Proc. Nati. Acad. Sci. USA Vol. 75, No. 7, pp. 3372-7376, July 1978 Genetics
Sites of integration of reticuloendotheliosis virus DNA in chicken DNA (RNA tumor virus/integrated provirus/restriction endonuclease/blotting/nucleic acid hybridization)
ELI KESHET AND HOWARD M. TEMIN McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706
Contributed by Howard M. Temin, May 5, 1978
ABSTRACT The pattern of integration of spleen necrosis virus (SNV) DNA in DNA from a large population of SNV-infected chicken cells was studied by nucleic acid hybridization with iodinated viral RNA by the blotting technique of Southern. SNV DNA was found to be integrated at multiple sites in acutely infected chicken cells. Concomitant with the transition from acute to chronic infection, a shift in the pattern of integration was observed. The majority of integrated SNV DNA found in acutely infected cells was absent from chronically infected cells. This result is consistent with the hypothesis that the cell death that occurs after infection of avian cells with reticuloendotheliosis viruses is a consequence of the multiple integrations of the provirus. Viral DNA was also integrated at multiple sites in chronically infected cells. However, infectious viral DNA molecules in chronically infected cells migrated in a uniform manner in agarose gel electrophoresis after EcoRI digestion (which does not cut viral DNA), indicating that not all integrated SNV copies are equally infectious.
consistent with the hypothesis that the cell death that occurs after infection of avian cells by reticuloendotheliosis virus is a consequence of these multiple integrations (6, 7). (iii) Viral DNA is also found integrated at multiple sites in chronically infected cells. However, consistent with our previous studies (6, 7), infectious viral DNA is only present at a restricted fraction of these sites.
MATERIALS AND METHODS The general sources and procedures for obtaining and propagating avian cells and avian reticuloendotheliosis viruses have been described (9-11). Chicken cells were infected with SNV at a multiplicity of less than 1 plaque-forming unit per cell. Data on the growth of SNV-infected avian cells have been presented (7, 9, 10). For analysis of acutely infected cells, cells were harvested 2 days after infection. For analysis of chronically infected cells, cells were transferred twice and harvested more than 3 weeks after infection. DNA was extracted from cells as described (11). Restriction endonuclease EcoRI, prepared according to Stimegi et al. (12), was a gift from S. Mizutani. Bgl II restriction endonuclease was a gift from J. Mertz. DNA was digested with EcoRI at 370 in 90 mM Tris-HCl, pH 7.5/10 mM MgCl2/50 mM NaCl, and with Bgl II in 20 mM Tris-HCI (pH 7.2) containing 6 mM MgCl2, 6 mM mercaptoethanol, and 100 jig of bovine serum albumin per ml. DNAs were electrophoresed through horizontal agarose slab gels under conditions specified in each figure legend. Electrophoresis buffer included 0.5 ,ug of ethidium bromide per ml. Blotting of DNA onto nitrocellulose filter paper was performed as described by Southern (8). Nitrocellulose filters were soaked for 16-20 hr at 650 in the buffer solution suggested by Botchan et al. (13) and hybridized in 3.0 ml of 6X SSC (SSC is 0.15 M NaCl/15 mM sodium citrate, pH 7) containing (wt/vol) 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 0.5% sodium dodecyl sulfate, and 1 mM EDTA. Hybridization mixtures included 100 ,ug of carrier yeast RNA per ml and 5-10 X 106 cpm of SNV 125I-labeled RNA. Hybridization was at 650 for 20-24 hr in sealed polyethelene bags. Filters were then washed extensively at 650 with 2X SSC containing 0.1% sodium dodecyl sulfate, treated for 1 hr with 20 ,g of RNase A per ml in 2X SSC at 370, rewashed with 2X SSC, and air dried. SNV RNA was extracted from sucrose-banded virions (14). RNA (60-70S) was isolated by centrifugation through 15-30% glycerol gradients, denatured by incubating 1 min at 85°, and further purified by two successive passages through oligo(dT)Abbreviations: SNV, spleen necrosis virus; Ch(SNV), chicken cells infected with SNV; SSC, standard saline-citrate solution (0.15 M sodium chloride/0.015 M sodium citrate, pH 7); Mdal, megadaltons; ID50,
