher-molecular-weightviral DNA-containing fragments, were consid-ered to represent endogenous ALV-related se-quences. We concluded that the liver was notextensively infected with ALV, whereas all ofthe other tissues contained easily detectablequantities ofexogenously introduced ALV DNA.Furthermore, comparison of the tissue-derivedviral DNA bands with those of linear DNA fromALV 5938 (lane HS) showed that most of pro-viral DNA in infected tissues, including the tu-mors, had the same EcoRI map as the originalinfecting virus.

Figure 1B demonstrates the results of EcoRIdigestion of DNA from the tissues of three morebirds. RBC and bursal DNA from an uninfectedcontrol bird (2407) lacked exogenous ALVEcoRI 1.65 x 106- and 0.9 x 106-dalton frag-ments, whereas DNA from RBC, bursa, andbursal nodule (Tf), but not from the liver, ofinfected bird 2999 contained the standard exog-enous ALV EcoRI fragments. This figure alsoshows the results of digestion ofDNA from RBCand an advanced widespread bursal lymphomawhich appeared in chicken 2902. In this case, inaddition to the standard viral EcoRI bands, anew viral DNA containing fragment of 2.3 x 106daltons is clearly seen in the EcoRI digest oftumor DNA, but not in the RBC DNA. Thisobservation with the metastasizing tumors wasnot repeated in analysis ofDNA from six bursalnodules from this experiment where unusualEcoRI fragments did not appear. For example,an EcoRI digest of bursal nodules T' and T andother tissues from bird 2993 yielded the standardALV-specific intemal fragments (Fig. 10). Inthis case, we probed the organization of ALVDNA in the tumors and other tissues further bydigestion with a second restriction endonuclease,BamHI. In all tissues, two small viral DNAfragments of 1.1 x 106 and 1.5 x 106 daltons weredetected which corresponded to the expectedinternal fragments released from ALV linear

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ALV DNA IN BURSAL LYMPHOMAS 181

81A-"

2-407

RC Bu

6> - 4*

Us.R--,.

6...4w4.'._

2902 2999RC T9 RC L

M ;:...%..

c2X3-`2 Ecor .rr,

-B:B.u

FIG. 1. Analysis of viral DNA with EcoRI andBamHI. DNA preparations from various tissues ofindividual AL V-infected birds were digested with therestriction endonuclease, subjected to electrophoresison agarose gels, and transferred to nitrocellulosemembranes. Viral DNA-containing fragments werelocated by hybridization to 32P-labeled ALV cDNAprobes and radioautography. (A) Outer lanes are 32plabeled size markers (x 106 daltons) of adenovirustype 2 DNA digested with either XbaI (left) or EcoRI(right). HS is an EcoRI digest ofALV linear uninte-grated DNA from a Hirt supernatant fraction ofinfected fibroblasts. In bird 2989, DNA was analyzedfrom two distinct neoplastic tumor nodules (Ta andTb), normal uninvolved bursa (Bu), RBC nuclei (RC),and liver (L). (B) Size markers used were HindIIIfragments ofphage DNA (not shown). Bird 2407 wasan uninfected control. Bird 2902 was infected andhad a metastasizing malignant lymphoma (Tw). Bird2999 had a single bursal nodule (Tf). The remainder

DNA by BamHI digestion (data not shown).Therefore, the three BamHI sites and the fourEcoRI sites present in the genome of the ALVused for infection appear to be preserved in theALV DNA detected in the various infected tis-sues and tumors studied in this experiment.Digestion of viral and cellular DNA with

