JOURNAL OF VIROLOGY, Oct. 1982, p. 269-275 Vol. 44, No. 10022-538X/82/100269-07$02.00/0Copyright C 1982, American Society for Microbiology

Molecular Cloning of Circular Unintegrated DNA of TwoTypes of the SEATO Strain of Gibbon Ape Leukemia Virus

EDWARD P. GELMANN, CECELIA D. TRAINOR, FLOSSIE WONG-STAAL, AND MARVIN S. REITZ*Laboratory of Tumor Cell Biology, National Cancer Institute, Bethesda, Maryland 20205

Received 10 March 1982/Accepted 18 June 1982

Closed circular unintegrated DNA of the SEATO strain of gibbon ape leukemiavirus (GaLV-S) was isolated from canine thymus fibroblasts after cocultivationwith chronically infected bat lung fibroblasts. Restriction endonuclease HindIIIcleaves GaLV-S DNA once, thus allowing isolation and cloning of HindIIl-digested unintegrated DNA in a permuted form. Two clones isolated in the vector,Charon 21A, were nearly identical by restriction enzyme mapping to each of thetwo types of GaLV-S previously observed. These two types differ at a single Sallsite. Unlike previous maps of GaLV-S proviral DNA, however, both clones lackSstI sites in the long-terminal-repeat units. Both the GaLV-S clones and the majorspecies of GaLV-S proviral DNA contain an EcoRI site in the long-terminal-repeat units. The presence of this EcoRI site and the absence of an SstI site in theGaLV-S long-terminal-repeat units differentiate it from all other known GaLVstrains and from the closely related nononcogenic simian sarcoma-associatedvirus. Heteroduplex comparisons of each of the two clones to clones of simiansarcoma-associated virus show no obvious deletion or substitution loops. Thissuggests that the ability of GaLV-S to induce myeloid leukemia in gibbon apes isnot due to an acquired onc gene.

The different strains of gibbon ape leukemiavirus (GaLV) and simian sarcoma-associatedvirus (SSAV) comprise a closely related groupof retroviruses which are horizontally transmit-ted among primates (3). SSAV, the nonpatho-genic helper virus for simian sarcoma virus(SSV), probably arose from a single horizontaltransmission of GaLV from a gibbon ape to awoolly monkey (3, 23). Two strains of GaLV,Hall's Island (GaLV-H) (2) and San Francisco(GaLV-SF) (11), are associated with lymphocyt-ic leukemia in gibbon apes (2, 11, 18) but havenot yet been shown to induce leukemia in vivo.The SEATO strain of GaLV (GaLV-S) (10) isunique among mammalian retroviruses in that itefficiently induces chronic myelogenous leuke-mia. Young gibbons inoculated with 105 infec-tious units of GaLV-S develop the disease with alatent period of less than 1 year (12). SSAV/SSVinduces fibrosarcomas, fibromas, and astrocyto-mas in marmosets (22). The oncogenic potentialof SSAV/SSV is due to the presence of the SSV-coded onc gene, v-sis, which originated from thehost woolly monkey chromosome (23). Someoncogenic retroviruses which cause subacutediseases, however, such as the avian leukosisviruses, do not have viral onc genes but arethought to cause cellular transformation by in-serting proviral DNA into a specific chromo-

somal site, thereby altering the expression of anormal cellular gene (7, 13).To study the molecular basis of the unusual

oncogenic properties of GaLV-S, we cloned theunintegrated viral genomic DNA. Two clonesisolated in the Charon 21A vector representedboth types of unintegrated viral DNA, whichwere previously reported to differ at a Sall site(21). Comparisons of restriction maps of theclones and unintegrated GaLV-S DNA and het-eroduplex comparisons of cloned SSAV (5) andGaLV-S are presented and discussed.

MATERIALS AND METHODSIsolation and cloning of unintegrated viral DNA.