Retrovirus DNA is stably integrated in host cell DNA (1, 2). However, little is known about the sites of integration and the mechanism by which viral DNA integrates into host cell DNA. Early after retrovirus infection of sensitive cells, many complete viral DNA molecules are synthesized and some are also found in the nucleus. Only a portion of these molecules become stably integrated in the host genome (2-5). The reason for the restricted number of integrated viral DNA molecules is unknown. A limited number of sites in host cell DNA that can integrate viral DNA molecules has been suggested (3). Previous studies from our laboratory have shown that infectious reticuloendotheliosis virus DNA is integrated at multiple sites in acutely infected avian cells (soon after infection) and at a unique site in chronically infected avian cells (late after infection) (6, 7). However, since the assay method used in these experiments detected only infectious viral DNA molecules, it is possible that noninfectious viral DNA is also present at additional sites in the host cell. We used nucleic acid hybridization techniques to test this possibility and to obtain more information about the pattern of integration of spleen necrosis virus (SNV) in chicken cell DNA. DNA from a large population of SNV-infected chicken cells was digested with restriction endonucleases. DNA fragments that contain viral nucleotide sequences were specifically detected by nucleic acid hybridization with iodinated viral RNA by the blotting technique of Southern (8) and were visualized by autoradiography. Size analysis and quantification of DNA fragments that contain viral nucleotide sequences yielded the following conclusions: (i) Viral DNA in acutely infected cells is integrated at multiple sites. (ii) The majority of this integrated viral DNA is absent from chronically infected cells, a result 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. §1734 solely to indicate this fact.
mean infective dose.
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cellulose columns. RNA was iodinated according to Teroland McCarthy (15), was further purified by Sephadex G-75 column chromatography and repeated extractions with phenol and chloroform/isoamyl alcohol (24:1), and was concentrated by ethanol precipitation. Specific activities of about 5 X 107 cpm/,gg were obtained. Preparations of SNV 125-Ilabeled RNA were assayed for the presence of contaminating ribosomal RNA by comparison of autoradiograms with autoradiograms obtained with iodinated ribosomal RNA. Contaminating ribosomal RNA was usually undetectable or hardly detectable. Autoradiography was carried out at -70° with Kodak X-Omat x-ray film with the use of Ilford fast-tungstate enhancing screens (16). Exposure time varied from 1 to 20 days in different experiments. Autoradiograms were scanned with a Joyce-Loebel microdensitometer. Preliminary experiments, performed under identical conditions to those reported in this manuscript, showed a linear relationship between densitometry tracings of autoradiograms and amounts of input, hybridizable, Hirt supernatant SNV DNA (data not shown). Infectious DNA was assayed by the calcium phosphate coprecipitation procedure (17) as described (7). Mean infective dose (IDso) was calculated according to Reed and Muench (18).