Sacl. We observed that Sacl (and its isoschizo-mer SstI) cleaved linear ALV DNA at a singlesite, yielding two fragments of 4.8 x 106 and 0.25x 106 daltons. Therefore, Sacl digestion of inte-grated proviral DNA which is coextensive withcytoplasmic linear viral molecules should yieldonly fragments covalently linked to host cellsequences. Figure 2 (panels B, C, and D, laneHS) shows the larger SacI-generated fragmentof unintegrated linear ALV fragment (thesmaller one migrated beyond the bottom of theillustration and required very long exposure ofthe radioautogram in order to visualize). Theposition of this band can be compared with viralDNA-containing fragments observed in SacI di-gests of DNA from the same tissues depicted inFig. 1 except 2989 RC (Fig. 2B), where the DNAwas not digested by Sacl. In contrast to theresults obtained with EcoRI digests, no bandswere observed in either bursa or RBC DNAwhich migrated at the same position as that ofthe SacI fragment of unintegrated ALV DNA.In fact, only endogenous viral DNA fragmentswere detected in normal tissues. We interpretedthis observation to indicate that the viral DNAmolecules detected in EcoRI digests of DNAfrom bursa, RBC, and kidney (not shown) areprobably integrated, but in such a large numberof sites that they cannot be visualized. In bursalnodules T', Tb (Fig. 2B), Tc, Tr (Fig. 2C), and Tf(Fig. 2A, bird 2999), SacI-generated fragmentscontaining viral DNA were detected which wereabsent in the digests of both linear unintegratedDNA and DNA from infected but untrans-formed tissues. A pair of such fragments wasdetected in four of these nodules as indicated bythe data in the various figures; only one distincttumor-specific fragment was identified in Tf.These fragments may represent both halves ofa single SacI-digested ALV provirus linked toflanking host cell sequences, or they may repre-sent distinct integration sites of ALV. It alsoseems reasonable to assume that we were ableto visualize these integration sites in tumors, as

of the lane designations are as for (A). The dotdenotes a unique tumor-specific fragment. (C) Anal-ysis with both EcoRIandBamHIofDNA from tissuesfrom bird 2993. There were two distinct bursal nod-ules in this bird, TC and Td. The markers and lanedesignations are as before. The dots denote tumor-specific fragments.

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182 NEIMAN, PAYNE, AND WEISS

A;z07 2902 2999- C Bu RC Tg RC L Bu T

42 .-...........

26-

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2892HS Tbb K RC L Bu

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FIG. 2. Analysis of viral DNA with Sac. DNA from the tissues of six different birds were digested withSacI and analyzed as described in Fig. 1. (A) DNA from tissues from birds 2407, 2902, and 2999 with markersand lane designations corresponding to Fig. lB. The dots again denote tumor-specific fragments. (B) DNAfrom tissues of bird 2989 corresponding to EcoRI digests depicted in Fig. IA. RBC DNA failed to digest withSac. (C) Same analysis for DNA from bird 2993 as in Fig. IC. (D) Analysis ofDNA from tissues ofa bird 2892with a large nephroblastoma (?*b). Lane K is from uninvolved kidney. The remainder of the designationscorrespond to our figures.

opposed to infected but untransformed bursaltissues, because the integration site ofALV pro-virus is identical in each cell in the tumor. Thesimplest explanation for this finding is that thesenodules are composed of the progeny of a singlecell (that is, are clonal growths).

Figure 2A also shows SacI digests of DNAfrom tissues of an uninfected control bird, 2407,and of DNA from RBC and tumor tissue fromthe bird (2902) with widspread metastatic bursallymphoma. In the latter case, there were a largenumber (about six of tumor-specific ALV DNA-containing fragments. Similarly, in bird 2892,

which developed a large nephroblastoma, fourviral DNA-containing fragments were observed(Tbb, Fig. 2D) which were clearly absent in theuninvolved kidney and other tissues. The stan-dard EcoRI viral DNA fragments were observedin all these tissues (not shown). Therefore othervirus-induced neoplasms, such as nephroblasto-mas, are probably clonal growths. Furthermore,the possibility is raised that in aggressive metas-tatic lymphomas like Tg, there is an amplifiednumber of integrated ALV proviruses. Sincethese tumors also yielded a new EcoRI fragment(Fig. 1B), at least one of these proviruses may

