viral dna and characterization of the endogenous viral

Vol. 41, No. 3JOURNAL OF VIROLOGY, Mar. 1982, p. 842-8540022-538X/82/030842-13$02.00/0

Colobus Type C Virus: Molecular Cloning of UnintegratedViral DNA and Characterization of the Endogenous Viral

Genomes of ColobusEDWARD H. BIRKENMEIER,1t TOM I. BONNER,'* KAY REYNOLDS,1 GEORGE H. SEARFOSS,2

AND GEORGE J. TODARO1Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 21701,1 and Frederick

Cancer Research Center, Frederick, Maryland 217012Received 28 August 1981/Accepted 21 October 1981

The unintegrated viral DNA intermediates of colobus type C virus (CPC-1)were isolated from infected human cells that were permissive for viral growth.There were two major species of DNA, linear molecules with two copies of thelong terminal repeat and relaxed circles containing only a single long terminalrepeat. In addition, there was a minor species (-10%) composed of relaxed circleswith two copies of the long terminal repeat. A restriction endonuclease map of theunintegrated DNA was constructed. The three EcoRI fragments of circular CPC-1DNA were cloned in the EcoRI site of XgtWESXB and then subcloned in theEcoRI site of pBR322. Using these subgenomic fragments as probes, we havecharacterized the endogenous viral sequences found in colobus cellular DNA.They are not organized in tandem arrays, as is the case in some other genefamilies. The majority of sequences detected in cellular DNA have the same mapas the CPC-1 unintegrated DNA at 17 of 18 restriction endonuclease sites. Thereare, however, other sequences that are present in multiple copies and do notcorrespond to the CPC-1 map. They do not contain CPC-1 sequences either in analtered form or fused to common nonviral sequences. Instead, they appear to bederived from a distinct family of sequences that is substantially diverged from theCPC-1 family. This second family of sequences, CPC-2, is also different from thesequences related to baboon endogenous type C virus that form a third family ofvirus-related sequences in the colobus genome.

An endogenous type C virus, CPC-1, isolatedfrom the Old World monkey Colobus polykomos(20) has substantial sequence homology with thetype C viruses MAC-1 and MMC-1 isolated fromthe stumptail monkey (Macaca arctoides) andthe rhesus monkey (Macaca mulatta), respec-tively (16, 20). Colobus cellular DNA contains50 to 70 copies of sequences which are closelyrelated to CPC-1. These multiple copies pose anumber of evolutionary questions such as howlong have they been endogenous in the primatesand how was this copy number obtained. Theendogenous primate viruses, including CPC-1,offer an advantage over a number of retrovirusesfor approaching these questions since they arexenotropic in their host range. Thus, the analy-sis of the cellular copies is not complicated bythe possibility of recent infection, as is the casewith ecotropic viruses. If xenotropic viral geneshave been endogenous in the primates for tens ofmillions of years, as has been suggested (3-5),

t Present address: The Jackson Laboratory, Bar Harbor,ME 04609.

then there must be a mechanism for main-taining homogeneity among the copies. Similarhomogeneity has been observed for a number offamilies of repeated cellular sequences. Thesefamilies include both expressed genes such asthe rRNA (12) and histone genes (10) as well asapparently unexpressed satellite sequences (6).All of these families are organized in tandemarrays, sometimes including repeated spacer se-quences. This organization may allow the mech-anism of unequal crossover to maintain homoge-neity (23). It would be useful to know whetherthe endogenous primate viral sequences have ananalogous organization.To study further the organization and evolu-

tion of endogenous primate retroviruses, wehave constructed a restriction endonucleasemap of unintegrated CPC-1 DNA and thencloned this DNA in lambda phage and plasmidvectors. Using subgenomic fragments of thecloned DNA, we have examined the organiza-tion of CPC-1-related sequences in colobus cel-lular DNA. Among the major bands detected incolobus cellular DNA, the majority correspond

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MOLECULAR CLONING OF UNINTEGRATED CPC-1 DNA

to fragments predicted from the CPC-1 restric-tion map. These bands have intensities indica-tive of multiple copies. There are, however,other bands of comparable intensity which arenot immediately explained by the CPC-1 map.One possibility is that they represent fragmentswith one end within the viral sequence and theother within a repeated spacer DNA. Such frag-ments would occur if the copies of CPC-1-related sequence were organized in the samemanner as other highly homogeneous repeated-sequence families. The data establish that this isnot the case. Instead, there appear to be twodistinct families of sequences which hybridize toCPC-1 DNA. One family has a restriction mapthat corresponds to CPC-1 and the second fam-ily (CPC-2) does not. CPC-2 is also distinct froma third family of sequences (CPC-3) detectedwith an endogenous baboon type C viral probe.However, when a cloned endogenous retroviralsequence from chimpanzee cellular DNA wasused as a probe, the CPC-2 bands were detectedwith much more intensity than the CPC-1 bands.The shift in relative intensity of CPC-1 and CPC-2 bands demonstrates that the CPC-2 sequencesare substantially diverged from the CPC-1 se-quences and do not result from a few simplechanges in the CPC-1 sequence.

MATERIALS AND METHODS

Isolation of uninterated viral DNA. The humancarcinoma cell line A549 was grown in Dulbecco'smodified Eagle medium supplemented with 10%o fetalcalf serum. Cells were infected with CPC-1 for 24 hand washed twice with phosphate-buffered saline, andthe unintegrated viral DNA was isolated by hydroxy-apatite chromatography (22).

Isolation of cellular DNA. Infected A549 cells sus-pended in SSC (SSC = 0.15 M NaCl and 0.015 Msodium citrate) or colobus cells in a homogenate ofcolobus liver tissue were lysed by incubating them for2 h at 37°C in SSC containing 0.5% sodium dodecylsulfate and 100 F^g of proteinase K per ml (E. Merck).The DNA was then purified as previously described(2).