RESULTS SNV DNA is integrated at multiple sites in acutely infected chicken cells The general design of the experiments to study the pattern of integration of SNV DNA in chicken cell DNA was as follows: DNA was extracted from a large population of chicken cells either acutely or chronically infected with SNV; the DNA was digested to completion with restriction endonuclease EcoRI, which does not cut viral DNA; and the resulting DNA fragments were separated according to size by electrophoresis through agarose gels. DNA fragments were then denatured in situ and immobilized on nitrocellulose filters by the blotting technique of Southern (8). DNA fragments that contain SNV DNA were specifically detected by nucleic acid hybridization with an iodinated viral RNA probe and were visualized by autoradiography. Information regarding the integration pattern of SNV in avian cells was obtained from the quantification and size analysis of the DNA fragments that contain viral DNA sequences. The predominant form of nonintegrated viral DNA in acutely infected cells is a linear double-stranded DNA of approximately 6 megadaltons (Mdal) (Fig. 1, lane A). Previous studies showing that EcoRI does not inactivate infectivity of SNV DNA were confirmed here by showing that extensive digestion with EcoRI did not change the mobility of nonintegrated SNV DNA (Fig. 1, lane B). Under the conditions used, phage X DNA included in the reaction mixture was completely
digested. No hybridization could be detected to DNA from uninfected chicken cells (Fig. 1, lane C). Since DNA from uninfected chicken cells contains nucleotide sequences homologous to only about 10% of the SNV genome (14), the failure to detect the endogenous SNV nucleotide sequences could be a result either of the limited sensitivity of the detection method or of a noncontiguous arrangement of these nucleotide sequences in the chicken genome. Therefore, when DNA from SNV-infected cells is digested with EcoRI, it is expected that every DNA fragment that is detectable by hybridization should contain all of the viral nucleotide sequences that are integrated in that
Proc. Natl. Acad. Sci. USA 75 (1978)
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FIG. 1. Digestion with restriction endonuclease EcoRI of DNAs from acutely or chronically SNV-infected chicken cells-size of SNV-containing DNA fragments. Ten micrograms of cellular DNA (from uninfected, acutely infected, or chronically infected chicken cells) were digested with 40 units of EcoRI for 2 hr under standard conditions. Preliminary experiments in which phage X DNA was included in the incubation mixture assured complete digestion of cellular DNA under this condition. DNA from the supernatant fraction of SNV-infected chicken cells after extraction by the Hirt procedure (19) was also digested to completion with EcoRI together with phage A DNA. Digestion reactions were terminated by incubation for 5 min at 650, and the complete reaction mixtures were electrophoresed through a 6-mm-thick, 0.4% agarose horizontal slab gel. Electrophoresis was at 70 V for 20 hr. DNA fragments were denatured and transferred to nitrocellulose filter paper by the blotting technique of Southern (8). Hybridization with SNV 1251-labeled RNA and autoradiography were as described in Materials and Methods. Exposure was for 3 days (lanes A-G) or 13 days (lanes H and I). (A) Hirt supernatant DNA from SNV-infected chicken cells [Ch(SNV)]; (B) Hirt supernatant DNA from Ch(SNV) digested with EcoRI; (C) DNA from uninfected chicken cells; (D) DNA from acutely infected Ch(SNV); (E) DNA from chronically infected Ch(SNV); (F) DNA from acutely infected Ch(SNV) digested with EcoRI; (G) DNA from chronically infected Ch(SNV) digested with EcoRI; (H and I) longer exposures of lanes F and G, respectively. Molecular size markers were Ava I and Sma I digests of phage A DNA run in parallel lanes and EcoRI-generated fragments of X DNA included in lane B.
