Proc. Nat. And. Sci. USA Vol. 70, No. 11, pp. 3067-3071, November 1973 Integration of Deoxyribonucleic Acid Specific for Rous Sarcoma Virus after Infection of Permissive and Nonpermissive Hosts (RNA tumor viruses/reassociation kinetics/duck cells) HAROLD E. VA4RMUS*, PETER K. VOGTt, AND J. MICHAEL BISHOP* * Department of Microbiology, University of California, San Francisco, Calif. 94122; and t Department of Microbiology, University of Southern California Medical School, 2025 Zonal Ave.. Los Angeles. Calif. 90033 Communicaled by Norman Dwidson, July 11, 1973 ABSTRACT A relatively simple but stringent tech- nique was developed to detect the integration of virus- specific DNA into the genomes of higher organisms. In both permissive (duck) and nonpermissive (mammalian) cells which normally contain no nucleotide sequences specific for Rous sarcoma virus, transformation by the virus results in the appearance of DNA specific for Rous sarcoma virus covalently integrated into strands of host- cell DNA containing reiterated sequences. Early after infection of mouse or duck cells by Rous sarwma virus, unintegrated DNA specific for the virus can be demon- strated. ~~ Replication of and transformation by RNA tumor viruses probably proceed by way of a DNA intermediate (1). Al- though the site of synthesis of oncornavirus DN.4 remains in dispute (2, 3), it is generally assumed that the DNA is syn- thesized immediately after infection by virion-associated RNA-directed DNA polymerase, integrated into the host- cell genome, and subsequently transcribed into viral RNA. Direct physicochemical observation of this sequence of events, however, has not been reported. Integration of genomes of DNA tumor viruses into host-cell DNA has been demon- strated by cosedimentation of viral DN.4 with high-molec- ular-weight cell DN9 in alkaline sucrose gradients (4). We have presented a preliminary report of a less cumbersome and more stringent method for detecting integrated viral DNA in cells (5). In this communication, we present details of this method and some estLmples of situations in which either inte- grated or unintegrated Rous sarcoma virus (RSV)-specific DN.4 is found. The integration test is based upon the observation by Britten and coworkers that the vast majority of high-molec- ular-weight DN.4 extracted from higher organisms contains reiterated sequences (6,7). They showed that when unsheared cell DNA is incubated to Cot values at which repeated, but not unique, sequences reassociate, "networks" of DNA are formed (Fig. 1). These networks can be separated from the remainder of the DNA by sedimentation. We now report that testing the DNA in networks for virus-specific nucleotide sequences by molecular hybridization constitutes a relatively simple asay for integration of viral DNA. The finding of virus-specific DNA in networks demonstrates covalent linkage of viral DNA to strands of cell DNA containing repeated sequences, and thus its integration into the host genome. We used this approach to study RSV-specific DNA in two different host cells after infection by RSV: (i) duck-embryo Abbreviations: ItSV, Rous sarcoma virus; Cpt, product of DNA concentration and time; gs, groupspecific antigen; SV40, simian virus 40. fibroblasts, which contain no detectable endogenous RSV- specific nucleotide sequences, are readily transformed by RSV, and support its replication; and (ii) mammalian cells, particularly BALB/c 3T3 cells, which also possess no endog- enous RSV-like genes, but are inefficiently transformed, and do not support replication of RSV. In both cases we show that under suitable circumstances unintegrated oncornavirus DNA can be demonstrated early after infection and that fully transformed cells have one or more copies of integrated virus- specific DNA. MATERIALS AND METHODS Viruses and Cells. BALB/c 3T3 cells and 3T3 cells trans- formed by the B77 strain of avian sarcoma virus (B77/3T3) were described (8). XC cells, derived from a tumor induced in a rat by the Prague strain of RSV (9), were kindly provided by Dr. Jay Levy. Normal Pekin duck cells prepared from 12- to 14day embryonated eggs (purchased from Reichardt Duck Farm, Petaluma, Calif.) and RSV-transformed duck cells were grown in Medium 199 (Grand Island Biologicals Inc.) supplemented with 10% tryptose phosphate broth, 570 calf serum, and 1% heat-in-activated chick serum. Prague strains (subgroups R and C) of RSV were grown in gs- chick-embryo fibroblasts. '077 strain of avian sarcoma virus was generally grown in cultures of