Restriction endonudease digestion. Restriction endo-nucleases were obtained from New England Biolabs orBethesda Research Laboratories, and digestions wereperformed under conditions recommended by themanufacturer. Double enzyme digests were performedsimultaneously since the buffers for enzyme combina-tions used in this study were compatible. The com-pleteness of digestion was monitored by adding phagelambda to the reaction mixtures. For figures in whichmore than one probe was used, a single digest wasdivided among three or four lanes of the gel. Thisprocedure guarantees that digests hybridized to eachprobe are precisely the same.

Gel electrophoresis and blotting. DNA was electro-phoresed in 0.7 or 0.8% agarose gels (Seakem, MEgrade) with the use of a Tris-acetate buffer, pH 7.8(19). The gels were poured to a 0.6 cm thickness in ahorizontal slab gel unit (Bethesda Research Labora-

tories, model Hi). Restriction fragiments of lambdaand 4X174 DNA were used as size markers. Labeledmarkers were made by using avian myeloblastosisvirus DNA polymerase to fill in the ends of HindlIlfragments of A and TaqI fragments of 4)X174 (26).[32P]dCTP was incorporated under conditions similarto those used to make cDNA. After electrophoresis,DNA was transferred from gels onto 0.45-,um nitrocel-lulose membranes (Schleicher & Schuell Co.) as de-scribed by Southern (24).Preparation of probes and hybridization of blots.

cDNA was prepared with viral 70S RNA as templatefor avian myeloblastosis virus DNA polymerase aspreviously described (2). Strong stop cDNA purifiedby acrylamide gel electrophoresis was provided byGeorge Mark. Nick translation of cloned DNA wasperformed as previously described (18). Cloned, unin-tegrated DNA from the endogenous baboon virus, M7,was provided by N. Battula. Baked nitrocellulosemembanes containing the blotted DNA were hybrid-ized by published procedures (2). Low-stringency hy-bridizations were used where indicated in the figurelegends. In this case, both hybridization and washingof the filter after hybridization were done in 4.5x SSCat 60°C.

Cloning of viral DNA. Unintegrated viral DNA waspartially digested with EcoRI. The DNA fragmentswere sedimented in an 11-ml 5 to 20%o (wt/wt) sucrosegradient in electrophoresis buffer. The gradients werecentrifuged in a Beckman SW41 rotor at 33,000 rpmfor 17 h at 4°C. Fractions of 0.5 ml were collected bypumping out the gradient from the bottom of the tube,and 50 ,d from each fraction was electrophoresed in anagarose gel, blotted, and hybridized to a cDNA probeto locate the large, partially digested viral DNA frag-ments. To these were added 2.3 ,ug of simian virus 40DNA form I and 1.0 ,ug of AgtWESAXB EcoRI arms(14) with 2 volumes of ethanol at -20°C. The DNAwas suspended in 25 iil of 50 mM Tris-hydrochloride(pH 7.6), 5 mM MgCl2, 5 mM dithiothreitol, and 1 mMATP; 2.5 U of T4 DNA ligase (Bethesda ResearchLaboratories) was added, and the reaction mixturewas incubated overnight at 120C. The DNA was pack-aged into phage particles as described by Enquist andStemnberg (11). Plaques were screened for CPC-1 DNAby the Benton and Davis plaque lift procedure (3) withviral cDNA as a probe. Phages were propagated inEscherichia coli strain DP50 supF in broth that con-tained 10 g of tryptone (Difco), 5 g of yeast extract(Difco), 5 g of NaCl, 2.5 g of MgSO4, 10 ml of 1.0%odiaminopimelic acid, 10 ml of 0.4% thymidine, and 10ml of 1.0 M Tris-hydrochloride (pH 7.4) per liter.Phage particles were purified in CsCl gradients asdescribed by Fred Blattner in his outline accompany-ing the Charon phage. The method ofDNA extractionhas been published (15).Phage DNA containing the CPC-1 DNA inserts was

digested with EcoRI, and the inserts were purified byelectrophoresis in low-melting-temperature agarose(Seaplaque) gels (27). Under the same reaction condi-tions described above, 1.0 ,u.g of insert DNA wasligated to 0.2 pg of pBR322 DNA. The pBR322 DNAhad previously been digested with EcoRI and bacterialalkaline phosphatase (Bethesda Research Labora-tories). E. coli strain HB101 was transformed (9), andampicillin-resistant colonies were screened for CPC-1DNA (13) with viral cDNA used as a probe.

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844 BIRKENMEIER ET AL.

Restriction mapping of cloned viral DNAs. The CPC-1 EcoRI fragments of 1.3, 3.1, and 3.4 kilobase pairs(kb) were cut out of the vector, purified by agarose gelelectrophoresis, and 32P labeled by nick translation.Single and double restriction enzyme digests wereperformed on approximately 5,000 cpm of labeledDNA in the presence of 1 ,ug of lambda DNA to serveas a control for the completeness of the digestions.The samples were then electrophoresed on 0.3 cmthick agarose gels (1.0 or 1.5% agarose). The gel wasplaced onto Whatman 3MM paper and dried. Autora-diography of the dried gel allowed the easy detectionof fragments as small as 0.1 kb.