particular site and the adjacent cellular sequences. In addition, the hybridization technique will detect nonintegrated DNA molecules, which contain only viral nucleotide sequences. When total DNA isolated from acutely infected chicken cells was electrophoresed and hybridized, integrated SNV DNA was found associated predominantly with very large DNA (Fig. 1, lane D). In addition, a large amount of nonintegrated SNV DNA was found. After digestion with EcoRI, integrated SNV DNA was found associated with a heterogeneous population of DNA molecules distributed over a wide size range (Fig. 1, lane F). This result confirms the finding of Battula and Temin (6, 7) that, in DNA from acutely infected chicken cells digested with EcoRI, infectious DNA is found in DNA fragments with a wide size distribution. These results indicate that SNV DNA is integrated at multiple sites in chicken DNA soon after infection (acute phase of infection). In addition to DNA fragments of 6 Mdal or larger, a low level of DNA fragments smaller than 6 Mdal that contain SNV DNA was also detected. When such fragments were eluted from the gel and again electrophoresed, they migrated to the same position as after the first electrophoresis (data not shown). The nature of these DNA fragments is not known. The size distribution of SNV-containing fragments in DNA from infected cells prior to EcoRI digestion (Fig. 1, lane D and Fig. 2, lane B) indicates that the DNA fragments of molecular size smaller than 6 Mdal are not degradation products of high molecular weight DNA. Also, the detection of roughly similar levels of these DNA
Genetics: Keshet and Temin
3374 Silrei~dali Origin
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FIG. 2. Resolution into size classes of DNA fragments that contain SNV DNA from EcoRI-digested DNA from chronically infected Ch(SNV). Fractionation of EcoRI-digested DNA from chronically infected Ch(SNV): 550 ,gg of DNA from chronically infected Ch(SNV) was digested with 1400 units of EcoRI for 2.5 hr under standard conditions, loaded into a 140-mm-long and 6-mm-thick slot of a 0.4% agarose gel, and electrophoresed. DNA fragments generated by Ava I and Sma I digestion of X DNA were run in parallel to serve as molecular size markers. Electrophoresis was at 70 V for 20 hr. Eight gel slices, 5 mm wide each, that collectively covered the size range of 20-S Mdal, were sliced from the gel. DNA was eluted from each of these fractions as described (7). The ethidium bromide was removed by repeated extractions with isoamyl alcohol saturated with water. The DNAs were then concentrated by ethanol precipitation and resuspended in 150 Ml of O.1X SCC. Re-electrophoresis of DNA fractions: 25 ,l of DNA from each fraction (3.7-5.2 Mig) was electrophoresed in an agarose gel along with an EcoRI digest of uninfected chicken DNA (lane A) and total DNA from chronically infected Ch(SNV) undigested (lane B) or digested with EcoRI (lane C). Lanes D-J correspond to DNA fractions of decreasing size. The size of the DNAs after re-electrophoresis, as seen by UV light, was the same as the initial size. Therefore, no significant degradation of DNA occurred. The DNAs were blotted, hybridized, and autoradiographed as described.
fragments in undigested and EcoRI-digested DNAs (compare lanes B and C of Fig. 2) indicates that they are not composed of integrated SNV DNA sequences. Previous studies from our laboratory have shown that acutely infected chicken cells contain more integrated infectious SNV DNA per cell than do chronically infected chicken cells (2). A comparison was made, therefore, of the content of integrated SNV DNA in acutely and chronically infected chicken cells. It was found that in the chronic phase of infection chicken DNA contained less integrated SNV DNA than in the acute phase of infection (compare, in Fig. 1, lanes D and E, F and G, and H and I). This difference in number of integrated copies was quantified by densitometry of appropriate autoradiograms (see Materials and Methods). The results showed about 7-fold more integrated SNV DNA per cell in acutely infected chicken cells than in chronically infected chicken cells (data not shown), indicating a loss of the majority of the integrated SNV DNA present in acutely infected cells. SNV DNA is integrated at multiple sites in chronically infected chicken cells Integration of SNV DNA in chronically infected chicken cells also is at multiple sites. This conclusion is evident from the broad size distribution of DNA fragments that contain SNV DNA sequences that are generated by EcoRI digestion of DNA from chronically infected cells (Fig. 2, lane C and Fig. 4, lane C). When the same experiment was carried out with DNA from chronically infected pheasant or turkey cells, similar results were obtained; that is, after digestion with EcoRI, the resultant DNA fragments that hybridized with SNV RNA were of a broad size range (data not shown). Nonintegrated SNV DNA (migrating as a linear DNA molecule with molecular size of 6 Mdal) was detected in all preparations of DNA extracted from chronically infected cells (Fig.
