A specific viral DNA sequence is stably integrated in herpesvirus oncogenically transformed cells

Cell, Vol. 32, 569-578,

February

1983,

Copyright

0 1983

by MIT

A Specific Viral DNA Sequence Is Stably Integrated in Herpesvirus Oncogenically Transformed Cells Robin A. Robinson* and Dennis Department of Microbiology University of Mississippi Medical Jackson, Mississippi 39216

J. O’Callaghan Center

Summary The integration patterns of viral DNA sequences in three hamster embryo cell lines independently derived by transformation with equine herpesvirus type 1 (EHV-1) have been investigated by DNA blot hybridization analyses for the restriction enzymes Eco RI, Bgl II, Xba I and Barn HI with 32P-labeled selected DNAs from a collection of cloned EHV-1 restriction enzyme fragments as probes. These EHV-1 -transformed cell lines contained subgenomic portions of the viral genome in an integrated state at multiple sites in the host genome. At least one copy of a viral DNA sequence mapping colinearly from 0.32 to 0.38 map units within the EHV-1 genome was common among these three EHV-1 transformed cell lines. The 0.32-0.38 viral DNA sequence was maintained stably even after 125 cell passages, whereas sequences from other positions in the EHV-1 genome were lost progressively during continued cell passage. The significance of the findings that these oncogenically transformed cell lines harbor a specific region of the EHV-1 genome is discussed with regard to stable maintenance of the oncogenically transformed state. Introduction Oncogenic transformation of permissive and nonpermissive mammalian cells by herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and by equine herpesvirus type 1 (EHV-1) has been documented, and several of the biological properties of these cell lines have been reported (see Rapp, 1980; see O’Callaghan et al., 1981, 1982; Robinson et al., 1981). The cytolytic properties of these viruses during infection of permissive cells has necessitated the circumvention of the cytolytic cycle by various means to achieve successful transformation; however, oncogenic transformation of permissive hamster embryo cells can be mediated by preparations of live, infectious EHV-1 enriched for defective interfering particles (Robinson et al., 1980b; O’Callaghan et al., 1981, 1982; Dauenhauer et al., 1982). In addition, several workers have succeeded in morphologically transforming mammalian cells with sheared herpesvirus DNA (Wilkie et al., 1974; Jariwalla et al., 1980) and with restriction endonuclease fragments of herpesvi* Present address: Department of Microbiology, New York, Stony Brook, New York 11794.

State

University

of

rus DNA (Camacho and Spear, 1978; Reyes et al., 1979; Jariwalla et al., 1980; Galloway and McDougall, 1981). Attempts to detect viral DNA sequences in UV-irradiated-herpesvirus-transformed and tumor cells have relied primarily on DNA-DNA reassociation analyses, and these studies have demonstrated the presence of several copies of subgenomic viral sequences in these cells (Frenkel et al., 1976; Minson et al., 1976; Robinson et al., 1980a, 1980b). Galloway et al. (1980) using 32P-labeled HSV-2 DNA restriction endonuclease fragments as probes in liquid hybridization studies, identified two major sets of viral sequences (0.21 to 0.33 and 0.60 to 0.65 in the HSV-2 genome) present in HSV-2-transformed hamster cells. Results from transfection studies with HSV-1 restriction endonuclease fragments indicated that the morphological transforming activity of this virus is located on a fragment mapping between 0.30 and 0.45 on the HSV-1 genome (Camacho and Spear, 1978; Reyes et al., 1979). Similar studies with HSV-2 DNA restriction endonuclease fragments have yielded quite different results. Hayward and coworkers (Reyes et al., 1979) have reported morphological transforming activity to reside at 0.584 to 0.628 map units on the HSV-2 (strain 333) genome. These results have been corroborated by Galloway and McDougall (1981) but other workers using restriction endonuclease fragments of HSV-2 DNA (strains S-l and 333) have described neoplastic transformation of hamster embryo cells with a viral DNA fragment bearing map coordinates 0.43 to 0.58 (Jariwalla et al., 1980). The identification and structural arrangement of viral DNA sequences integrated into host chromosomes can be discerned by Southern blot hybridization techniques. Moreover, the availability of herpesvirus DNA restriction endonuclease fragments cloned into recombinant plasmid vectors provides a source of probes in blot hybridization experiments that can detect accurately a single copy of subgenomic herpesvirus sequences present in host cells (Robinson et al., 1981 b). By using this technique with a collection of EHV-1 restriction fragment probes, we have been able to determine the genomic arrangement of herpesvirus DNA sequences in hamster cells oncogenitally transformed by UV-irradiated EHV-1. In this paper, we demonstrate that in EHV-1 -transformed cells, subgenomic viral sequences are covalently linked to cellular sequences and thus are probably integrated into host cell chromosomes. Integration of multiple copies of-these viral sequences appears to occur at different sites within the host genome. Construction of restriction enzyme maps of the integrated viral sequences and the adjoining cellular sequences has revealed that a specific viral sequence (0.32 to 0.38) is common to all EHV-l-transformed cell lines examined to date.