RESULTSRestriction endonuclease map of CPC-1. Hu-

man tissue culture cells were infected with CPC-1, and the unintegrated viral DNA was isolated24 h later. Unintegrated viral DNA was digestedwith restriction endonucleases, and the DNAfragments were separated by electrophoresis inan agarose gel. After Southern blotting, the

fragments were detected by use of a cDNAprobe representative of the CPC-1 genome (Fig.1). The undigested DNA consisted of two majorforms. On the basis of the DNA forms foundwith the baboon virus M7 (2) and our datapresented below, we concluded that these formswere linear DNA molecules with two copies ofthe long terminal repeat (LTR) sequence andrelaxed circular molecules with one LTR. Inaddition, there was a minor form (-10%) thatconsisted of relaxed circular molecules with twocopies of the LTR. The presence of three formsof unintegrated DNA resulted in complicatedrestriction patterns in Fig. 1. However, the mostintense bands were either internal fragments ofthe linear map which were generated from allthree forms or fragments specific to the one LTRcircle. Less intense bands were either terminalfragments from the linear form or fragmentsspecific to the two LTR circle.To identify the internal fragments, we digest-

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FIG. 1. Restriction endonuclease fragments of unintegrated CPC-1 DNA. Unintegrated viral DNA wasisolated 24 h after infection. The DNA was digested with restriction endonucleases and electrophoresed in 0.8%agarose gels. The viral DNA fragments were transferred to nitrocellulose paper and detected by hybridization toa 32P-labeled viral cDNA probe. Circle 1 and circle 2 refer to the undigested circular DNA forms with one andtwo LTR, respectively. Linear refers to the undigested linear form with two LTR; kb refers to the size of thefragments in kilobase pairs determined by using lambda DNA fragments as size markers. Panels A and Brepresent two different gels.

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MOLECULAR CLONING OF UNINTEGRATED CPC-1 DNA 845

ed cellular DNA from chronically infected hu-man cells that contained CPC-1 proviruses inte-grated at numerous sites and hybridized theDNA fragments to cDNA as described above.The internal fragments should occur in all inte-grated copies, resulting in intense bands, where-as the terminal fragments should be different foreach integration site, resulting in a continuum offaint bands. SalI, BamHI, and KpnI each cutwithin the virus twice to generate one internalviral band (Fig. 2). The lengths were 1.2, 3.8,and 2.8 kb, respectively. EcoRI cut three timesto generate two internal fragments of 1.3 and 3.4kb. BglII cut four times to generate three inter-nal fragments of 0.7, 2.0, and 3.2 kb. In Fig. 2the 0.7-kb fragment has migrated beyond the endof the blot and is not seen. There are no intenseinternal bands with Hindlll and XhoI, and there-fore they cut only once. However, minor bandsof greater than 4 kb were apparent in the Hin-dIII, XhoI, Sall, and KpnI digests. These areprobably due to unintegrated viral DNA beingentrapped in the high-molecular-weight cellularDNA during extraction since they migrate as thebands from unintegrated DNA seen in Fig 1.To confirm these results, we digested uninte-

grated viral DNA as before, but hybridized it to

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FIG. 2. Internal restriction endonuclease frag-ments of CPC-1 DNA. Cellular DNA from chronicallyinfected human cells was digested with restrictionendonucleases and electrophoresed in an 0.8% agarosegel. Viral DNA fragments were detected and their sizewas determined as described in the legend of Fig. 1.Shorter exposure of the HindIll lane reveals a singlemajor band of 7.7 kb which probably results fromunintegrated viral DNA. This band is not as intense asthe bands which occur between 2 and 4 kb.

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FIG. 3. Restriction endonuclease fragments ofCPC-1 DNA that contain strong stop DNA sequences.Unintegrated viral DNA was digested with restrictionendonucleases and electrophoresed in an 0.8% agarosegel. Viral DNA fragments were detected by hybridiza-tion of a Southern blot to 32P-labeled strong stopDNA.

strong stop DNA (Fig. 3). Since the strong stopsequence is within the LTR, the bands which aredetected should be the terminal fragments of thelinear form or the fragments specific to thecircular forms. HindIII and XhoI cut the linearvirus once, and each generated two fragments,3.5 and 4.9 kb or 1.3 and 7.1 kb, respectively.They also generated a 7.7-kb linear molecule bycutting the major circular form which containedone LTR. Sall and BamHI cut the linear formtwice to generate terminal fragments of 2.4 and4.8 kb or 2.0 and 2.5 kb, respectively. The 6.6-kbSalI (= 2.4 + 4.8 - 0.6 kb) and 3.9-kb BamHI(= 2.0 + 2.5 - 0.6 kb) fragments were generatedfrom the major circular form. EcoRI generatedterminal fragments of 1.7 and 1.9 kb as well as a3.1-kb fragment from circles with one LTR.These results are precisely complementary tothose of Fig. 2. Taken together, they allow theidentification of the bands of Fig. 1 and confirm

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the interpretation of Fig. 1 which was basedsolely on band intensity.The fragments were oriented relative to one

another by digesting unintegrated viral DNAwith two enzymes simultaneously (Fig. 4). Themap of restriction endonuclease sites (Fig. 5)was derived as follows. The single XhoI site wasarbitrarily positioned at 1.3 kb from the left endof the linear map. Since the Sail 4.8-kb fragmentwas cut by XhoI to give 1.3- and 3.5-kb frag-ments, it is at the left end. The 2.4-kb terminalfragment must be at the right end, and the 1.2-kbfragment is in the middle. Since BamHI diges-tion reduced the size of the 1.2-kb Sall fragmentby 0.1 kb, there must be a BamHI site between2.4 and 3.6 kb from the right end of the map.