1, lanes E, G, and I, and Fig. 2). Other experiments showed that infectious nonintegrated SNV DNA persists in chicken cells as late as 6 weeks after infection (data not shown). To demonstrate further that SNV integrates at multiple sites in DNA from chronically infected chicken cells, an attempt was made to resolve the broad band of SNV-specific hybridization into discrete bands of different molecular sizes. DNA from chronically infected chicken cells was digested with EcoRI and electrophoresed through an agarose gel. The gel was sliced into segments, and DNA was eluted from each slice, re-electrophoresed through an agarose gel, transferred to nitrocellulose paper, and hybridized to labeled viral RNA. The results (Fig. 2) show that SNV-containing DNA fragments can be resolved into discrete size classes. Furthermore, each one of the hybridizable fractions shown in Fig. 2 is itself probably composed of more than one DNA species, judged from the broadness of the bands in respect to the broadness of a band composed of a single molecular species of comparable intensity (for example, in lane H of Fig. 2 both a sharp band of nonintegrated SNV DNA and a broader band of integrated SNV DNA molecules are seen). To establish that the hybridizable DNA fragments that are larger than 6 Mdal that are visualized in different fractions of the autoradiogram shown in Fig. 2 are indeed integrated SNV DNA sequences flanked by cellular DNA sequences, we carried out the following experiment: DNA from a large population of chronically infected cells was digested with EcoRI and the DNA fragments were separated into size classes as described in the legend of Fig. 2. Samples of DNA recovered from each of these fractions were then digested with restriction endonuclease Bgl II, and the resulting fragments were assayed by the blotting technique for their content of SNV DNA. Preliminary experiments have shown that Bgl II introduces two cuts in SNV DNA, generating fragments of 3.5, 1.4, and 1.2 Mdal. These fragments were placed in order, and the 3.5Mdal fragment was found to be the middle one (unpublished data). The rationale of the experiment was that if the hybridization in the original DNA fragments from the experiment of Fig. 2 is of SNV DNA that is flanked by cellular DNA sequences of different lengths, a discrete fragment of 3.5 Mdal will appear after Bgl II digestion. The results of this experiment are shown in Fig. 3. A 3.5-Mdal band was observed in the three DNA fractions that were tested. In each of the fractions tested, additional hybridization was also observed over a wide range of molecular sizes. The latter hybridization is most likely the result of that fraction of the SNV genome that corresponds to the two terminal Bgl II fragments. The detection of this hybridization spread over a wide range rather than as discrete hybridization bands is what one expects from integration at multiple sites. Little hybridization was detected in the region between 3.5 and 4.9 Mdal. This result may indicate that in the population of different DNA fragments that contain SNV DNA produced by EcoRI digestion certain molecular species are more abundant than others. For example, from the pattern shown in lane C of Fig. 2 (where DNA fragments of molecular size 9.5-12.0 Mdal were digested with Bgl II), it may be that the provirus is more abundantly integrated at a distance of 3.5-3.7 Mdal from one of the EcoRI sites. SNV DNA molecules integrated at different sites in
chronically infected chicken cells are not equally infectious
Previous experiments from this laboratory have shown that when DNA from chicken cells chronically infected with SNV
Genetics: Keshet and Temin A
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Proc. Natl. Acad. Sci. USA 75 (1978) C
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FIG. 3. Digestion with BgI of EcoRI-generated DNA fragments of different sizes derived from DNA of chronically infected Ch(SNV). Samples of DNA from three different fractions of EcoRI-digested DNA from chronically infected Ch(SNV) were digested with Bgl II. The DNA samples were from the same fractions shown in Fig. 2 (prepared as described in the legend of Fig. 2). The DNAs used in lanes B, C, and D correspond to DNAs from lanes E, F, and G of Fig. 2, respectively. Three micrograms of DNA was digested with 5 units of Bgl II for 4 hr. Electrophoresis, nucleic acid hybridization, and autoradiography were done as before. DNA from Ch(SNV) from the supernatant fraction of a Hirt extraction and digested with Bgl II was electrophoresed in lane A. The DNA fragment of 4.9 Mdal in lane A is a product of incomplete digestion.
is digested with EcoRI, electrophoresed, and assayed for infectivity, the infectious molecules are distributed in a narrow range of molecular size (6, 7). On the other hand, when DNA from chicken cells chronically infected with SNV is digested with EcoRI and assayed for the presence of SNV DNA sequences, SNV DNA sequences are distributed in a wide range of molecular sizes (Fig. 2). This difference indicates that there might exist, in chronically infected chicken cells, integrated SNV copies that are not infectious or that are much less infectious than SNV copies integrated at other sites of the chicken genome.