Cell 570

Results Reconstruction Experiments and Rationale of Blot Hybridization Approach The size and the complexity of intact herpesvirus genomes preclude their use as hybridization probes to determine the arrangement of viral DNA sequences within host DNA in blot hybridization approaches as described for SV40-transformed cells by Ketner and Kelly (1976) and Botchan et al. (1976). DNA-DNA reassociation analyses of some of the EHV-1 transformed cell lines indicated that these cells harbor subgenomic viral sequences that represent 2.2% to 20.9% of the 92 megadalton (Md) genome (Robinson et al., 1980a). Therefore, reconstruction experiments were designed to determine the minimal size of an EHV-1 DNA probe that could detect less than a single copy per cell of a viral DNA sequence with a molecular weight of 2.0 Md by blot hybridization analysis with standard radiolabeling and blotting methods (see Experimental Procedures). LSH hamster DNA and selected EHV-1 restriction fragments that had been cloned into the plasmid vector pBR322 (Robinson et al., 1981 b) and were approximately 2.0 Md in size were mixed and digested to completion with the restriction enzymes Eco RI or Bgl II as described in Experimental Procedures. The restriction digests were fractionated by electrophoresis through agarose gels, visualized by ultraviolet light after ethidium bromide staining to ensure complete enzyme digestion, alkali denatured in situ, neutralized and transferred onto nitrocellulose filters (Southern, 1975). EHV-1 DNA restriction fragments that varied in molecular weight (6.3 to 24.5 Md) and that contained all of the sequences in the viral DNA fragment which was mixed with cell DNA were labeled by nick translation with CY3’P-deoxynucleotides and were hybridized to individual blots as given in the legend to Figure 1. As seen in Figure 1, viral DNA sequences of 2 Md size could be detected readily with 32P-labeled viral probes with molecular weights of 6.3 Md (lane l), 8.3 Md (lane 2) and 13.7 Md (lane 3) after only a four day autoradiographic exposure. Under these conditions for hybridization, the 2 Md viral sequence was not detected immediately by viral DNA probes with molecular weights progressively greater than 13.7 Md (lanes 4 to 7); however, if the exposure period for autoradiography was increased to 10 or more days, the 2 Md viral sequence could be detected with the 18.6 Md and 24.5 Md probes. The results from these reconstruction experiments indicated that EHV-1 DNA probes with molecular weights of less than 14 Md would readily detect a 2 Md viral sequence present in amounts of only 5 pg within 25 pg of cellular DNA. Since all LSEH-transformed and tumor cells had been shown to contain only a few copies per cell of subgenomic viral DNA sequences having complexities of less than 21% of the viral genome (Robinson et al.,

Figure

1. Reconstruction

Experiment

Various EHV-1 DNA restriction fragments (5 pg) of similar molecular weight (-2.0 Md) were mixed with 25 fig of a Eco RI digest of normal LSH hamster DNA, fractionated by electrophoresis through 0.7% agarose gels at 1.5 V/cm for 48 hrs. stained with ethidium bromide (0.5gg/ml), visualized by UV light source, transferred to nitrocellulose filters by the method of Southern (1975) and hybridized with selected 32P-EHV-1 DNA restriction fragments under conditions described in Experimental Procedures. The viral DNA fragments contained in the viral-cellular DNA mixture and the corresponding viral DNA probes were (lane 1) Xba I L, 2.5 Md; 32P-Bgl II F, 6.3 Md: (lane 2) Xba I 0, 1.7 Md; Bgl II C. 8.3 Md; (lane 3) Xba I 0, 1.7 Md; ‘*P-Barn HI A, 13.7 Md; (lane 4) Bgl II M. 1.9 Md; 32P-Xba I A, 18.6 Md; (lane 5) Xba I N, 2.1 Md; 32P-Bgl II A, 24.5 Md; (lane 6) Xba I N, 2.1 Md; 3zPgenomic DNA, 92 Md; (lane 7) 32P-Eco RI fragments of the genome as markers.

1980a), and since the above reconstruction experiments indicated that restriction fragments with a size ~14 Md could be used as probes to detect these viral sequences, a collection of EHV-1 DNA restriction fragments, as listed in Table 1, was selected from our library of cloned EHV-1 restriction fragments. Their selection was based on the following lines of reasoning. First, these EHV-1 restriction fragments were cloned into plasmid vectors, and thus represented highly purified viral sequences. Second, the coding capacities of many of these restriction fragments overlapped, and thus their use allowed a higher degree of mapping refinement. Third, the sum coding capacities of these restriction fragments equaled the entire EHV-1 genome, and thus provided adequate screening for all possible viral sequences. Fourth, the size of all restriction fragments was well within the limits demarcated in the reconstruction experiments de-

Herpesvirus 571

Oncogenic

Table 1. Cloned EHV-1 Hybridizations to Detect Transformed Ceils Fragmenta

DNA Sequences

Restriction Fragments Used in Blot Viral Sequences Present in EHV-I-

MW x IO6

Kilobases

Map Units

1. Barn HI-E

6.2

9.3

0.00-0.06

2. Barn HI-H

4.3

6.5

0.07-0.11

3.