Thus, the 2.5-kb BamHI terminal fragment is atthe right end. The remaining terminal fragment,2.0 kb, must be at the left end, and the internal

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fragment of 3.8 kb is in the middle. The 1.7-kbEcoRI fragment was cut by XhoI, placing it atthe left end and leaving the 1.9-kb fragment atthe right end. The 3.4-kb EcoRI fragment wascut by Sall to generate fragments of 0.5, 1.2 (theinternal Sall fragment), and 1.7 kb. The positionof the two Sal sites implies that the 1.3-kbEcoRI fragment is between the 1.7- and 3.4-kbfragments. Thus, the order of EcoRI fragmentsfrom left to right is 1.7, 1.3, 3.4, and 1.9 kb. TheHindIII site is 4.9 kb from one end. SinceHindlll decreased the size of the 1.2-kb Sallfragment by 0.1 kb, the site must be 4.9 kb fromthe left end. KpnI cut the virus twice to generate0.8-, 2.8-, and 4.8-kb fragments, as well as a 4.9-kb fragment from circles with one LTR. XhoIcut the KpnI 2.8-kb fragment to generate 0.5-and 2.3-kb fragments. HindIII cut the 4.8-kbKpnI fragment. Thus, the KpnI 2.8-kb fragment

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FIG. 4. Digestion of unintegrated CPC-1 DNA with two restriction endonucleases simultaneously. Uninte-grated viral DNA was digested with two enzymes in each reaction mixture. The viral DNA fragments were

electrophoresed in agarose gels and were detected on Southem blots as described in the legend of Fig. 1. PanelsA and B represent two different gels. Fragments mentioned in the text that are less than 1.0 kb can be seen on theoriginal film but not in the printed figure.


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A.0 1 2 3 4 5 6 7 8Kb

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FIG. 5. Restriction endonuclease cleavage sites in linear and circular unintegrated CPC-1 DNA. Panel Amaps the cleavage sites in linear DNA. The 5' to 3' orientation is with respect to viral RNA. The 5' probe and 3'probe are the regions of the CPC-1 genome that were hybridized to the Southern blot in Fig. 7. Panels B and Cmap the EcoRI sites with slashes and the XhoI sites with arrows in circles with one and two LTR, respectively.Numbers refer to the size of the DNA fragments in kilobase pairs. Shaded boxes indicate the LTR.

is internal, with the 0.8- and 4.8-kb fragments asthe left and right terminal fragments, respective-ly. All of the restriction endonuclease sites inFig. 5 have been confirmed by additional doubledigests (data not shown). The 5' to 3' orientationwas not determined from the unintegrated DNAbut from sequence analysis of the cloned CPC-1DNA (see below).

Cloning of unintegrated CPC-1 DNA. Uninte-grated linear and circular viral DNA was partial-ly digested with EcoRI. The reaction productswere sedimented in a sucrose gradient. A samplefrom each fraction of the gradient was assayedfor viral DNA fragments by agarose gel electro-phoresis, Southern blotting, and hybridization toa viral cDNA probe. One fraction had fragmentslarger than the EcoRI 3.4-kb fragment but hadonly trace amounts of the 3.4-kb fragment. TheDNA in this fraction was cloned in AgtWES-XB.Approximately 104 plaques were screened forviral sequences, and 10 positive plaques werefound. None of these clones contained all theEcoRI fragments of a complete viral genome.Three clones were further characterized. Twoclones had three DNA inserts of 1.3, 3.1, and 5.0kb, of which the 1.3- and 3.1-kb fragments

hybridized to CPC-1 cDNA. The 3.1-kb frag-ment was not cut by HindIll or BamHI but wascut by XhoI to give a 2.6-kb fragment. This isconsistent with the 3.1-kb fragment being theEcoRI fragment from circular viral DNA. The1.3-kb fragment was not cut by HindIII or XhoIbut was cut by BamHI to give a 1.1-kb fragment.This is consistent with the 1.3-kb fragment beingthe EcoRI internal 1.3-kb viral fiagment. Pre-sumably, for each of the two clones, the 1.3- and3.1-kb viral fragments were ligated into thelambda vector as a 4.4-kb fragment from apartially digested circular viral DNA molecule.The 5.0-kb fragment of one of the clones had arestriction endonuclease map consistent with itsbeing the EcoRI 4.9-kb internal lambda fragmentof XgtWES-XB.The third clone had inserts of 1.3, 3.1, and 3.4

kb, of which the 1.3- and 3.4-kb fragmentshybridized to CPC-1 cDNA. The 3.4-kb frag-ment was not cut by XhoI. It was cut by BamHIto give a 2.9-kb fragment and by HindIII to give1.6- and 1.8-kb fragments. This is consistentwith the 3.4-kb fragment being the EcoRI inter-nal 3.4-kb viral fragment. The 1.3-kb fragmentwas cut by BamHI and not by XhoI or HindIII,

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consistent with its being the viral 1.3-kb frag-ment. Presumably, a 4.7-kb EcoRI fragmentfrom partial digestion of either circular or linearviral DNA was ligated into the lambda vectorwith a 3.1-kb fragment of host cellular DNA.The 1.3- and 3.1-kb EcoRI viral fragments