Chronically infected bat lung fibroblasts (CCL88) wereused as a source of GaLV-S. The virus from this cellline has also been referred to as GaLV-3M (20). High-molecular-weight DNA, purified from these cells bypronase-sodium dodecyl sulfate digestion and phenol-chloroform extraction (24), was the source of integrat-ed GaLV-S provirus. To establish an acute infection,canine thymus fibroblasts (Cf2th) (8 x 109 cells) werecocultivated for 48 h with 2 x 109 chronically infectedCCL88 cells. Low-molecular-weight DNA was ex-tracted from these cells by the procedure of Hirt (8)and used as a source of unintegrated viral DNA.Where indicated, viral DNA was further purified bypreparative slab gel electrophoresis with 0.8% low-melting-temperature agarose (Bio-Rad Laboratories,

269

270 GELMANN ET AL.

Richmond, Calif.). The areas of the gel that weredetermined by an analytical Southern blot to containunintegrated viral DNA were excised, and the DNAwas extracted. The presence of open circular viralDNA was confirmed by restriction endonuclease di-gestion with Hindlll, which cuts the unintegrated viralgenome once. On a Southern blot, the digested DNAmigrated slightly faster than the circular DNA andcomigrated with linear viral DNA at 9.1 kilobases (kb).The extracted DNA was then cleaved with HindlIland ligated to HindlIl-cleaved Charon 21A phage X,whose cloning capacity is 0 to 12.0 kb (1). The ligationmixture was transferred directly to an in vitro packag-ing reaction (9). The packaged phage (4 x 104 plaquesper 0.5 ,u.g of input DNA) were screened with 32p_labeled GaLV-S cDNA (5). Two plaques were isolat-ed, and each was purified three times.

Restriction enzyme analysis and heteroduplex forma-tion. Phage DNA was isolated from large-scale lysatesby pelleting particles and banding twice in CsCl (4).Restriction enzyme analyses and hybridization condi-tions for Southern blots have been described previous-ly, as have the conditions for heteroduplex analysis(4). [32P]cDNA was prepared by transcription ofGaLV-S 70S RNA with avian myeloblastosis virusreverse transcriptase (Life Science, St. Petersburg,Fla.) with DNase-digested calf thymus DNA as arandom primer (15).

RESULTSTypes of unintegrated viral DNA. Linear unin-

tegrated GaLV-S DNA is a 9.1-kb molecule witha single Hindlll restriction site which lies 5.6-kbfrom the 5' end of the molecule. Restrictionmaps of linear unintegrated GaLV-S DNA arepresented in Fig. 1.There are two major types of GaLV-S DNA

(21). Thus far, these two types have been shownto differ only by the presence of an extra Sallsite in one (type 1). Digestion of linear uninte-grated DNA with Sall yields fragments of 6.2,4.0, 2.9, and 2.2 kb (Fig. 3, lane A). The 6.2- and2.9-kb bands are fragments of type 2 GaLV-S. Intype 1, however, the 6.2-kb fragment is cleavedby Sall into 4.0- and 2.2-kb components. The

Bg~I I

,1. 1a

2.9-kb fragments at the 5' end of both types ofGaLV-S DNA are identical. This fragment canbe identified by the presence of an XhoI cleav-age site (Fig. 3, lane B). Unintegrated GaLV-SDNA also contains circular molecules. Thesewere digested with the single-cut enzyme Hin-dIII and cloned into Charon 21A.GaLV-S recombinant clones. The phage clones

were isolated by using GaLV-S [32P]cDNA as aprobe to screen 5 x 104 plaques as describedpreviously (5). These were designated X-GaS-1and X-GaS-2. HindlIl digestion ofDNA from thepurified recombinant phages showed that the A-GaS-1 cloned viral insert measured 8.5 kb, andthe X-GaS-2 insert measured 9.1 kb (Fig. 2). x-GaS-1 also contains a nonviral 2.25-kb HindIIIfragment, or coclone, which does not hybridizeto the GaLV-S [32P]cDNA. The coclone is posi-tioned between the GaLV-S insert and the rightX phage arm (XR; see below).