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VOL. 34, 1980

be structurally altered.Finally, fragments of ALV containing DNA

were observed of about 3.4 x 106 daltons whichwere variably present in SacI digests of DNAfrom different tissues. For example, in Fig. 2,this specific fragment appears in bursal nodulesand normal bursa, but not in other tissues indigests of DNA from birds 2989, 2993, and 2892(Fig. 2B to D). In contrast, a fragment of thissize was detected in all tissues of bird 2999 andno tissues from bird 2407 (Fig. 2A), as well as inother infected birds (not shown). The signifi-cance of this finding is, at present, unclear.The data from SacI digests of DNA from

tissue of six different birds with bursal nodulesor tumors are summarized in Table 1. All viralDNA-containing fragments observed were clas-sified into two broad groups. Those fragments

ALV DNA IN BURSAL LYMPHOMAS 183

appearing in all normal and neoplastic tissues ofany given bird were assigned to the category ofendogenous viral information. Some of thesefragments were common to all of these birdsstudied and presumably came from the inbredwhite leghorn parent. For example, bands of 13.0x 106, 6.0 x 106, and 3.9 x 106 daltons have beencharacterized in line 15I (1). Other fragmentswere variably present in different birds and wereprobably segregating within the brown leghornflock. The 3.4 x 106-dalton variable fragmentwas assigned to this endogenous category. Theremainder of the observed SacI-generated viralDNA-containing fragments were tumor specific.The table lists the molecular weights of thesefragments for six distinct bursal nodules, one

metastasizing bursal lymphoma, and two ne-phroblastomas. If these fragments define the

TABLE 1. Analysis of viral DNA with SacEEndogenous' Tumor specificC

Bird no. Bursal nodulesd Bursal Nephro-AR,tno. Bursal lymphoma' blastomaAll tissues Bursa__

a b c d e f g aa bb

2989 13.0 8.5 Same 6.0 7.36.0 plus 2.1 4.63.9 3.4

2993 13.0 8.5 Same 7.3 5.16.0 2.1 plus 4.8 4.03.9 1.4 3.4

3000 13.0 8.5 Same 4.55 5.06.0 2.1 4.50 4.53.9 1.4

2999 13.0 1.1 Same 5.56.0 8.53.9 2.13.4

2902 13.0 8.5 NAf Multiple6.0 2.1 5.5-4.83.9 1.45

2892 13.0 2.1 Same 7.06.0 1.4 plus 5.43.9 3.4 4.5

3.4aData indicate the molecular weight (x 10-6) of SacI-generated viral DNA-containing fragments. All

fragments could be classed in two categories, endogenous or tumor specific. Most of the data were derived fromthe experiments depicted in Fig. 2.

'Defined as bands observed in DNA from all tissues (tumors, bursa, liver, kidney, RBC nuclei) in a particularbird.

C Detected only in DNA from tumor tissue.d Individual small neoplastic bursal nodules dissected free of surrounding normal bursal tissue. Designations

correspond to Fig. 2.'Main mass of a widespread bursal lymphoma.f NA, Not applicable.

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184 NEIMAN, PAYNE, AND WEISS

integration site of ALV in the neoplastic cells,then their different sizes suggest different inte-gration sites in each tumor. This observationappears to hold both for fragments compared on

the same gel (see Fig. 2) and for distinct tumornodules arising in the same bursa (e.g., nodulesT5 and Tb in bird 2989 as well as Tr and Td inbird 2993).Analysis with other restriction endonu-

cleases. One of the potential conclusions arisingfrom the preceding analysis with SacI is that thepair of tumor-specific fragments observed rep-

resented a single exogenously introduced ALVprovirus in each bursal nodule examined. Thisconclusion might be challenged on the groundsthat the small (0.25 x 10' daltons) SacI fragmentof linear ALV DNA might not be readily detect-able even when covalently attached to adjacenthost sequences, using our probe. If that weretrue, then all of the SacI tumor-specific frag-ments might contain only the larger (4.8 x 106daltons) viral segment, indicating at least twoproviral copies per cell in each of the tumornodules. Table 1 and Fig. 2 show several exam-ples of tumor-specific fragments which aresmaller than 4.8 x 106 daltons and thereforecannot be composed of a viral DNA segment ofthat size plus additional host cell sequences.Either these fragments must represent thesmaller viral segment linked to flanking hostsequences, or else some altered arrangement ofALV DNA sequences must be present. An al-tered arrangement was not detected in theEcoRI digests. To resolve this tissue, DNA fromthe tissues of bird 2993 was digested with two