from one clone and the 3.4-kb viral fragmentfrom the third clone were purified by electropho-resis in an agarose gel. They were then individ-ually subcloned at the EcoRI site of pBR322. Arestriction endonuclease map was made for eachfragment. The maps of the 1.3- and 3.4-kb frag-ments were identical to that predicted from theunintegrated linear viral DNA map (Fig. 5).XhoI and KpnI digests of the 3.1-kb fragmentwere consistent with its having sequences fromthe left end of the linear map. Since no enzymeswere mapped which cut within the 1.9-kb EcoRIfragment of the linear map, three additionalenzymes, BglII, HpaI, and SacI, were mappedin the three cloned fragments. These additionalenzyme sites were placed on the linear map byassuming that the 3.1-kb fragment was derivedfrom a circle containing only one LTR. Thesemap positions were completely consistent withrestriction digests of the unintegrated DNA(data not shown). Thus, the 3.1-kb fragmentmust contain both 3' and 5' ends, as assumed.The size of the LTR was estimated to be 0.5 kbby comparing the sum of the sizes of the 1.7- and1.9-kb ends of the linear molecule with the 3.1-kb size of the circular fragment lacking oneLTR. Since Sacl cuts the LTR near the 5' end ofthe CPC-1 strong stop sequence (George Mark,personal communication), we were able to de-termine the 5' to 3' orientation of the restrictionmap with respect to viral RNA. The 1.72-kbdistance between the EcoRI and SacI sites in the3.1-kb fragment implies that a SacI site exists atapproximately 0.18 kb from the right end of themap. Since the DNA sequencing shows that theSacI site is 132 nucleotides from the 3' end of theLTR, the right end of the map must be the 3'end.Endogenous CPC-1 sequences of colobus. Nu-

cleic acid hybridization studies have shown thatthere are 50 to 70 copies of the CPC-1 genome incolobus cellular DNA (20). We have examinedthe restriction endonuclease fragments from theendogenous CPC-1 genomes. Colobus cellularDNA was digested with restriction endonucle-ases, and the fragments were separated by elec-trophoresis in agarose gels. Southern blots werehybridized to CPC-1 cDNA or nick-translated,cloned CPC-1 DNA (Fig. 6). The dominantbands observed included the internal viral frag-ments generated by EcoRI (1.3 and 3.4 kb),BamHI (3.8 kb), and BglII (2.0 and 3.2 kb). Ofthe 12 internal bands defined on the viral map,10 were observed as major bands. The 1.40- and

4.65-kb HpaI bands were not observed althoughthere was instead a 6.0-kb band. This bandprobably resulted from the absence of the HpaIsite which separates the two fragments on theviral map. At least some of the fainter bandsobserved in the various digests should representfusion fragments that have one end within theviral sequence and the other within the flankingcellular sequence. This was most easily seenwith the HindIII digest where all the fragmentsshould be fusion fragments since HindlIl cutsCPC-1 only once. However, a 2.2-kb HindIIIfragment was observed even though the smallestpredicted fusion fragment should have been atleast 3.5 kb. In addition, there were severalintense bands which were not explained fromthe map. For example, there were BamHI frag-ments of 2.5 and 3.5 kb and EcoRI fragments of2.8 and 3.8 kb.One plausible explanation is that the unex-

plained BamHI and EcoRI bands (but not the2.2-kb HindIII band) reflect a common flankingsequence. If the endogenous copies were orga-nized in a tandem array with a constant spacersequence, as is observed with some multigenefamilies, then we would expect, for enzymeswhich cut within both the viral and the spacersequences, two major virus-spacer fusion bands,one containing the 5' end and one containing the3' end of the viral genome. To test this possibili-ty, we used probes specific for each end of theviral genome. A 5'-specific probe was made bynick translation of the 1.0-kb KpnI-EcoRI frag-ment of the cloned CPC-1 DNA. The 3'-specificprobe was made by using the 0.7-kb Bgll andthe 0.4-kb BglII-EcoRI fragments. When theseprobes were hybridized to restriction digests ofcolobus cellular DNA (Fig. 7), a large number offaint bands were detected which did not corre-spond to the major bands detected with therepresentative probe. The only bands whichcould represent multiple copies appear to havefewer than five copies. These bands probablyresult from a fortuitous clustering of bands withunrelated flanking sequences. Thus, there is noconvincing evidence for common flanking se-quences.

Additional experiments (data not shown) wereperformed to determine whether the 3.8- and2.8-kb EcoRI bands could be derived from theCPC-1 sequences by insertions, deletions, orscattered point mutations, as has been observedfor baboon endogenous viral sequences (7).These experiments included separating the 3.8-and 2.8-kb fragments by preparative gel electro-phoresis and digesting them with additional re-striction enzymes whose positions on the CPC-1map were known. The results failed to supportany reasonably simple model. In addition, hy-bridization of the three cloned EcoRI fragments

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A

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FIG. 6. Endogenous CPC-1 sequences of colobus. Colobus cellular DNA was digested with restrictionendonucleases and electrophoresed in 0.7 or 0.8% agarose gels. Viral DNA was detected on Southern blots byhybridization to a cDNA probe (panel A) or to a nick-translated, cloned DNA probe (panel B). Both of the probeswere representative of the entire CPC-1 genome. The blot in panel B was hybridized at lower stringency.

to cellular DNA revealed that the two EcoRlbands, as well as the 3.5- and 2.5-kb BamHIbands, hybridized exclusively to the 3.4-kbEcoRI fragment.A second family of CPC-1-related sequences.

Because the data did not support an insertion,deletion, and point mutation model, we consid-ered the hypothesis that there was another fam-ily of sequences which was substantially di-verged from the CPC-1 family. It might beexpected to share homology primarily within the3.4-kb EcoRI fragment of CPC-1 since this is themost conserved region of CPC-1 when com-pared with reticuloendotheliosis virus (REV),M7 virus, and AKR virus (17; T. I. Bonner,unpublished data). Clones of CPC-1-related se-quences from chimpanzee cellular DNA (T. I.Bonner, E. H. Birkenmeier, M. A. Gonda, G. E.Mark, G. H. Searloss, and G. J. Todaro, manu-script in preparation) were used as probes toprove this hypothesis. One of the clones, 5CH3,

contains an 8.3-kb sequence which extends froman EcoRI site in its 5' LTR to the correspondingEcoRI site in its 3' LTR and which is homolo-gous to CPC-1 in a colinear fashion over most ofits length. The LTR sequences in 5CH3 weredefined by nucleotide sequence comparison withthe strong stop sequence of CPC-1. Althoughrelated to CPC-1, this 8.3-kb sequence is 20 to25% diverged from CPC-1 on the basis of melt-ing temperature differences.When digests of colobus DNA were hybrid-

ized to the 5CH3 probe (Fig. 8A), the majorunexplained bands were detected much morestrongly than were the characteristic CPC-1bands. In contrast, when hybridized to the CPC-1 probe (Fig. 8B), the same digests showed thecharacteristic CPC-1 bands as slightly to sub-stantially more intense than the unexplainedbands. In addition, the 5CH3 probe detected avery strong 2.2-kb EcoRI band which was de-tected as a very weak band with the CPC-1