chick-embryo cells not tested for gs antigen. B77 virus was also grown in gs- chick cells after rescue from B77-transformed 3T3 cells by cultivation with chick cells. Short-Term Infections. Virus-containing medium from RSV- transformed chick-cell cultures was clarified by low-speed centrifugation (2000 rpm for 10 min), and 2-4 ml was added to each of several petri dishes (100 mm in diameter) con- taining about 3 X lo6 normal duck or 3T3 cells. Polybrene (Aldrich) was present at 2 pg/ml, and multiplicities of infec- tion varied from 0.1 to 5 focus-forming units (assayed with chick cells) per cell. After incubation at 38" for defined periods, the medium was removed by suction, and the cells were scraped into 4 ml of "DNA buffer" [containing 20 mM Tris.HCI (pH 8.0)-IO mM EDTA-O.1 M NaCl], collected by centrifugation (2000 rpm, 10 min), and resuspended in DKA buffer at 5 to 10 X 1W cells per ml. DNA Purification. The technique has been described (8) and was designed to avoid loss of nonintegrated viral DNA and preserve high-molecular-weight DNA. In brief, cells were lysed with 0.5% Na dodecyl sulfate, incubated for at least 1 hr at 37" with 500 pg/ml of Pronase (digested for 2 hr at 3067 3068 Microbiology: Varmus et al. Proe. Nut. Ad. Sci. USA 70 (197.9) AFTER DENATUIAIION @ AFlEI INCUIAlION llowc,lI ~:KAi!&ENCES AFILR CENTRlFUGATION -UNIQUE CELL SEQUENCES - RSV3PECIFIC SEQUENCES -LAMBDA PHAGE DNA Fro. 1. Preparation of network DNA from unsheared cellular DNA. 37'), and gently extracted twice with phenol at room tem- perature. Two volumes of ethanol were added and, after 1-2 hr at -20°, the precipitated nucleic acids were centrifuged at 2000 rpm for 20 min, suspended in 10 mhf Tris.HC1 (pH 7.4)-10 mM EDTA, and treated for at least 3 hr at 37' with 100 wg/ml of pancreatic ribonuclease (Worthington; boiled for 10 min to inactivate DNase). Two phenol extractions at room temperature were followed by extensive dialysis against 15 mM NaCl-1.5 mM Na-citrate. After determination of absorbance at 260 and 280 nm, a portion of the DNA was sheared at 50,000 lbelinch' for assay of virus-specific DNA and the remainder was used for preparation of networks. Network Preparation. Unsheared cellular DNA in 15 mM NaCl-1.5 mM Na-citrate was denatured by heating at 100' for 3 min, then incubated at 2-4 A units in a 68' bath in the pres- ence of 0.6 M NaCl for 1 hr (Fig. 1). The solutions were chilled to 4' and centrifuged for 15 min at 40,000 rpm in a Spinco fixed-angle 40 rotor at 4'. The supernatant was removed and the visible pellet was vigorously suspended in 15 mM NaCl- 1.5 mM Na-citrate. The amount of DNA entering networks was determined by reading the Ala of the supernatant and of the resuspended DNA pellet. These fractions were then sheared at 50,OOO Ibs/inchs, precipitated with ethanol, and FRAW Fro. 2. Alkaline sucrose gradient of unsheared DNA from XC cells. 100 pg of DNA extracted from XC cella was mixed with ['HIDNA from lambda bacteriophage, incubated at 37" for 15 min in 0.6 M NaOH, and layered onto a 520% sucme gradient containing 0.9 M NaOH-1 M NaC1-IO mM EDTA. After centrifugation at 64,000 rpm for 75 rnin at 15O, fractions were collected, measured for absorbance at 260 nm (o), and precipitated with trichloroacetic acid for determination of radioactivity (0). assayed for FtSV-specific sequences. Occasionally, sheared supernatant DNA was freed of oligonucleotides by passage over a Sephadex G-50 column equilibrated with 0.0 M NaCl- 10 mM EDTA-1 mM Tris - HC1 (pH 7.4). Assay for RSV-Specijic DNA. We have described our proce- dure for determining the number of copies per diploid cell of RSV-specific DNA (5, 8, 10). The method, originally devised by Gelb et al. for detection of SV40 DNA (ll), is based upon the ability of virus-specific sequences present in unlabeled cell DNA to accelerate the reassociation of labeled, double- stranded, virus-specxc DNA synthesieed by RSV poly- merase. Reassociation reactions were performed in 100 pl containing heat-denatured, 'H-labeled polymerase product; denatured, sheared cell DNA; and 0.4 M phosphate buffer (equimolar NaHtPOd and NkHPOS. Mixtures were incubated at 68' for up to 80 hr and samples were periodically removed into 1OmM phosphate buffer for analysisof secondary structure by elution from hydroxyapatite (Biorad). Raw data were cor- rected according to the behavior of single-and doublestranded standards and plotted against Cat for the labeled DNA. Re- duction in Gtl/, caused by the unlabeled cell DNA is propor- tional to the amount of