Viral DNA and Characterization of the Endogenous Viral

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  • 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


    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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    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.


    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 37C 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 60C.

    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 4C. 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 -20C. 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