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FIG. 7. Endogenous CPC-1 5' and 3' sequences of colobus. Colobus cellular DNA was digested withrestriction endonucleases and electrophoresed in an 0.8% agarose gel. Viral DNA fragments were detected byusing a 5'-specific probe (panel A), representative of the entire CPC-1 genome (panel B), and a 3'-specific probe(panel C). The probes were made by nick translation of fragments of cloned CPC-1 DNA as described inthe text. Location of the 5' and 3' fragments on the CPC-1 map is shown in Fig. 5. Marker lanes contain 32P-labeled fragments of lambda and 4OX174 DNAs.

probe. Hybridization with M7 probe (Fig. 8C)detected an entirely different set of major bands.These results imply that the unexplained bandscontain sequences which are more homologousto the 5CH3 probe than to the CPC-1 probe.Thus, these bands are not CPC-1 sequences witha few deletions or modified restriction sites.Conceivably, the unexplained bands might beCPC-1 sequences with an inserted sequencewhich has a homolog inserted in the 5CH3sequence. However, the increase in hybridiza-tion with the 5CH3 probe would require theputative insert to be a major portion of most ofthese bands. This is clearly not the case for the2.2-kb EcoRI band which gives an approximate-ly fivefold greater signal with the 5CH3 probe.As shown below this band is detected primarilyby the B fragment of 5CH3, a fragment that hasno insertions relative to CPC-1 as determined byheteroduplex analysis. Thus, the data indicatethat the unexplained bands are derived from asecond family of CPC-1-related sequenceswhich are substantially diverged from the CPC-1family. This family has (i) much greater homolo-gy to SCH3 than to CPC-1 and a number ofcopies comparable to CPC-1, (ii) equal homolo-

gy to CPC-1 and 5CH3 but many more copiesthan CPC-1, or (iii) a combination of being moreclosely related to 5CH3 than CPC-1 and havingsubstantially more copies than the CPC-1 fam-ily. Furthermore, this family is not the M7-related sequences which were previously shownto exist in colobus DNA (4, 5).We have partially mapped the new family of

sequences which we have named the CPC-2family. This was done by hybridizing four sub-cloned regions of the 5CH3 viral sequence torestriction digests of colobus DNA (Fig. 9). The2.2-kb EcoRI fragment was detected by the Bprobe and weakly by the A and C probes. Thus,its homology to 5CH3 extends to within at least2.5 kb of the 5' end. Similarly, the 3.8- and 2.8-kb EcoRI fragments were detected by both the Cand D probes. Therefore, they must be overlap-ping, probably with the 2.8-kb fragment contain-ing less of the D sequence than the 3.8-kbfragment. The 3.5- and 2.5-kb BamHI fragmentswere detected strongly by the B and C probesand weakly by the D probe, indicating that theyoverlap and extend across the junction betweenthe 2.2-kb EcoRI fragment and the 3.8- and 2.8-kb EcoRI fragments. Finally, a 1.7-kb HpaI

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band was detected strongly by the A and Bprobes and not at all by the C or D probes,whereas a 3.25-kb HpaI band was detectedstrongly by the C and D but not by the A and Bprobes. Taken together, these results indicatethat there is a 5-kb region of homology betweenSCH3 and CPC-2 sequences. At the 5' end thishomology must include approximately 0.5 kb ofregion A since the 1.7-kb HpaI band hybridizedabout equally well to the A and B regions. At the3' end the homology must extend at least 0.5 kbinto region D since the 2.8-kb EcoRI fragmenthad a minimum of 0.5 kb mapping into the Dregion and the 3.8-kb band hybridized better tothe D probe, indicating that its homology ex-tends even farther. The MMC-1 viral genomealso contains a 2.2-kb EcoRI fragment and a 2.6-kb BamHI fragment (25). However, they are notthe same as the CPC-2 fragments since they mapin different portions of the genome. The MMC-12.6-kb BamHI fragment contains the whole 2.2-kb EcoRI fragment within it and is locatedprecisely in the middle of the genome.

DISCUSSIONWe have isolated the unintegrated viral DNA

resulting from an acute infection of human cells

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with CPC-1. Two major forms of DNA werefound. These were a linear form and a relaxedcircular form containing one copy of the LTR. Inaddition, there was a minor circular form con-taining two copies of the LTR. This generalcharacteristic of unintegrated retrovirus DNAhas also been found for the endogenous type Cvirus isolated from baboons (2, 8). By comparingrestriction endonuclease fragments of uninte-grated viral DNA with fragments from integrat-ed viral DNA of infected human cells, we havegenerated a restriction endonuclease map of theunintegrated CPC-1 DNA. The EcoRI fragmentsof CPC-1, including a 3.1-kb fragment from themajor circular form, were cloned in AgtWESAXBby partial digestion of the unintegrated viralDNA with EcoRI. The restriction endonucleasemap of the cloned virus corresponded exactly tothe map of the unintegrated viral DNA.There are 50 to 70 copies of the CPC-1 genome

in colobus cellular DNA (20). We used subge-nomic fragments of the cloned virus as probes todetermine the restriction map of these endoge-nous genomes. Endogenous viral fragments thatcorresponded to the internal fragments of CPC-1were found, and the amount of hybridizationindicated that multiple copies were present. Of