Bgl II-D

7.3

11 .o

0.08-0.16

4.

Barn HI-N

3.2

4.8

0.15-0.19

5.

Barn HI-D

7.2

10.8

0.1 g-0.27

6. Eco RI-F

8.2

12.3

0.21-0.30

7.

Barn HI-F

5.4

8.1

0.27-0.32

8.

Barn HI-B

9.8

14.7

0.32-0.43

9.

Bgl II-B

9.2

13.8

0.38-0.48

10.

Barn HI-A

13.7

20.6

0.48-0.63

11.

Xba I-Jb

2.9

4.4

0.52-0.55

12.

Bgl II-C

8.3

12.5

0.57-0.66

13.

Bgl II-L

2.4

3.6

0.66-0.68

14.

Barn HI-K

3.6

5.4

0.67-0.71

15.

Bgl II-J

3.8

5.7

0.69-0.73

16.

Eco RI-B

12.4

18.6

0.73-0.87

17.

Eco RI-M

3.4

5.1

18.

Eco RI-D

9.1

13.7

0.86-0.90 0.90-l

.oo

EHV-I DNA restriction fragments were cloned into the plasmid pBR322 and propogated in E. coli strains HBl 01 and C600 SF8 as described in Experimental Procedures under P2 containment conditions described by the recent NIH Guidelines for Recombinant DNA Research. A more detailed description of the construction and the properties of these recombinant DNA molecules is given in Robinson et al., 1981 b. a These restriction fragments were 32P-labeled by nick translation (Rigby et al., 1977) and denatured at 100°C for IO min in a 0.3 M NaOH solution followed by neutralization prior to use in hybridizations. b The source of this fragment was purified bands resolved by electrophoresis in agarose gels. The procedure used for removing fragments from gels was as described in Experimental Procedures.

scribed above, and thus ensured the detection of viral sequences in small amounts within these ceils. Finally, the number of restriction sites within these fragments for the four restriction endonucleases used to digest cellular DNAs permitted us to map the arrangement of EHV-1 -specific DNA sequences in virus-transformed cells. The forthcoming experiments used all 18 fragments listed in Table 1 as probes to determine the presence as well as the arrangement of herpesvirus DNA sequences within the host genomes of the EHV-1 -transformed cells. Restriction Fragments of LSEH-Transformed Hamster DNA Containing EHV-1 Specific Sequences By reassociation of an EHV-1 specific genomic DNA probe in the presence of molar excesses of cellular DNA, we estimated that hamster cells transformed by

UV-irradiated EHV-1 contain approximately one to two copies of subgenomic viral sequences per haploid genome (Robinson et al., 1980a). Analysis of the kinetics and the extent of reassociation does not discern which viral genes are present in these cells, and whether the viral copies exist as a colinear entity comprised of repeating subunits or as random viral sequences dispersed widely throughout the hamster genome. Our approach to map viral genes associated with oncogenesis has been to digest transformed cell DNA with various restriction endonucleases and to identify DNA fragments containing EHV-1 -specific sequences by molecular blot hybridization. Using probes with the specificity of EHV-1 restriction fragments having molecular weights less than 14 Md (Figure 1), with specific activities of greater than 1 x lo8 cpm/ pg, and with the selectivity of EHV-1 restriction fragments cloned into plasmid vectors (Table l), we have identified specific EHV-1 DNA sequences in these cells and have elucidated their distribution in the hamster genome. The restriction enzymes chosen for this study were among those whose position within cleavage maps of the EHV-1 genome had been discerned previously (Figure 2; Henry et al., 1981). These four enzymes recognize multiple, one, or no sites in the respective restriction fragments used as probes, and the number of sites and their map location in these probes are presented in Table 1. Digestion of high-molecular-weight DNA from LSEH3, -4, or -8 transformed hamster cell lines with Eco RI generated two, four and two fragments, respectively, all of which annealed with the Barn HI-B (0.32-0.43) probe (Figure 3, lane 8); the molecular weights of these fragments differed from one cell line to another. Viral DNA sequences were detected with the Barn HI-F (0.27-0.32) probe among the LSEH-4 and -8 cell DNA Eco RI fragments, but not among the LSEH-3 DNA fragments (Figure 3, lane 7). The molecular weights (12.4 and 10.1 Md) of the fragments detected in the LSEH-8 cells with the Barn HI-B and -F probes were identical, and thus these LSEH-8 Eco RI bands probably represented the same fragments and contained viral sequences that map at the junction of these probes (0.32 map units). The greater number of fragments detected in the LSEH-4 cells and the greater number of probes sharing homology with the LSEH-4 cell DNA indicated that the complexity of the viral sequences in this cell line was greater, as expected, than that of viral sequences present in LSEH3 or -8 cells. The distribution pattern of virus-specific fragments detected with this collection of viral probes varied among these three transformed cell lines, since the molecular weight and the number of bands resolved with each viral probe differed from one cell line to another. These results suggested that the subgenomic fragments of the EHV-1 genome were not present at the same site(s) within the hamster genome. The virus-specific fragments detected by several of