Since SalI digestion allowed discrimination ofthe two types of GaLV-S DNA, we digested thephage DNA with Sall and compared it to Sall-digested unintegrated viral DNA. SalI cuts onlyonce in the X arms, 10 kb to the right of theinsert. Thus, Sall digestion also allowed us toorient the clones with respect to the phage Karms. A Southern blot of Sail-digested A-GaS-1hybridized to GaLV-S [32P]cDNA reveals twomajor hybridizing fragments, 18 and 2.2 kb,indicating that K-GaS-1 has the internal 2.2-kbSall fragment and is similar to GaLV-S type 1(Fig. 3, lanes A and C). By referring to therestriction map of the unintegrated viral DNA(Fig. 1) we were able to orient the 3'-derivedsequences of the GaS-1 insert next to the leftphage arm (XL). Similarly, a Southern blot ofSall-digested A-GaS-2 yielded two hybridizingbands, 30 and 12.8 kb, but no small SalI frag-ment internal to the viral genome (Fig. 3, lane 4).Thus, the A-GaS-2 insert is similar to GaLV-Stype 2 and is oriented with its 5'-derived regionadjacent to XL.

E

HlBI I A tk tP S Sa St

K

I

I I I I I I I I I1 2 3 4 5 6 7 8 9

KbFIG. 1. Restriction endonuclease map of GaLV-S unintegrated viral DNA. Ten enzymes are shown in the

illustration. XbaI did not cleave GaLV-S DNA. GaLV-S type 2 lacked the Sall site at 5.3 kb. Abbreviations: P,PstI; E, EcoRI; S, SmaI; K, KpnI; Bg, BglII; Xh, XhoI; Sa, Sail; B, BamHI; H, HindIl; St, SstI.

J. VIROL.

MOLECULAR CLONING OF GIBBON APE LEUKEMIA VIRUS 271

-GaS-2

4

-- 9 1 kt

FIG. 2. Agarose gels of DNA from X-GaS-1 and X-

GaS-2 digested with HindIII. After electrophoresis thegels were stained with ethidium bromide (lanes 1 and3) and analyzed by Southern blotting (lanes 2 and 4) byusing GaLV-S [32P]cDNA. The HindIlI digest of X-

GaS-1 contains a 2.25-kb fragment which does nothybridize to GaLV-S [32P]cDNA. This nonviral co-clone is situated between the GaLV-S insert and XR.

Further restriction mapping of the clones wasdone with eleven enzymes (Fig. 4). The restric-tion sites were nearly identical to those that havebeen mapped on the unintegrated GaLV-S DNA(21) (Fig. 1). Two SstI sites are located 0.4-kbfrom one another, on either side of the HindIIIsite. Because of their proximity, one of these

sites was missed in mapping the unintegratedviral DNA. This was also the case for the twoBamHI sites near XL in X-GaS-1 and XR in A-GaS-2. The 0.6-kb size difference between thetwo clones can be accounted for by the presenceof a single long-terminal-repeat (LTR) unit (0.6kb in length) in X-GaS-1 as compared to twotandem LTRs in X-GaS-2. This was demonstrat-ed by digestion with PstI, KpnI, and SmaI,which are known to cleave within the LTR. Twoadjacent repeat units allowed each of theseenzymes to excise from X-GaS-2 a full-length,permuted terminal repeat unit. The excised 0.6-kb fragment and its neighboring fragments canbe detected on a Southern blot by a radiolabeledprobe of cloned SSAV LTR (pLTR) (5) (Fig. 5).Since PstI cleaves the LTR near its 3' end andKpnI and SmaI cleave near the 5' end, X-GaS-1can be shown to contain one LTR by PstI-KpnIor PstI-SmaI codigestion. These excise 0.5-kbfragments from either clone which hybridize to32P-labeled pLTR (Fig. 5).The restriction enzyme map illustrates two

other points of interest regarding the GaLV-SLTR. First, there is an EcoRI site located about130 base pairs to the right of the KpnI site.Restriction digestions designed to isolate thepermuted LTR fragment of X-GaS-2 and hybrid-ization of Southern blots with 32P-labeled pLTRplaced the EcoRI cleavage site within the LTR(Fig. 6). This cleavage site in the GaLV-S LTRsets this virus apart from the other members ofthe SSAV-GaLV group. Its possible significance