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FIG. 9. Restriction endonuclease map of CPC-2. Southern blots of colobus cellular DNA were hybridized atlow stringency to nick-translated subgenomic fragments (A, B, C, and D) of the CPC-1-related chimpanzeeDNA, 5CH3. Each of the subgenomic fragments was individually cloned in pBR322. Their location on the 5CH3map is indicated in the middle portion of the figure. The heavy lines on the 5CH3 map indicate homology of5CH3with CPC-1 as determined by heteroduplex analysis. The boxes indicate LTR sequences. In the bottom portionof the figure, CPC-2 fragments are aligned with the 5CH3 map as determined by the Southern blots. Marker lanescontain 32P-labeled fragments of lambda and (X174 DNAs.

the 18 restriction enzyme sites which defineinternal fragments on the unintegrated DNAmap, 17 are present in many if not most of theendogenous genomes. Only the HpaI site at 2.35kb from the 5' end of the map is missing in the

majority of cellular copies. Thus, there is afamily of viral genomes with a restriction mapthat corresponds to the map of CPC-1. Howev-er, there were intense bands that did not corre-spond in size to CPC-1 fragments. This result, as

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well as the homogeneity of endogenous copies,could be explained by the hypothesis that theendogenous viral copies are organized in a tan-dem array with constant spacers between theviral sequences. However, this hypothesis wasdisproved by the results of Fig. 7 in which 5' and3' specific probes failed to detect the EcoRIband of 3.8 and 2.8 kb and the BamHI bands of3.5 and 2.5 kb. The detection of many otherbands with these probes demonstrates that thereare no extended regions of immediately flankingsequence which are common to more than a fewviral copies.

Since the viral copies are not organized intandem arrays, the homogeneity of the viralgenes requires an explanation other than un-equal crossover. One possible mechanism is theprocess of gene conversion (1). It is not knownwhether this mechanism is more frequent fortandem genes than for randomly distributedgenes. Thus, it is not clear whether this processwould result in most of the copies being in atandem array. Another possibility is that most ofthe copies arose from recent amplification. Nu-merous studies have shown that infection ofcells with a retrovirus results in the stable inte-gration of only a few viral copies. To obtain theapproximately 50 copies typically observed forendogenous primate viruses, some process ofamplification is necessary. Since the viral copiesare probably dispersed, the amplification proc-ess does not resemble the gene amplificationprocesses responsible for the large copy numberof the rRNA or histone genes or satellite DNAsequences. However, there are clearly cellularamplification processes which produce suchnontandem genes as the hemoglobin genes. Theother alternative is that the amplification resultsfrom a specific property of the viral sequenceseither through their ability to produce virus orepisomal DNA which reintegrates at additionalsites or through their possible ability to act astransposable elements (21).Anomalous bands have been obtained with

the baboon endogenous virus and rhesus virus(MMC-1) genes which appear to be similar to theanomalous colobus bands. Some of the endoge-nous viral genomes related to MMC-1 in rhesusDNA have restriction maps that differ from themap of integrated proviruses in infected caninecells (25). No further experiments were per-formed to clarify their origin. There are alsobaboon endogenous virus sequence variations inbaboon cellular DNA (7). The major variationsare accounted for by missing or modified BamHIrecognition sequences at two sites. Both varia-tions are in the gag gene region of baboonendogenous virus. Since the BamHI site wasalso missing in one provirus cloned in lambdaphage, the BamHI site probably contained an

altered DNA sequence. The anomalous CPC-1-related fragments which we observed in colobuscellular DNA could have a similar origin. How-ever, a number of experiments failed to supportthis hypothesis. By cloning CPC-1-related se-quences from chimpanzee cellular DNA andhybridizing them to colobus cellular DNA, wewere able to characterize the major anomalousbands. Comparison of the relative intensities ofthe predicted CPC-1 bands with the anomalousbands by using 5CH3 and CPC-1 probes re-vealed that the major anomalous bands repre-sent a distinct family of sequences, the CPC-2family. This family is related to CPC-1 but issubstantially diverged from CPC-1. Its homolo-gy with 5CH3 maps to the central 4.5 kb of5CH3. The homology of CPC-2 with CPC-1 islimited to the 3.4-kb EcoRI fragment of CPC-1.Thus, the CPC-2 sequences have more homolo-gy to the chimpanzee sequences than they haveto the CPC-1 sequences. In addition, they aredistinct from the baboon virus-related sequencesthat form a third family of type C virus-relatedsequences in colobus.The presence of diverged yet related families

of viral sequence in the colobus genome may beof considerable importance in the interpretationof data on the evolution of endogenous viralsequences. Previous experiments (4, 5, 20) haveattempted to correlate the divergence of the viralsequences within various primates with the di-vergence of the species themselves. Althoughthe data for many primates showed a high degreeof correlation, the viral sequences in a numberof species appeared to diverge more rapidly thanpredicted. Implicit in the analysis of these data isthe assumption that there is a single family ofsequences related to the viral probe in each ofthe primate genomes. However, the occurrenceof two families of CPC-1-related sequences incolobus could be indicative of a more generalsituation in which other species contain multiplerelated families. Preliminary results suggest thatboth the baboon and rhesus cellular genomesalso contain more than one family of CPC-1-related sequences.