Cell 572

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Map Unit6 I : : 0.0 0.1

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7.2

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2. Eco RI, Bgl II, Xba I and Barn HI Restriction

These

maps are from Henry

6.i

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Cleavage

and O’Callaghan

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13.7

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et al. (1981)

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DNA

et al. (1981).

the viral probes had molecular weights in excess of those predicted by cleavage of viral DNA only, and were concluded to contain sites of viral integration or of rearranged viral sequences. To delineate more precisely the boundaries of the viral sequences detected in these cells, we digested DNAs from these three transformed hamster cell lines with Bgl II, Xba I or Barn HI, and hybridized them to the same set of viral probes (Figures 4, 5, 6). Digestion of these cell DNAs with Bgl II yielded results similar to those of the Eco RI digests, as viral sequences were detected with the same viral probes in both experiments. However, the molecular weights of the detected fragments for the same cell line differed between the Eco RI and the Bgl II digests, and thus indicated that a specific and unique distribution pattern of viral DNA sequences was exhibited in each of these cell lines. The results of hybridizations of Bgl II digests of LSEH-4 cell DNA with the Barn HI-D, Eco RI-F and Barn HI-F viral probes (Figure 4, lanes 5, 6, and 7) suggested that copies of viral sequences mapping from 0.28 to 0.38 map units were organized as two separate insertion units of single copy number and a third insertion comprised of multiple copies. There were two bases for this conclusion. First, four fragments (5.1, 4.3, 3.1 and 1.9 Md) that were expected from Bgl II cleavage of viral DNA mapping at the 0.26 to 0.43 region of the EHV-1 genome were detected by the three probes described above, and this finding was consistent with the colinearity of these viral sequences in the cell DNA. Second, the intensity

of these bands as measured by scanning autoradiographs of these hybridizations with a soft laser scanning densitometer, as outlined in the Experimental Procedures, was two to four fold greater than that demonstrated by other fragments detected in the same experiment. Since three (5.1, 4.3 and 1.9 Md) of the LSEH-4 Bgl II fragments detected with the Barn HI-F and -B probes (Figure 4, lanes 7 and 8) were predicted by cleavage of viral sequences only from the 0.27-0.44 region of the EHV-1 genome, and since three large fragments (21.4, 18.8 and 14.0 Md) also detected with the Barn HI-B fragment were considerably larger than the probe, the rightmost boundary (toward the S region terminus; see Figure 2) of each of the viral copies comprising this region mapped at approximately 0.42 map units. Consistent with this result was the finding of a 10.6 Md repeating Eco RI subunit mapping at 0.30 map units and proceeding rightward on the EHV-1 genome in LSEH-4 cell DNA (Figure 3, lanes 5-9). Bgl II digests (Figure 3, lane 8) of LSEH-3 DNA hybridized with only the Barn HI-B probe and yielded results similar to those of the Eco RI digests. Digestion of DNA from these three transformed hamster cell lines with Xba I yielded results similar to those described above, with regard to the viral DNA sequences represented in each line (Figure 5). Although no major findings were discovered to be inconsistent with the Eco RI and Bgl II results, the Xba I digests of LSEH-4 cell DNA produced fragments mapping at 0.25-0.45 that were fewer in number and

Herpesvirus 573

Oncogenic

DNA Sequences

Figure 4. Blot Hybridizations of Bgl II Digests of LSEH-8-, LSEH-4-, and LSEH-3-Transformed Cell DNA with 32P-Labeled Cloned Restriction Fragments of EHV-I DNA

Figure 3. Blot Hybridization of Eco RI Digests of LSEH-8-, LSEH-4-, and LSEH-3-Transformed Cell DNA with 32P-labeled Cloned Restriction Fragments of EHV-1 DNA Transformed cell DNA (25 pg) was digested with Eco RI, separated by electrophoresis in 0.7% agarose gels, transferred to nitrocellulose filters by a modification of the blotting method of Southern (1975), and hybridized separately to each of the EHV-I restriction fragments 32P-labeled by nick translation (listed in Table 1). The cellular fragments containing EHV-1 DNA were detected by autoradiography of hybridized filters for 4-10 days. The viral DNA probe used in each hybridization is indicated by the numeral (1 to 18) above each lane, and corresponds with the number for each cloned viral restriction fragment in Table 1. The lane designated MW contained 32P-labeled EHV-1 DNA digested with Eco RI as molecular weight markers. This figure and succeeding figures of autoradiographs are composites of the eighteen separate hybridization reactions.