J. VIROL.

additional enzymes: BamHI, which should yieldboth junction fragments and internals, andHpaI, which cuts linear ALV DNA once near

the middle of the molecule (data not shown).The results with BamHI (Fig. 1C) and HpaI(not shown) are summarized in Table 2. Withboth enzymes, a distinct pair of tumor-specificfragments was detected in each of the two bursalnodules (TC and T) present in the bird. Thisresult is also most consistent with the notionthat a single provirus is present in each of thecells making up the bursal tumor nodules.

It was also of interest to determine whetherDNA from the metastatic tumor (T9) whichappeared to contain several ALV proviruseswhen analyzed with Sacl contained multipletumor-specific bands when analyzed with otherenzymes. Digestion with HpaI also generatedmultiple tumor-specific viral DNA-containingfragments (Table 2). We therefore conclude thatthis metastatic neoplasm is also probably a

clonal growth carrying about three exogenousALV proviruses integrated in distinct sites in thehost cell genome. The simple explanation thatthe multiple copies are the result of the fusionof several bursal nodules in the formation of thetumor might be challenged on the groups thatthe SacI-generated tumor-specific fragments inTg shown in Fig. 2A (as well as those producedby HpaI listed in Table 2) appear to be roughlyof the same intensity as the endogenous viralbands in the same DNA. We would have ex-

pected that a mixture of cells originating fromdifferent tumor nodules would have resulted indilution of the tumor-specific bands in compar-

TABLE 2. Analysis with other restriction endonucleasesaTumor specific Exogenous

Endonuclease Endogenous intemnals2993 DNAHpaI 13.0 5.5 3.9 3.3

11.0 4.2 2.8 2.58.0 2.4

BamHI 5.1 2.4 12.5 15.5 1.53.9 1.43.4 1.0 6.4 6.6 1.12.9

2902 DNAHpa I 13.0 4.8 14.0

11.0 3.6 7.56.5 2.0 5.5

4.03.32.4

a Molecular weight (x 10-6) of viral DNA-containing fragments from tissues in birds 2993 and 2902 afterdigestion with HpaI and/or BamHI. Designations correspond to those in Fig. 2 and Table 1.

'Correspond to the fragments released from linear ALV DNA.

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ALV DNA IN BURSAL LYMPHOMAS 185

ison with the endogenous band present in allcells in the tumor.

DISCUSSION

These observations have led us to a numberof conclusions. First, the provirus of ALV caneasily be detected in the target tissues (RBC,kidney, bursa) of infected, susceptible birds. In-fection of the liver appears to be less efficientthan that of other tissues. Second, the provirusappears to be integrated in distinct sites in virus-induced tumors arising in these tissues. TheALV integration sites appeared different foreach of nine neoplasms, six bursal nodules, onemetastatic bursal lymphoma, and two nephro-blastomas we examined. These findings are sim-ilar to the results of studies carried out withother retrovirus-induced neoplasms such asmammary adenocarcinomas induced by murinemammary tumor virus (4) and thymic lympho-mas induced by murine leukemia virus (18). Weshare with these authors the conclusion thatsuch observations suggest a clonal origin forthese relatively long-latency-type tumors in-duced by viruses apparently lacking (at least onthe basis of present knowledge) unique trans-forming genes.Aided by the fact that the endogenous viral