A B C D

SALSALI -. SAL SAL

XHO

LINEAR UNINTEGRATEDVIRAL DNA TYPE6.2 -

4.0 -

2.9 --

2.2 -

2.9 2.2 4.0

LINEAR UNINTEGRATEDVIRAL DNA TYPE 2 --2 6.2

GAS-1

GAS-2

2 2

--244

FIG. 3. Southern blot analysis of GaLV-S unintegrated viral DNA (lanes A and B) and the DNA of X-GaS-1(lane C) and X-GaS-2 (lane D) by using Sall and XhoI. Sail restriction maps of these DNAs are illustrated. Theprobe was GaLV-S [32P]cDNA. The four Sall fragments visible in lane A indicate that there were two types ofGaLV-S unintegrated viral DNA as discussed in the text. Both types were cloned. Sall digestion of X-GaS-1yielded two hybridizing fragments, the 2.2-kb internal viral fragment and an 18-kb fragment consisting of a 6.1-kbregion ofGaLV-S, the coclone, and the 10-kb fragment of XR. The segment of GaLV-S-derived sequences linkedto XL were of insufficient size to label this fragment. The Sail digest of X-GaS-2 contained 30- and 12.4-kbfragments corresponding to type 2 of GaLV-S. Lanes C and D are separate experiments, and a size comparisonof the upper bands in these lanes cannot be made from this figure.

.I-G aS--kc. 1 2

85- EPd

2 25--.. 1i.",Jr

VOL . 44, 1982

272 GELMANN ET AL.

K EBB Bg Bg KE

1----,. II I &t-

St: SP Sa Xh SPSa

K E E EBm Bg IBg

StI I I

St Si Sa Xh SPSP

E

KA RIGHT

S St

EKlI w A LEFT

I ISS St

1 2lHl3 4

I I I 15 6 7 8 9

Kb

FIG. 4. Restriction endonuclease maps of the viral inserts of X-GaS-1 and X-GaS-2. There were no XbaIsites within this region of either clone. As previously discussed X-GaS-1 contained a 2.25-kb coclone of unknownorigin positioned between the GaLV-S viral insert and XR, and it is not shown here. Abbreviations are describedin the legend to Fig. 1.

is discussed below. Second, we fdoes not cleave in the LTRs of eaddition, the LTRs of the unirDNA used for cloning did not c

GAS-I

P -Kj 4 C,;,. 1

..:1 Fr

FIG. 5. Southern blot analysis ofGaS-2 DNA utilizing three restricticwith sites within the LTR of GaLV-SSmaI). The probe was 32P-label(pLTR). The absence of a 0.6-kb fra2, and 3 of the X-GaS-1 autoradiogrzthis clone did not have two tandem I

fragment in lanes 4 and 5 generatewith two of the above enzymes estaence of a single LTR. In contrast, X

two tandem LTRs, as indicated by thkb fragments generated by each of thThe multiple faint bands in the Kpnpartial digestions. Abbreviations arelegend to Fig. 1.

round that SstI site. This contrasted with previous maps ofither clone. In integrated GaLV-S proviral DNA molecules,itegrated viral some of which were found to have SstI sites inontain an SstI the LTRs (21).

Heteroduplex comparison of GaLV-S andSSAV. Since GaLV and SSAV have a highdegree of nucleic acid homology and because

>-- GaLV-S is an oncogenic virus (12) but SSAV isnot, we compared the two by heteroduplexformation to try to locate any region of theGaLV-S genome for which there is no SSAVcounterpart. We have previously described cir-cular SSAV molecules which were cloned attheir single EcoRI sites in Charon 21A (5). Thiscleavage site is located 5.8 kb from the 5' end ofthe SSAV genomic map, a location analogous tothe single HindIII site of GaLV-S which is 5.6 kbfrom the 5' end of the GaLV-S map. We formedheteroduplex molecules between X-GaS-1 and X-

C50, an SSAV clone containing one LTR andoriented 3' -* 5' within the arms (4), and

f X-GaS-1 and A- between X-GaS-2 and X-B11, an SSAV clone)n endonucleases with two tandem LTRs and oriented 5' -- 3'