Transformed as described the eighteen

cell DNA (25 pg) was digested with Bgl II and analyzed in the legend to Figure 3. The figure is a composite of separate hybridizations.

that had molecular weights much greater than had been observed previously with the other enzyme digests (Figure 5, lanes 5-S). This result was expected, since most of the ethidium-bromide-stained hamster cell DNA fragments generated by Xba I digestion migrated in gels to a distance corresponding to 20 Md or greater (unpublished observation). The results of analysis of Xba l-digested LSEH-4 cell DNA confirmed the conclusion that viral sequences mapping from 0.26 to 0.42 were integrated into the hamster genome as three separate insertions. Barn HI generated a

Cell 574

Figure 5. Blot Hybridizations of Xba I Digests of LSEH-8-, LSEH-C. and LSEH-3-Transformed Cell DNA with 32P-Labeled Cloned Restriction Fragments of EHV-1 DNA Transformed as described the eighteen

cell DNA (25 pg) was digested with Xba I and analyzed in the legend to Figure 3. The figure is a composite of separate hybridizations.

modest number of viral-specific fragments (Figure 6) that were in accordance with Eco RI, Bgl II and Xba I results for each of these three cell lines. One interesting observation supported by results of each enzyme digest was the finding of viral sequences mapping between 0.32 and 0.38 map units in all three cell lines. These viral sequences, designated as “consensus transformation sequences,” were present as two insertion elements in the LSEH-3 and -8 cell lines and as three insertions in the LSEH-4 cell line. Viral sequences representing regions of the EHV1 genome other than the consensus transformation

Figure 6. Blot Hybridizations 4-. and LSEH-3-Transformed striction Fragments of EHV-1

of Barn HI Digests of LSEH-8-, LSEHCell DNA with 32P-Labeled Cloned ReDNA

Transformed cell DNA (25 pg) was digested with Xba I and analyzed as described in the legend to Figure 3. The figure is a composite of eighteen separate hybridizations.

sequence were identified only in the LSEH-4 cell line and mapped at 0.08-0.10 (L region) and 0.79-0.82 (S region). These latter S region sequences existed as three separate insertion elements, as demonstrated by the detection of three fragments having molecular weights larger than the Eco RI-B or -D probes in several enzyme digests (Figures 4, 5, and 6; lanes 16 and 18). In all cases, fragments from normal hamster embryo tissues failed to anneal reproducibly with any of the EHV-I restriction fragment probes used in these experiments.

Herpesvirus 575

Oncogenic

DNA Sequences

m I = G m

m s 0 lz

Figure 7. Blot Hybridization of Bgl II Digests of Transformed Cell DNA from Different Passages of LSEH-4 Cells with “P-Labeled Cloned Restriction Fragments of EHV-1 DNA Twenty-five micrograms of several different passages of LSEH-4. transformed cells was digested with Bgl II and examined for viral sequences by blot hybridization as described in the legend to Figure 3, except that a limited number of 32P-labeled restriction fragments of EHV-I DNA, indicated above each set of lanes, was used. Lanes 1, 2 and 3 for each probe represent hybridizations with DNA from cell passages 25, 66 and 125, respectively, of LSEH-4-transformed cells. The figure is a composite of eight separate hybridizations

and thus would provide more instructive mapping data. Only those probes that had successfully detected viral DNA in low passages of LSEH-4 cells were used to analyze the distribution of viral and adjoining cellular sequences. The data presented in Figure 7 demonstrated that selective changes had occurred in the pattern of distribution of viral DNA sequencesnamely, those mapping at 0.08-0.16 map units in the L region and those at 0.79-0.82 map units within the S region. Viral DNA sequences mapping at 0.08-0.16 map units.were present in transformed cells at passage 25 (lane a), but were not detectable in cells of greater passage number, such as passage 66 (lane b) or passage 125 (lane c). S region sequences (0.730.87) present in low passages of LSEH-4-transformed cells were either deleted or present in amounts below the sensitivity of this technique in cells of prolonged passage (lanes b and c). Interestingly, viral DNA bordering the consensus transforming sequence, such as Eco RI-F (0.21-0.30) and Bgl II-B (0.38-0.43) were detected in cells grown to low passage (P-25, lane a) and intermediate passage (P-66, lane b), but were not found in cells of high passage (P-l 25, lane c). In contrast, the consensus transformation sequence detected by the Barn HI-F probe, Barn HI-B probe and Bgl II-B probe was retained in an integrated state in cells passaged extensively in culture. Thus it was concluded that only certain regions of the EHV-1 genome were maintained stably during oncogenic transformation of hamster embryo cells. Discussion