This hypothesis was tested by a direct comparison of the size distribution of DNA molecules that hybridize with SNV DNA (quantified by a densitometry tracing of an autoradiogram) and of the infectivity of DNA fragments eluted from the corresponding gel slices (quantified by an endpoint dilution assay and determination of ID50 values). DNA from chronically infected chicken cells was first purified in an agarose gel to remove unintegrated SNV DNA; it was then digested with EcoRI and electrophoresed in two parallel lanes. The lane used for infectivity assays was sliced into 30 2-mm-wide slices. DNA was eluted and used in a standard transfection assay. DNA from the parallel lane was blotted and hybridized with labeled SNV RNA. The results are shown in Fig. 4. DNA fragments that contain SNV nucleotide sequences (evident by nucleic acid hybridization) are distributed over a wide size range. However, infectivity is restricted to a narrow size range. This pattern of size distribution of infectious DNA molecules with a peak at 10-11 Mdal was reproducibly obtained in this study and is essentially the same pattern obtained by Battula and Temin in previous studies (6, 7). Thus, these results
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FIG. 4. Digestion of DNA from chronically infected Ch(SNV) with EcoRI-comparison of the size distribution of hybridizable fragments with the size distribution of infectious DNA fragments. DNA from chronically infected Ch(SNV) was purified by electrophoresis in a 0.4% agarose gel in order to remove unintegrated SNV DNA. High molecular size DNA (over 50 Mdal) was eluted and recovered from the gel as described in the legend of Fig. 2. DNA was digested with EcoRI under the conditions described in the legend of Fig. 1, and two aliquots of 25 gg of EcoRI-digested DNA were electrophoresed in two parallel lanes of a 0.4% agarose gel. The size distribution of the infectious DNA fragments was determined by slicing one of the gel lanes into 2-mm-wide slices. DNA was eluted from each of the slices and assayed for infectivity as described (6). The results are expressed as ID50 values (0). The size distribution of the hybridizable DNA was determined by transfer of the DNA fragments from the parallel gel lane (lane C) onto a nitrocellulose filter, nucleic acid hybridization, and autoradiography. DNAs from uninfected chicken cells (lane B) and unintegrated SNV DNA (lane A) were also treated the same way. Densitometry tracings of the autoradiograms are shown for the relative quantification of integrated SNV DNA of each molecular size. For direct comparison, infectivity and hybridization profiles were superimposed on the same scale of molecular size (deduced from the mobility of size markers electrophoresed in parallel).
indicate that not all integrated SNV copies are equally infectious, and that the infectious molecules after EcoRI digestion migrate in a uniform manner. DISCUSSION The production of avian reticuloendotheliosis viruses in avian cells.occurs in two phases. In the first phase, acute infection, virus production is accompanied by cell death and cytopathic effects. In the second phase, chronic infection, virus is produced without cell death or cytopathic effects. Concurrent with the transition from the acute to the chronic phase of infection, there is a shift in the pattern of integration of SNV DNA. The results reported here show that the majority of the integrated viral DNA present in acutely infected cells is absent from chronically infected cells. This finding is consistent with the previous finding of a loss in infectivity of the integrated viral DNA during the transition from the acute to the chronic phase of infection (2) and indicates that this loss is not a result of inactivation of integrated viral DNA, but is primarily a loss of integrated DNA molecules from the DNA of the infected cells. Two mechanisms can account for such a loss: excision of viral DNA from the cell genome or a selection process in which those cells that have a large number of integrated viral DNA copies die. Under the latter hypothesis, the cells that survive and es-