(PstI, KpnI, and within the A arms. The Charon 21A HindlIl siteled SSAV LTR is 1.9 kb to the right of the EcoRI site. Thisgment in lanes 1, misalignment of the phage arms formed 1.9-kbam indicated that deletion loops, which were landmarks at eitherLTRs. The 0.5-kb end of the insert DNA. Under the heteroduplexd by codigestion conditions of these experiments, we were able toLblished the pres- detect substitutions or deletions as small as 300eGpresenceofO0.6- base pairs. No regions of nonhomology werete three enzymes. seen in either molecule (Fig. 7). The heterodu-I lanes represent plex between X-GaS-1 and X-C50 illustrates adescribed in the deletion loop at the left end of the inserts, a 7.9-

kb region of homology, and a substitution loop

GAS-1

A LEFT

GAS-2

A RIGHT_

J. VIROL.

MOLECULAR CLONING OF GIBBON APE LEUKEMIA VIRUS 273

K P K-E P-ES E E P-K

t

a _ib * --_*a- ---do

"-420BASESI]

'-420BASES

K f-- -

P Ki

E

.130BASES

p

-130BASES

FIG. 6. LTR EcoRI site in GaLV-S DNA. South-ern blots of X-GaS-2 DNA are shown that used fourrestriction enzymes with sites within the LTR ofGaLV-S (KpnI, SmaI, PstI, and EcoRI) with SSAVpLTR as the probe. Codigestion with combinations ofthese four enzymes was used to localize the EcoRI sitewithin the LTR. The smallest band in each of the firstfour lanes migrates at 0.6-kb and is a single permutedLTR. The KpnI-EcoRI, and the KpnI-PstI fragmentsare both about 420 base pairs, whereas the PstI-EcoRIfragment is about 390 base pairs. This localized theEcoRI site to the position indicated in the illustration.The size of the PstI-EcoRI fragment indicated that theEcoRI site occurred between the KpnI and PstI sites.The size similarity of the two larger fragments indicat-ed that the left EcoRI site must be the same distancefrom the left KpnI site as the left PstI site is from theright KpnI site. As shown in the above map, thisdistance is about 130 base pairs. The illustrationrepresents two tandem permuted LTRs (straight line)flanked by the internal sequences of GaLV-S (curvedline) oriented 3' -- 5'. The right hand lane is a markerof 250 bases. Abbreviations are described in the legendto Fig. 1.

formed by the XR of X-C50 opposite the cocloneand a small stretch of GaS-1 (Fig. 7A, B, and E).The X-GaS-2/X-B11 molecule displays 8.4 kb ofhomology between GaLV-S and SSAV. At eachend of the inserts, the minor components of thesubstitution loops (0.37 kb on the left and 0.12kb on the right; Fig. 7) represent viral DNA notannealed due to misalignment of the respectiveviral EcoRI and HindIll sites. We can concludethat there is probably no unique gene in GaLV-Swhich directly confers leukemogenicity.

DISCUSSION

Unintegrated circular GaLV-S viral DNA wascleaved at a unique HindlIl restriction site andthen inserted into phage vector Charon 21A.

The two clones obtained represent two previ-ously described types of GaLV-S which appar-ently differ only at a single SalI site (21). Onerecombinant clone, X-GaS-1, contained oneLTR and, like type 1 of GaLV-S, had two Sallrestriction sites. The other clone, X-GaS-2, con-tained two LTRs and, like type 2 of GaLV-S,had only one SalI site. The two clones wereotherwise identical to each other and to uninte-grated and integrated GaLV-S DNA (21), exceptthat they lacked an SstI site in the LTRs.GaLV-S is unique in its capacity to induce

myelogenous leukemia in young gibbon apes(12). The proviral DNAs of the GaLV-SSAVgroup differ at many restriction sites within thecoding sequences, although they have a highdegree of nucleic acid homology (3, 14). TheLTRs, however, have characteristic sites forfour restriction enzymes (KpnI, SmaI, SstI, andPstI), which seem to be identical for all membersof this virus group (21) and which are alsopresent in many strains of murine leukemiavirus. The two cloned types of GaLV-S de-scribed here and the unintegrated viral DNA oforigin differ from all of the other GaLV strainsand SSAV in that they lack SstI LTR sites andcontain an EcoRI LTR site near the location ofthe SstI sites in the other viral LTRs. Sequenc-ing the SSAV LTR has shown that the region ofthese changes is located just to the left of the U3-R junction (S. Josephs, unpublished data).GaLV-S is the only member of this group ofviruses shown to have an EcoRl site and to lackan SstI site in this region. Although these char-acteristics could result from two single-basechanges, the conservation of these sites in theLTRs of all other infectious primate retrovirusesas well as in many murine retroviruses suggestthat these changes may be significant. Since thearea encompassing the SstI and EcoRI sites isnear the viral transcriptional promoter and othercontrol elements, it is intriguing to speculate thatthese differences may reflect alterations in theLTR which allow GaLV-S to induce myeloidleukemia.The absence of SstI sites in the LTR of