Effect of Prolonged Passage of Transformed Cells on the Distribution Pattern of Integrated Viral Sequences The occurrence of excision of viral sequences from transformed cells under selective pressure in animals (tumorigenesis) was suggested by results from Cot analysis, which revealed that the genetic complexity of EHV-1 sequences was reduced in LSEH tumor ceils as compared with that of the parent transformed cell (Robinson et al., 1980a). To determine whether other means of selection, such as extended passage in culture, affected the pattern of integration of viral DNA in the LSEH-transformed hamster cells, we extracted DNA from LSEH-4 cells at different passage levels (as great as 800 cell divisions), and digested it to completion with the restriction endonuclease Bgl II. LSEH-4 cells were chosen because they contained greater amounts of viral DNA (2.2 genome equivalents of 20.9% of the EHV-1 genome; Robinson et al., 1980a), and because a considerable loss of viral DNA was observed in the tumor cells (2.5 genome equivalents of 5.1% of the EHV-1 genome; Robinson et al., 1980a). Bgl II was used because there are more restriction sites within the 0.26-0.42 region of the EHV-1 genome for Bgl II than for the other enzymes,

This report represents one of the first studies to characterize the structure of integrated herpesvirus sequences involved in oncogenic transformation by direct mapping techniques. Two major conclusions from these investigations are that only a specific portion of the EHV-1 genome persists stably in host genomes throughout herpesvirus oncogenesis and that these viral sequences experience several different types of recombination events affecting their organization during transformation and subsequent passage in culture. Other conclusions supported by these studies can be summarized as follows. First, the results of these blot hybridization analyses with the DNAs of LSEH-transformed cells confirmed the findings from reassociation kinetic studies (Robinson et al., 1980a) that a limited portion of the EHV-1 genome is harbored in these cells. Second, EHV-1 -specific DNA sequences appeared to be integrated into the hamster genome as colinear tracts of subgenomic viral DNA. That EHV-1 DNA was integrated into host DNA was indicated by the findings of offsize bands that were larger than probe DNAs (Figures 3-6) and findings of fragments that map at the terminus of a viral DNA unit and have a molecular weight unpredicted by digestion with a

Cell 576

particular restriction enzyme (Figures 3-6). This inference has been substantiated by our recent isolation of several h Charon 4A recombinant phages containing both viral and cellular sequences arranged colinearly from Eco RI digests of LSEH-4-transformed cell DNA (O’Callaghan et al., in press). Third, the patterns of integration of EHV-1 -specific DNA differed among the transformed cell lines examined and for the reqtriction enzymes used, suggesting that multiple sites exist for the integration of EHV-1 DNA sequences. Fourth, the viral sequence found to be common in all cell lines examined to date mapped from 0.32 to 0.38 map units on the EHV-1 genome. This finding considered with the results of HSV-I and HSV-2 transfecti,on studies (see Introduction) suggests that a single specific or a selected few herpesvirus genes are responsible for transforming normal cells to an oncogenic state. Finally, viral sequences exclusive of the 0.320.38 sequence were not maintained stably in LSEHtransformed cells, since restriction enzyme mapping of LSEH-4 cell DNAs indicated the loss of viral sequences originating from 0.08-0.16 of the L region and 0.79-0.82 of the S region subsequent to extended passage of transformed cells (Figure 7). The nature of the mechanism for diminution’of viral sequences form virally transformed cell genomes is unknown at present. The fact that all of these EHV-l-transformed cells, even those that contain only the 0.32-0.38 DNA sequence, are oncogenic in animals suggests that this specific viral sequence is associated with the maintenance of an oncogenic transforming phenotype. That this portion of the EHV-1 genome was retained in these transformed cell lines during prolonged passage in culture, while EHV-1 DNA exclusive of the EHV-1 consensus transformation sequence was excised by passage 66, offers further support for this hypothesis. In addition, blot hybridization studies of DNA from tumor tissues and tumor cell lines derived from these fibrosarcomas in animals inoculated with these EHV1 -transformed cell lines have revealed that the 0.320.38 EHV-1 sequence is retained selectively (R. Robinson and D. O’Callaghan, manuscript submitted). Thus we conclude that the morphological transforming genes of EHV-1 reside between the 0.32-0.38 map coordinates of the viral genome. The restriction enzyme mapping results of viral insertions and flanking cellular sequences presented in this paper do not provide a definitive basis for supporting either a random or a site-specific mechanism for integration of herpesvirus DNA. Indeed, it will be interesting to discern whether herpesvirus DNA integration during oncogenic transformation conforms to the nonspecific or random integration processes ascribed for several retroviruses (Shimotohno and Temin, 1981) and for SV40 and adenovirus type 2 (Sambrook et al., 1980; Stringer, 1981; see Sharp,