virus-specific DNA per cell. Determi- nation of the number of complements of labeled virus-specific sequences and of diploid-cell genomes in each reaction mix- ture permits calculation of the copy number for those viral sequences per diploid cell. For example, if a reaction mixture containing one set of labeled viral sequences per genomic com- plement of cell DNA demonstrates a reduction of the Cotl,r by half, the doubling of the rate indiestea a doubling of the concentration of relevant sequences, or one copy per diploid cell. The accuracy of copy numbers is infiuenced by the mod- erate heterogeneity of polymerase products, by determination of the concentration of labeled DNA from its specific activity, and by uncertainties involved in estimating sequence com- plexity of polymerase products from their reassociation kinetics (8, 12). In addition, redundancy or absence of a minor ck of viral sequences in the cell may be overlooked with a technique that measures principally the Cat,/,; however, no data in support of this possibility have been obtained. cot (rocwc/W Fro. 3. Integration of Rsy DNA in XC cella. Reassociation kinetics of slowly reassociating ['HIDNA prepared with B77 virus polymerase (3.8 ng/ml, specific activity 5ooo cpm/ng) were followed during incubation in the presence of &-thymus DNA (A, Ch/, = 1 X IO-' mol-sec/liter), unfractionated'XC DNA (m, Ch/, = 5 X lo-'), networks formed with XC DNA (0, Coti/, = 2.8 X lo-'), or DNA remaining in the supernatant after formation of networks'with XC DNA (0, Ch/, = 2.8 X lo-'). (A) 3.1 mg/d of cell DNA; (B) 4.0 mg/ml of cell DNA. Reactions in B were correspondingly faster in the presence of XC DNA. Experiments not shown here demonstrate simple second-order kinetics throughout annealing in the presence of XC DNA. Proc. Nat. Acad. Sei. USA 70 (1973) Integration of RSV DNA 3069 In experiments reported here, all assays were performed with the slowly reassociating fraction of doublestranded product synthesized by B77 virus polymerase (slowly re- associating DNA) (8, 12). The Cot,/, of this fraction is about 1 x 10-2 mol-sec/fter, and 1040% of its reassociation oc- curs over two logarithmic units of Cot. On the basis of com- parison with DNAs of known composition, we estimate the complexity of this fraction to be about 6 X 1od daltons and thus representative of at least 30% of the 705 RNA genome of RSV (8, 12). Because of our unpublished evidence that shared sequences comprise at least 85% of 705 RNAs and the slowly reassociating DNAs of B77, Schmidt-Ruppin, and Prague strains of RSV, we have used B77 slowly reassociating DNA in all experiments reported here. Detmminalion of Size of Cell DNA in Alkaline Sucrose Gradients. Cell DNA was denatured in 0.6 M NaOH at 37" for 15 min and layered on top of a 5-20'35 alkaline sucrose gradient (containing 0.9 M NaOH-1 M NaCl-10 mM EDTA). Sedimentation values were determined in relation to lambda bacteriophage DNA or SV40 DNA kindly provided by Dr. H. Boyer. RESULTS Preparation of DNA Networks. Unsheared mammalian DNA prepared as outlined in MeW has a major high-molecular- weight component sedimenting at 80-1 10 S in alkaline sucrose gradients (Fig. 2). This size corresponds to a molecular weight of about 30 to 50 X lod of single-stranded DNA. Similarly purified DNA from duck cells sediments at 40-60 S (data not shown). In accord with an earlier report (5), we found that generally 7595% of DNA prepared from mammalian cells participates in network formation, whereas 60-7574, of duck- cell DNA forms networks. The variations for any one kind of DNA in the fraction forming networks are presumably due in part to random nicking of the DNA with production of frag- ments containing insufficient reiterated sequences. In addi- tion, viscous preparations of high-molecular-weight DNA often retain appreciable amounts of oligonucleotides incapable of reassociating. These are apparent in alkaline sucrose gra- dients (Fig. 2) and can be considerably reduced by dialyzing the DNA against 150 mM NaC1-15 mM Na-citrate before network formation or removed at a later step by passing the fraction of DNA that does not form networks (supernatant DNA) through a Sephadex G-50 column. The relative reduction in the fraction of duck DNA- forming networks may also be iduenced by the amount or distribution of repeated sequence DNA in the avian genome. Although estimates of repeated sequence fractions are ap proximate and readily affected by reaction conditions, com- parison of published values for mammalian and avian genomes