DNA-containing fragments generated by SacI(or SstI) from white leghorn chickens have beenextensively catalogued (1), we were able toclearly separate all of the SacI viral bands ob-served into either endogenous or tumor-specificcategories (Table 1). This, in turn, allowed us tocount the apparent integration sites for ALV ineach tumor. There appeared to be only a singlesuch site per locus in each of the bursal nodules.Furthermore, on the basis of the analysis withEcoRI and BamHI, the ALV provirus occupyingthis site appears to have the same restrictionmap as the genome of bulk of the virus particlesin the stock used to infect the chickens. Inter-estingly, the metastasizing bursal lymphomastudied in this experiment appeared to haveseveral proviral copies integrated in differentsites in the host genome. Futhermore, EcoRIdigestion suggested a mutation in the EcoRI sitepresent in proviral DNA in this tumor. Such amutation might involve either the addition of anEcoRI site within the proviral DNA segment orpossibly the loss of a recognition site. The pos-sibility is thus raised that this amplification ofviral DNA copies, and perhaps other changes inprovirus structure, is associated with the acqui-sition of metastatic capability by the neoplasticbursal nodules.We mentioned in the introduction that the

mechanisms underlying induction of bursal lym-phomas (or either tumor) by ALV remain ob-

scure. The conclusions reached in this studymay be examined with respect to their impacton at least some classes of mechanisms whichmight be involved in viral oncogenesis in thissystem.For example, the idea that ALV might induce

neoplastic change by integrating at a specific sitein target cells is compromised by the lack of anyevidence of a similarity in the integration site ofALV in the various tumors examined in thisstudy (as well as the evidence cited in otherretrovirus systems). Furthermore, the presenceof a single exogenously introduced provirus forALV in each of the bursal nodules with anapparently orthodox restriction map for EcoRIand BamHI would seem to exclude the possibil-ity of a large host cell sequence insert within theprovirus of the kind identified with sarcomaviruses and defective acute leukemia viruses, atleast at the stage of formation of bursal nodules.The situation in metastasizing lymphomas maybe different. In this study, multiple ALV ge-nomes were detected as well as alterations in-volving the EcoRI recognition site. Although theconsistency of this phenomenon remains to beestablished, it is of interest that similar obser-vations of viral gene amplification have beenmade in Moloney leukemia virus-inducedthymic lymphomas in inbred BALB/Mo mice(7-9). If, indeed, metastasizing lymphomas con-taining several viral genomes arise from lessmalignant clonal tumors with single exogenousproviruses, then it seems reasonable to proposethat tumor progression in this system involvesone or more rounds of clonal proliferation ofaltered cells beyond the initial clonal expan-sion(s) responsible for the formation of bursalnodules. The additional proviral copies mayarise simply by superinfection proceding clonalovergrowth. It is unclear at present whether thisapparent viral gene amplification is a cause oftumor progression or simply a marker of clonalevolution. Continued examination of the struc-ture of the proviral DNA in these tumors atvarious stages in the development of bursal lym-phomas, coupled with assessment of the viralRNA and protein gene products in these cells,should continue to narrow down the list of pos-sibilities and provide further insight into themechanisms of oncogenesis by these agents.

ACKNOWLEDGMENTSWe thank L. Jordan and D. Macdonnell for excellent tech-

nical assistance and M. Ross for typing the manuscript.P.E.N. is a scholar of the Leukemia Society of America and

was supported by a faculty scholarship from the Josiah P.Macy Jr. Foundation. This work was also supported by PublicHealth Service grant CA 12895 from the National CancerInstitute.

LITERATURE CITED1. Astrin, S. M. 1978. Endogenous viral genes of the white

VOL. 34, 1980

on January 29, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from

186 NEIMAN, PAYNE, AND WEISS

leghorn chicken: common site of residence and sitesassociated with specific phenotypes of viral gene expres-sion. Proc. Natl. Acad. Sci. U.S.A. 75:5941-5946.