1980). Direct evidence of EHV-1 DNA integration at specific cellular sites can be provided by nucleotide sequence analysis of host-viral junctures located in DNA fragments in recombinant phages constructed from cell DNA of the EHV-l-transformed cells. A pivotal point that must be ascertained to understand fully the mechanism by which herpesviruses render permissive cells oncogenically transformed instead of cytolytically infected is whether integration of the whole genome or specific portions are requisite for initiating the transformation pathway. A common feature of tumor viruses that has emerged within recent years is the association of specific genes of viral and/or cellular origin with the oncogenic transformation of susceptible cells. In HSV1 and HSV-2 morphological transformation, specific viral sequences on the respective genomes have been reported to be the transforming regions of these viruses (Reyes et al., 1979; Galloway and McDougall, 1981). In this report, a specific region of the EHV-1 genome located at 0.32-0.38 map units has been demonstrated to be maintained stably in oncogenically transformed hamster cell lines. To determine whether other equine herpesviruses have a characteristic gene(s) coding for a function(s) leading to oncogenic transformation, we have shown that equine cytomegalovirus (EHV-2; Wharton et al., 1981) and equine veneral disease virus (equine coital exanthema virus, EHV3) are capable of oncogenically transforming hamster embryo fibroblasts (O’Callaghan et al., 1981; 1982), and presently these cells are being examined for virus-specific DNA. It will be of considerable interest to determine whether EHV-1 oncogenic DNA sequences are among those that exhibit homology with the EHV-2 and EHV3 genomes (Staczek et al., 1983). The demonstration of extensive homology of viral genes coding for oncogenic functions by these three viruses would lend substantial support to a unifying principle of a common oncogene residing in the genome of various herpesviruses. Experimental

Procedures

Cells and Viruses The three transformed hamster cell lines (LSEH) used in this work were established by Robinson et al. (1980a) by abortive infection of primary monolayers of LSH hamster embryo cells with preparations of UV-irradiated EHV-1. Methods to culture these cells and the phenotypic properties of these transformed cell lines have been described elsewhere (Robinson et al., 1980a; O’Callaghan et al., 1982). Equine herpesvirus type 1, Kentucky A strain, was propagated in suspension cultures of L-M mouse fibroblasts. quantitated and purified by methods previously described by Perdue et al. (1974). Virus preparations were monitored for purity by restriction endonuclease analysis of virion DNA and electron microscopy of virions. Isolation of Viral and Cellular DNA Standard EHV-1 DNA was extracted from purified virions by the pronase-SDS-phenol method as described by Henry et al., 1979; 1981). Viral DNA was purified further by CsCl equilibrium gradient

Herpesvirus 577

Oncogenic

DNA Sequences

centrifugation (Cohen et al., 1979). and was examined for purity by restriction endonuclease analysis and isopycnic banding in the analytical ultracentrifuge (Henry et al., 1981). High-molecular-weight DNA from LSH hamster embryo cells or EHV-l-transformed cells was extracted and purified as described previously (Gross-Bellard et al., 1973). Cells were removed from the surface of 1720 cm2 glass roller bottles by scraping with a rubber policeman, pelleted by centrifugation, washed twice with ice-cold phosphate-buffered saline and repelleted by centrifugation. After resuspension of cell pellets in a DNA extraction buffer (0.01 M TrisHCI [pH 8.01, 0.001 M EDTA, 0.12 M NaCI), proteinase K and SDS were added to final concentrations of 0.5 mg/ml and 0.5%, respectively. The cell lysates were incubated at 37°C for 12 hr, extracted twice with equal volumes of phenol and once with chloroform-nbutanol (24:i ; V:V), followed by extensive dialysis against DNA dialysis buffer (5 mM Tris-HCI [pH 7.41, 0.1 mM EDTA). The RNA in the nucleic acid solutions was digested by treatment with pancreatic RNAase A (50 pg/ml) for 1 hr at 37°C. The DNA solution was extracted subsequently with phenol, dialyzed against several changes of DNA dialysis buffer, measured by optical spectroscopy (A260,P80) to determine concentrations, and ethanol-precipitated to a final concentration of 1 mg/ml.

Blot Hybridization Prior to hybridization, filters with transferred cell DNA were incubated at 41°C for 6-12 hr in a preannealing mixture containing 50% formamide, 1 x Denhardt’s buffer (0.02% each bovine serum albumin, polyvinylpyrrolidone, and Ficoll; Denhardt, 19661, 3 X SSC (1 X SSC = 0.15 M NaCI, 0.015 M sodium citrate), 0.05 M HEPES buffer (pH 7.0). 200 pg/ml yeast RNA and 50 pg/ml single-stranded, sheared salmon sperm DNA. After preannealing, excess mixture was removed by blotting between two sheets of Whatman 3 MM filter paper, and the filter was hybridized with 3-6 X 10’ cpm/ml alkalidenatured 32P-labeled viral DNA probe for 48-72 hr at 41°C in a volume of 3-5 ml of the preannealing mixture. After incubation, the filters were washed in successive changes of 2 X SSC, 0.1 X SSC and 0.1% SDS, and 0.1 X SSC at 23°C and 51 “C. After air drying, the filter was exposed to Kodak RP-Royal X-Omat film at -7OOC in the presence of DuPont Cronex “Lightening Plus” intensifying screens (Swanstrom and Shank, 1978) for 4-10 days. Autoradiographs were scanned in an LKB Zeineh soft laser scanning densitometer which was capable of integrating intensities of individual bands.