suggests that avian cells may contain as little as one-third as much reiterated DNA as mammalian cells (13). In general, we observed about 2045% of chick or duck DNA reassociating at low Cot values (under 100 mol-sec/liter) at which 40% of mammalian DNA has reassociated, supporting the possi- bility that less extensive network formation with duck DNA may be due in part to a decrease in content of repeated se- quence DNA. We investigated the possibility that networks of high- molecular-weight DNA might trap unintegrated viral DNA and cause it to appear in the pellet after centrifugation. Labeled DNA extracted from lambda bacteriophage (molec- ular weight 30 X lo6) or SV40 virus (form 11, molecular weight 3 X lo6) was added to unsheared DNA from B77/3T3 cells and networks were prepared. Although over 80% of the cell DNA (as measured by absorbance) was found in the pelleted network fraction, 8540% of either species of viral DNA remained in the supernatant (Fig. 1). Therefore, the cell DNA remaining in the supernatant fraction was about 50-fold richer in viral DNA than was the network fraction. Since even a %fold enrichment of the supernatant fraction is detectable with the reassociation kinetics assay, it is clear that these minor degrees of trapping of viral DNA cannot account for results observed with RSV-infected cell DNA. Inkgrated Viral DNA in Stably Transformed Cells. We used the network technique to assess integration of RSV-specific DNA into the genome of three types of cells transformed by RSV: (1) XC cells, widely-used progeny of a tumor produced in a rat line with Prague strain of RSV (9): (2) B77/3T3 cells, derived from a single clone of BALB/c 3T3 cells trans- formed by the B77 virus (8); and (9) duckembryo fibroblasts transformed by infection with Prague C strain of RSV. In contrast with other lines of RSV-transformed mamma- lian cells we have studied, the XC cell line has a large number of copies of DNA homologous to the sequences represented in the slowly reassociating fraction of RSV polymerase product. Sheared DNA from these cells markedly accelerates the reassociation of labeled slowly reassociating DNA (Fig. 3A); the calculated copy number is 20 per diploid cell. When the preparation of unsheared XC cell DNA analyzed in the alkaline sucrose gradient in Fig. 2 was used to form networks, both the network DNA and the 25% of the DNA remaining in the supernatant possessed the same capacity as the total cell DNA to influence reassociation of slowly reassociating DNA (Fig. 3B). This result demonstrates that most, if not all, of the viral DNA present in the XC cells is covalently integrated into strands of cell DNA containing repeated sequences. It is probable that viral DNA remaining in the supernatant is integrated into strands of DNA that failed to join the networks on the basis of size or sequence composi- tion. However, we cannot exclude the possibility that a small fraction of viral DNA is not integrated. Similar results have been obtained with the B77/3T3 cell line. These cells, like most of the other RSV-transformed mouse, rat, or hamster cells we have studied (5, 8), contain one to two copies of RSV-specific DNA sequences per diploid cell. Network and supernatant DNA from these cells are equally efficient in accelerating the reassociation of RSV slowly reassociating DNA (Fig. 4). Their effect is similar to that of unfractionated DNA' upon the reassociation of slowly reassociating DNA (5, 8), and the supernatant fraction shows no enrichment, as would be expected if unintegrated viral DNA were present. As for XC cells, we interpret these results to mean that most or all of the RSV-specific DNA is cova- lently integrated in B77/3T3 cells, and that the virus-specific DNA in the supernatant fraction is linked to randomly broken strands of cell DNA. Duck cells, unlike other permissive avian cells we have tested (IO), are free of endogenous RSV-specific nucleotide sequences, as tested by reassociation kinetics (Fig. 5) and by hybridization of large excesses of duck DNA with labeled RSV 70s RNA (unpublished observations). -4fter transformation by B77 virus or by Prague C strain of RSV, duck cells contain four to six copies of RSV DNA se- 3070 Microbiology: Varmus et al. I Proc. Nal. Acad. Sn'. USA 70 (197.9) FIG. 4. Integration of RSV DNA in B77-transformed 3T3 cells. B77 slowly reassociating DNA (4.7 ng/ml) was reassociated in the presence of 3.1 mg/ml of calf-thymus DNA (A) or DNA from network (0) and supernatant (0) fractions prepared from B77/3T3 cell