LITERATURE CITED1. Baltimore, D. 1981. Gene conversion: some implications

for immunoglobulin genes. Cell 24:592-594.2. Battula, N., and G. J. Todaro. 1980. Physical map of

infectious baboon type C viral DNA and sites of integra-tion in infected cells. J. Virol. 36:709-718.

3. Benton, W. D., and R. W. Davis. 1977. Screening A gtrecombinant clones by hybridization to single plaques insitu. Science 196:180-182.

4. Benveniste, R. E., and G. J. Todaro. 1976. Evolution oftype C viral genes: evidence for an Asian origin of man.Nature (London) 261:101-108.

5. Bonner, T. I., and G. J. Todaro. 1980. The evolution ofbaboon endogenous type C virus: related sequences in theDNA of distant species. Virology 103:217-227.

6. Brown, S. D. M., and G. A. Dover. 1980. Conservation of

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segmental variants of satellite DNA of Mus musculus in arelated species: Mus spretus. Nature (London) 285:47-49.

7. Cohen, M., N. Davidson, R. V. Gilden, R. M. McAlister,M. 0. Nicolson, and R. M. Stephens. 1980. The baboonendogenous virus genome. II. Provirus sequence varia-tions in baboon cell DNA. Nucleic Acids Res. 8:4423-4440.

8. Cohen, M., M. 0. Nicolson, R. M. McAllister, M. Shure,N. Davidson, N. Rice, and R. V. GUden. 1980. Baboonendogenous virus genome. I. Restriction enzyme map ofthe unintegrated DNA genome of a primate retrovirus. J.Virol. 34:28-39.

9. Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972. Nonchro-mosomal antibiotic resistance in bacteria: genetic trans-formation of Escherichia coli by R-factor DNA. Proc.Natl. Acad. Sci. U.S.A. 69:2110-2114.

10. Cohn, R. H., J. C. Lowry, and L. H. Kedes. 1976. Histonegenes of the sea urchins (S. purpuratus) cloned in E. coli:order, polarity and strandedness of the five histone-coding and spacer sequences. Cell 9:147-161.

11. Enquist, L., and N. Stemnberg. 1979. In vitro packaging ofA Dam vectors and their use in cloning DNA fragments.Methods Enzymol. 68:281-298.

12. Forsbeit, A. B., N. Davidson, and D. D. Brown. 1979. Anelectron microscope heteroduplex study of the ribosomalDNAs of Xenopus laevis and Xenopus mulleri. J. Mol.Biol. 90:301-304.

13. Grunstein, M., and D. S. Hogness. 1975. Colony hybrid-ization: a method for the isolation of cloned DNAs thatcontain a specific gene. Proc. Natl. Acad. Sci. U.S.A.72:3961-3965.

14. Hager, G. L., E. H. Chang, H. W. Chan, C. F. Garon, M.A. Israel, M. A. Martin, E. M. Scolnick, and D. R. Lowy.1979. Molecular cloning of the Harvey sarcoma virusclosed circular DNA intermediates: initial structural andbiological characterization. J. Virol. 31:795-809.

15. Manlatis, T., R. C. Hardison, E. Lacy, J. Laver, C.O'ConneUl, D. Quon, G. K. Sim, and A. Efstratladls. 1978.The isolation of structural genes from libraries of eucary-otic DNA. Cell 15:687-701.

16. Rabin, H., C. V. Benton, M. A. Tainsky, N. R. Rice, andR. V. Gilden. 1979. Isolation and characterization of an

endogenous virus from Rhesus monkeys. Science204:841-842.

17. Rice, N. R., T. I. Bonner, and R. V. Gilden. 1981. Nucleicacid homology between avian and mammalian type Cviruses: relatedness of reticuloendotheliosis virus cDNAto cloned proviral DNA of the endogenous colobus virusCPC-1. Virology 114:286-290.

18. Rigby, P. W., M. Dieckmann, C. Rhodes, and P. Berg.1977. Labeling deoxyribonucleic acid to high specificactivity in vitro by nick translation with DNA polymeraseI. J. Mol. Biol. 113:237-251

19. Sharp, P. A., B. Sugden, and J. Sambrook. 1973. Detec-tion of two restriction endonuclease activities in Haemo-philus parainfluenzae using analytical agarose-ethidiumbromide electrophoresis. Biochemistry 12:3055-3063.

20. Sherwin, S. A., and G. J. Todaro. 1979. A new endoge-nous primate type C virus isolated from the Old Worldmonkey Colobus polykomos. Proc. Natl. Acad. Sci.U.S.A. 76:5041-5045.

21. Shinotohno, K., S. Mizutani, and H. M. Temin. 1980.Sequence of retrovirus provirus resembles that of bacteri-al transposable elements. Nature (London) 285:550-554.

22. Shoyab, M., and A. Sen. 1978. A rapid method for thepurification of extra-chromosomal DNA from eukaryoticcells. J. Biol. Chem. 253:6654-6656.

23. Smith, G. P. 1976. Evolution of repeated DNA sequencesby unequal crossover. Science 191:528-535.

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

25. Talnsky M. A. 1981. Analysis of the virogenes related torhesus monkey endogenous type C retrovirus in monkeysand apes. J. Virol. 37:922-930.

26. Wahl, G. M., M. Stern, and G. R. Stark. 1979. Efficienttransfer of large DNA fragments from agarose gels todiazobenzylomethyl-paper and rapid hybridization by us-ing dextran sulphate. Proc. Natl. Acad. Sci. U.S.A.76:3683-3687.

27. Wleslander, L. 1979. A simple method to recover intacthigh molecular weight RNA and DNA after electrophore-sis in low gelling temperature agarose gels. Anal. Bio-chem. 98:305-309.

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