Preparation of Cloned EHV-1 Restriction Fragments EHV-1 DNA restriction fragments cloned into the plasmid vector pBR322 and propagated in appropriate E. coli hosts have been described elsewhere by Robinson et al.. 1981 b). To isolate the viral DNA inserts, we digested recombinant plasmids with the appropriate restriction endonuclease. and the insert and vector DNAs were resolved by electrophoresis through 0.8% Sea-Plaque agarose gels (Marine Colloids Div., Rockland, Maine). After visualization of DNA by ethidium-bromide staining, gel slices containing insert viral DNA were excised, heated at 7O’C for 1 hr, and frozen at -70°C for 3-4 hr. After repeated freezing and thawing of gel samples, agarose was

We thank Dr. J. Craig Cohen for technical advice and helpful discussions about blotting methods, and Dr. Philip W. Tucker for technical assistance and advice with the construction of recombinant clones. We are grateful to L. Devine and M. Minyard for patient typing of the manuscript.

pelleted by centrifugation: viral DNA was recovered in the supernatant. phenol-extracted several times, concentrated by ethanol precipitation and resuspended in 10 mM Tris-HCI (pH 7.4)-0.1 mM EDTA.

Acknowledgments

The research was supported by grants from the Grayson Foundation, the National Science Foundation, and the National Institutes of Health, and an institutional grant awarded by the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

Nick Translation of Viral DNA Cloned EHV-1 DNA restriction fragments wre radiolabeled to high specific activities (f-4 X 10’ cpm/gg) by the introduction of o~-~‘Pdeoxynucleotides by an adaptation of the nick translation method (Rigby et al., 1977). After phenol extraction of the reaction mixture, unincorporated radiolabeled nucleotides were separated from labeled DNA by passage of reaction mixtures on a Sephadex G-l 00 column (1 x 10 cm) equilibrated with 10 mM Tris-HCI (pH 8.01, 2 mM EDTA, 120 mM NaCI. Probe DNA was alkali-denatured just prior to hybridization. Restriction Endonuclease Digestion A 5-10 fold excess of restriction enzyme (Eco RI, Xba I, Barn HI. or Bgl II) was included in the digestion of all cellular DNAs, and reaction conditions were as described previously (Henry et al., 1981). The completeness of all reactions involving cellular DNAs was monitored routinely by analysis of a proportional reaction with h DNA and cellular DNAs prior to electrophoresis of restricted cellular DNA samples, Gel Electrophoresis and DNA Transfer Restriction fragments of cell DNA were fractionated by electrophoresis through 0.7% horizontal slab agarose gels (0.5 X 13 x 30 cm) in 0.02 M sodium acetate (pH 7.0), 0.018 M NaCI, 0.002 M EDTA (Helling et al., 1974). Bromophenol blue was included as a tracking dye, and samples were subjected to electrophoresis at 50-60 volts until the dye had migrated 25-30 cm. EHV-1 DNA labeled with “P in vivo (Henry et al., 1981) and digested with Eco RI or other enzymes was included as molecular weight and transfer markers in parallel lanes. DNA in gels was visualized with a UV light source after ethidiumbromide (0.5 pg/ml) staining of gels. The transfer of cell DNA onto nitrocellulose filters was carried out by a modification of the method of Southern (1975) as described previously (Wahl et al., 1979).

July 26, 1982;

revised

November

5, 1982

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O’Callaghan. D. J.. Gentry, G. A. and Randall, C. C. 1982. The equine herpesviruses. In Herpesviruses, B. Roizman, ed. Series 2. Comprehensive Virology, H. Fraenkel-Conrat and R. Wagner, eds. (New York: Plenum Press), in press. O’Callaghan, D. J., Henry, B. E., Wharton, J. H.. Dauenhauer, S. A., Vance, R. B., Staczek, J., Atherton, S. S. and Robinson, R. A. (1981). Equine herpesviruses: biochemical studies on genomic structure, DI particles, oncogenic transformation, and persistent infection. In Developments in Molecular Virology. I. Herpesvirus DNA. Y. Becker, ed. (The Hague: Nijhoff), pp. 387-481. Perdue, M. L.. Kemp, M. C., Randall, C. C. and O’Callaghan, D. J. (1974). Studies on the molecular anatomy of L-M cell strain on equine herpesvirus type 1. Proteins of the nucleocapsids and intact virions. Virology 50, 201-216. Rapp, F. (1980). Oncogenic Raton. Florida: CRC Press).

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