DNA. Ct.tl/, is reduced from 1 X lo-* to 5 X 10-3 mol-sec/liter by DNA from B77/3T3 cells. Over SO% of the cell DNA entered networks. quences per diploid cell (14). Fig. 5 displays the equal effects of unfractionated, network, and supernatant DNA from RSV- transformed duck cells upon the reassociation of slowly reassociating DNA, consistent with the presence of four copies of RSV sequences per diploid cell. Since only two-thirds of the duck DNA participates in network formation, we can conclude with certainty only that at least two-thirds of the viral DNA is covalently linked to cell DNA. It seems likely, however, that the remainder is integrated into strands in- capable of forming networks. Uninlegrated DNA in Recently Znfecbd Cells. The ease of testing for integration of viral DNA with the network assay facilitates study of the kinetics of integration. We therefore measured the appearance of newly synthesized virus-specific DNA in networks prepared from freshly infected cells. These experiment9 further validate the network test as a measure of integrated DNA, since they demonstrate that viral DNA may be present in a nonintegrated state soon after infection. This situation is portrayed most dramatically in 3T3 cells 12 hr after infection with RSV (Fig. 6). Despite the relatively low efficiency with which mammalian cells are transformed by RSV (15), we discovered that readily detectable levels of RSV DNA may be observed in 3T3 cells within 12 hr after infec- tion with RSV rescued from RSV-transformed mammalian cells (14). No DNA synthesis is observed after infection by I I C.t(mOlaec/Hter) 10 = 10-2 to-= 10- I FIG. 6. Unintegrated RSV DNA in acutely infected 3T3 cells. Growing cultures of BALB/c 3T3 cells were infected with B77 virus rescued from B77-transformed 3T3 cells. 12 hr later, the DNA was extracted and a portion waa used to prepare networks. B77 slowly reassociating DNA (3.6 ng/ml) was then reassociated in the presence of (A) unfractionated DNA (m, 4 mg/ml); (B) network DNA (e, 4 mg/ml), DNA remaining in the supernatant after network preparation (0, 3.1 mg/ml); (A and B) calf-thymus DNA (A, 4 mg/ml). Unfractionated 3T3 DNA lowers the Cotl/, from 1.0 X lo-* to 3.5 X lo-* mol-sec/ liter; supernatant DNA reduces the Cotl/2 from 1.3 X lo-* to 1.7 X lo-) mol-secfliter. various RSV strains grown only in chick cells (14). This phenomenon is probably related to the increased efficiency of transformation of rat cells by B77 virus observed with virus rescued from transformed rat cells (15). In the experiment in Fig. 6, B77 virus rescued from B77-trans- formed 3T3 cells was added to growing cultures of 3T3 cells at a multiplicity of about 1 focus-forming particle (as assayed on chick cells) per 3T3 cell. After 12 hr, cell DNA was ex- tracted, a portion was sheared and tested for its content of RSV DNA (Fig. 6A), and the remainder was subjected to net- work formation. About 80% of the DNA formed networks. The network and supernatant DNAs were then also sheared and assayed for RSV DNA (Fig. 6B). The acceleration of reassociation observed in Fig. 6A indicates that an average of about 0.8 copies of RSV slowly reassociating DNA sequences are synthesized per diploid cell within 12 hr after infection. Since the efficiency of transformation of mammalian cells by RSV is low [10-t10-6 relative to transformation of avian cells (15)], and the number of RSV DNA copies in trans- formed clones of mammalian cells is only one to two (5, 8), it is likely that a large percentage of cells are infected (as seen I lo-' 10-3 10-1 CJ md-=c/w FIQ. 7. Unintegrated DNA in acutely infected duck cells. Growing secondary cultures of duck-embryo fibroblasts were infected for 10 hr with Prague C strain of RSV. The influence of 2 mg/ml of unfractionated DNA (m, A), network DNA (0, B), and supernatant DNA (0, B) upon the reassociation of B77 slowly reassociating DNA (5.1 ng/ml) was then determined. Normal duck-embryo DNA (A) served as a control. Unfractionated DNA reduces the Cotl/, from 1.2 X lo-' to 6 X mol-sec/liter; the Cotl/, is lowered from 1 X lo-* to 8 X lo-* by network DNA and to 2 X lo-' mol-sec/liter by supernatant DNA. Proc. Nat. Acad. Sci. USA 70 (1973) Integration of RSV DNA 3071 by synthesis of RSV DNA) but not transformed. Detection of RSV DNA in clones of infected nontransformed cells will be required to confirm this hypothesis. Networks prepared from this DNA are devoid of detectable RSV-specific sequences (Fig. 6B). The supernatant DNA however, contains 4.1 copies of slowly reassoriating