2. Burmester, B. R., and N. F. Waters. 1955. The role ofthe infected egg in the transmission of visceral lympho-matosis. Poultry Sci. 34:1415-1429.

3. Coffin, J. M., M. Champion, and F. Chabot. 1978.Nucleotide sequence relationships between the ge-nomes of an endogenous and an exogenous avian tumorvirus. J. Virol. 28:972-991.

4. Cohen, J. C., P. R. Shank, V. L Morris, R. Cardiff,and H. E. Varmus. 1979. Integration of the DNA ofmouse mammary tumor virus in virus infected normaland neoplastic tissue of the mouse. Cell 16:333-341.

5. Cooper, M. D., L. N. Payne, P. B. Dent, B. R. Burmes-ter, and R. A. Good. 1978. Pathogenesis of avianlymphoid leukosis. I. Histogenesis. J. Natl. Cancer Inst.41:373-389.

6. Hirt, B. 1967. Selective extraction of polyome DNA frominfected mouse cultures. J. Mol. Biol. 26:365-369.

7. Jaeni8h, R. 1976. Germ line integration and Mendeliantransmission of exogenous Moloney leukemia virus.Proc. Natl. Acad. Sci. U.S.A. 73:1260-1264.

8. Jaenish, R. 1979. Moloney leukemia virus gene expres-sion and gene amplffication in preleukemic and leu-kemic Balb/Mo mice. Virology 93:80-90.

9. Jahner, D., H. Stuhlman, and R. Jaenish. 1980. Con-formation of free and of integrated Moloney leukemiavirus proviral DNA in preleukemic and leukemicBALB/Mo mice. Virology, in press.

10. Motta, J. V., L. B. Crittenden, H. G. Purchase, H. A.Stone, and P. L. Witter. 1975. Low oncogenic poten-tial of endogenous RNA tumor virus infection or expres-sion. J. Natl. Cancer Inst. 55:685-689.

11. Neiman, P. E. 1978. Mapping by competitive hybridiza-

tion of sequences which differ between endogenous andexogenous chicken leukosis viruses. Virology 85:9-16.

12. Neiman, P. E., S. Das, D. Macdonnell, and C. Mc-Millin-Helsel. 1977. Organization of shared and un-shared sequences in the genomes of chicken endogenousand sarcoma viruses. Cell 11:321-329.

13. Neiman, P. E., C. McMillin-Helsel, and G. M. Cooper.1978. Specific restriction of avian sarcoma viruses by aline of transformed lymphoid cells. Virology 89:360-371.

14. Neiman, P. E., H. G. Purchase, and W. Okazaki. 1975.Chicken leukosis virus genome sequences in DNA fromnormal chick cells and virus-induced bursal lymphomas.Cell 4:311-319.

15. Peterson, R. D. A., B. R. Burmester, T. M. Frederick-son, H. G. Purchase, and R. A. Good. 1964. Effect ofbursectomy and thymectomy on the development ofvisceral lymphomatosis in the chicken. J. Natl. CancerInst. 32:1343-1354.

16. Shank, P. R., S. H. Hughes, H. J. King, J. E. Majors,N. Quintrell, R. V. Guntaka, J. M. Bishop, and H.E. Varmus. 1978. Mapping unintegrated avian sarcomavirus DNA: termini of linear DNA bear 300 nucleotidespresent once or twice in two species of circular DNA.Cell 15:1383-1395.

17. Southern, E. M. 1975. Detection of specific sequencesamong DNA fragments separated by gel electrophore-sis. J. Mol. Biol. 98:503-517.

18. Steffen, D., and R. A. Weinberg. 1978. The integratedgenome of murine leukemia avirus. Cell 15:1003-1012.

19. Taylor, J. M., R. Illmenee, and J. Summer. 1976.Efficient transcription of RNA into DNA by aviansarcoma virus polymerase. Biochim. Biophys. Acta 442:324-330.

J. VIROL.

on January 29, 2018 by guesthttp://jvi.asm

.org/D

ownloaded from