unintegrated viral DNA and the clones differsfrom previous mapping data for integratedGaLV-S proviral DNA (21). Since at that time itwas difficult to obtain sufficient unintegratedviral DNA from GaLV-S, we used integratedDNA from chronically infected cells. Since di-gestion of integrated proviral DNA with SstIyielded two fragments which together corre-sponded in size to a complete viral genome withone LTR, a third type of GaLV-S (type 3) mustbe present as a provirus in the chronically infect-ed bat lung fibroblast line which was our sourceof GaLV-S. This third type was not detectable inthe unintegrated viral DNA used for cloning and

3,-u-

VOL. 44, 1982

274 GELMANN ET AL.

Cf

Mi

4

PI00 AB

'NJ-A

I',,

D*--/ .--' ..: C...

:- : -.: >,*0 : . : - . : :* .. .. .. ..... . - . ..... .. .. . e ......... : . g. - . ..... .. . .l, ;' S /S % 4 wE

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FIG. 7. Heteroduplex comparison ofGaLV-S and SSAV. (A) Heteroduplex between A-GaS-1 and X-C50. Thesmall arrow shows the 2.1-kb deletion loop and the large arrow shows the substitution loop formed by thecoclone and XR DNA of X-C50. Original magnification, x42,000. (B) Tracing of the molecule in (A) with sizemeasurements shown in kilobases. Measurements are the average of 10 molecules with standard deviationsgiven. (C) Heteroduplex between X-GaS-2 and k-Bit. The small arrow denotes the right-hand substitution loopand the large arrow denotes the left. Original magnification, x42,000. (D) Tracing of the molecule in (C).Measurements were made as described for (B). (E) Schematic drawings of heteroduplex molecules with clone,coclone, and k DNA represented by different symbols to show the contribution of each to the heteroduplexstructures.

may not be expressed at as high a level as that oftypes 1 and 2. Types 1 and 2 are not detectableas integrated proviruses after SstI digestion be-cause of the lack of large internal fragments andthe heterogeneity of junction bands.

One of the purposes in cloning GaLV-S was toaid in elucidating its mechanism of leukemogen-esis. Several mechanisms of leukemogenesishave been proposed for retrovirus-induced leu-kemias. Some avian retroviruses (16, 17, 19) and

E

J. VIROL.

MOLECULAR CLONING OF GIBBON APE LEUKEMIA VIRUS

Abelson murine leukemia virus (6) are replica-tion-defective recombinant viruses which haveacquired transforming genes by recombinationwith cellular c-onc genes. These genes replaceviral sequences and are responsible for thetransformed phenotype after infection of suscep-tible cells. Some of these acutely transformingviruses cause acute leukemias in vivo. Otherretroviruses, such as avian leukosis virus (7, 13),are thought to induce leukemia indirectly byinserting part of the viral genome, including anLTR, into the vicinity of certain c-onc genes andaltering their expression. In this study, we havelooked for the presence of an onc gene in GaLV-S by typing heteroduplex molecules between ourclones and clones of SSAV. Whereas we hadpreviously located an onc gene, v-sis, by hetero-duplex formation between SSAV and SSV, webelieved that a comparable experiment betweenSSAV and GaLV-S would reveal an onc gene ifone were present. Since no regions of nonho-mology were detected, we conclude that types 1and 2 of GaLV-S do not contain an onc genelarger than 300 base pairs. We also did notobserve any defective provirus in either infectedCCL88 cellular DNA or in unintegrated viralDNA of acutely infected cells. Since it seemsthat the various types of GaLV-S do not containan onc gene, the virus must induce the diseaseby indirect mechanisms. In this regard, theuniqueness ofGaLV-S LTR sequences in the U3region may have relevance.

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