DNA per weight of DNA from one diploid cell. The supernatant DNA is, therefore, &fold enriched for RSV DNA compared to the unfractionated DNA. The degree of enrichment is consistent with the frac- tion of cell DNA (20%) remaining in the supernatant. We conclude that little, if any, RSV DNA is integrated 12 hr after infection of 3T3 cells. This conclusion is strengthened by the additional observations that integration of RSV DNA occurs in these cells (as shown by the appearance of RSV DNA in network fractions) within 22 hr after infection and that the viral DNA persists in an integrated state for at least six subse- quent generations (14). Similar results have been obtained with infected duck cells. Using either B77 virus or Prague C strain of RSV, we can de- tect virus-specific DNA within 3 hr after infection (14). No viral DNA is found 16 hr after infection with Prague B strain of RSV to which duck cells are not susceptible (14, 16). Using the network assay to follow integration of viral DNA, we ob- serve increasing amounts of RSV DNA present in networks from 6-24 hr after infection at which point the networks con- tain the four to six copies found in fully transformed cultures (Fig. 5). 10 hr after infection of normal duckembryo fibro- blasts with Prague C strain of RSV, about 0.8 copies of slowly reassociating DNA sequences have been synthesized per diploid cell (Fig. 7A). 0.25 Copies have been integrated, as computed from the experiment in Fig. 7B, and the super- natant fraction is over %fold enriched for RSV DNA in com- parison with unfractionated DNA. These data indicate that about onethird of the RSV DNA synthesized during the first 10 hr of infection is integrated into the duck genome. DISCUSSION The ease, efficiency, and stringency of the network technique recommend it for the study of integration of both DNA and RNA tumor virus genomes. The degree of trapping of non- integrated DNA by the networks is small, as shown by trapping controls with labeled viral DNAs and by the detec- tion of unintegrated viral DNA early after infection (Figs. 6 and 7). The single potential limitation of the technique resides in the possibility that viral DNA might, under wme circum- stances, be integrated specifically into an uncommon region of the cell DNA that was devoid of reiterated sequences and did not form networks. In this case, integrated viral DNA would not be present in the networks, and the supernatant DNA would be enriched for viral sequences. However, in practice we have always found network DNA from transformed cells to be as rich in viral sequences as the unfractionated DNA, and the supernatant DNA to be no further enriched (Figs. 3-5). It seems probable that the supernatant DNA is composed prin- cipally of a random selection of the cell genome that has been sufficiently nicked during extraction to prevent entry into networks. In this report we illustrated the usefulness of the network assay for integrated viral DNA in mammalian and duck cells infected with Rous sarcoma virus. In contrast to studies of RSV-specific DNA in the natural host for RSV (chick cells), these experiments are not complicated by the presenceof endog- enous RSV genetic information (10). Consequently, the viral DNA detected appears in the cell after infection, presumably as a product of the intracellular activity of virus-sssociated RNAdirected DNA polymerase. The assay, therefore, con- stitutes an assay for in vivo polymerase activity and may per- mit detailed analysis of the mechanism of RNAdirected DNA synthesis in the host cell. Although the experiments reported here establish that RSV- specific DNA is integrated in transformed duck and mamma- lian cells and that the kinetics of integration can be studied in detail, the extent of representation of the viral genome in our hybridization probe, slowly reassociating DNA, may be as little as 30% (17). Therefore, we cannot claim that the entire viral genome is integrated as DNA in the host cells we have studied. Moreover, we cannot describe the location or specific- ity of the integration sites. We conclude that there is now substantial support for the notion that the genomes of both RNA and DNA tumor viruses are integrated into host-cell DNA, but the sites and mechanism for integration remain un- known. Dr. Robin A. Weisa suggested the duck cell as a permissive host likely to be free of endogenous RSV-specific DNA. Ms. Suzanne Heasley and R. Howard provided excellent technical assistance. 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