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tablish a chronic infection are those that have integrated a smaller number of viral DNA copies. We favor the latter mechanism, since there is a correlation between the decrease in number of integrated viral DNA molecules and cell death, and since this mechanism does not necessitate a specific excision mechanism. The decrease in number of integrated SNV copies per cell during the transition from acute to chronic infection that is reported here (about 7-fold) is roughly the same as the decrease in the amount of infectious DNA molecules per cell that was previously reported (2). The finding of a similar loss of infectivity and of hybridizable DNA combined with the finding that in chronically infected cells not all integrated SNV DNA molecules are infectious indicates that also in acutely infected cells not all integrated SNV copies are equally infectious. Both infectious and noninfectious integrated SNV DNA molecules were found in chronically infected chicken cells. Previous studies have shown that all chronically infected chicken cells produce infectious virus (10). From other experiments using nucleic acid hybridization in liquid it was estimated that in chronically infected chicken cells there is an average of five SNV DNA copies per genome (14). It seems likely, therefore, that most of the chronically infected cells have multiple integrated viral DNA copies and that only one of them leads to production of infectious virus. It also seems likely that the noninfectious molecules are integrated in different sites in different cells. The difference in specific infectivities of integrated SNV DNA molecules may have several causes. The host DNA sequences that are covalently linked to the viral DNA may play a role in determining the infectivity of the fragment. An example of a lack of infectivity of DNA fragments that contain a complete nondefective endogenous provirus is the RAV-0 in DNA from V + tvbr chicken cells (20). It was suggested there that the cellular DNA sequences that flank the provirus and that determine its activity in a transfection assay are acting as a control element for viral expression (20). If the situation in SNV-infected chicken cells reflects an analogous situation for an exogenous virus, then viral DNA integrated at particular sites might have different biological effects from viral DNA integrated in other sites. It is also possible that the occurrence of noninfectious integrated viral copies is a result of integration of defective genomes, either incomplete genomes or rearranged DNA molecules. Another possibility is integration of viral genomes in the wrong orientation. Experiments that can distinguish between these possibilities are now feasible.
Infectious DNA fragments generated by digestion with EcoRI of chronically infected cell DNA migrate in a uniform manner when electrophoresed in agarose gels. However [as was previously discussed (7)], different DNA fragments of the same length or DNA fragments that differ slightly in length cannot be resolved under these conditions. It is not certain, therefore, that infectious SNV DNA is integrated at one site in chronically infected cells. Moreover, SNV DNA integrated at additional sites might also be slightly infectious, but escape detection as a result of the limited sensitivity of the assay. In any case, it is clear that there is a shift from the complex distribution of infectivity in acutely infected cells to the simple distribution of infectivity found in chronically infected cells. We thank Drs. B. Sugden, L. Estis, and J. O'Rear for comments on the manuscript, Drs. J. Mertz and S. Mizutani for enzymes, and S. Hellenbrand and S. Kuo for excellent technical assistance. This investigation was supported by Research Grant CA-07175 from the National Cancer Institute. H.M.T. is an American Cancer Society Research Professor. 1. Varmus, H. E., Vogt, P. K. & Bishop, J. M. (1973) Proc. Natl. Acad. Sci. USA 70,3067-3071. 2. Fritsch, E. & Temin, H. M. (1977) J. Virol. 21, 119-130. 3. Khoury, A. T. & Hanafusa, H. (1976) J. Virol. 18,383-400. 4. Ali, M. & Baluda, M. A. (1974) J. Virol. 13, 1005-1013. 5. Dastoor, M. N., Shoyab, M. & Baluda, M. A. (1977) J. Virol. 21, 541-547. 6. Battula, N. & Temin, H. M. (1977) Proc. Natl. Acad. Sci. USA 7. 8. 9. 10.
74,281-285. Battula, N. & Temin, H. M. (1978) Cell 13,387-398. Southern, E. M. (1975) J. Mol. Biol. 98,503-517. Temin, H. M. & Kassner, V. K. (1974) J. Virol. 13,291-297. Temin, H. M. & Kassner, V. K. (1975) J. Gen. Virol. 27, 267-
274. 11. Kang, C.-Y. & Temin, H. M. (1973) J. Virol. 12, 1314-1324. 12. Sumegi, J., Breedveld, D., Hossenlopp, P. & Chambon, P. (1977) Biochem. Biophys. Res. Commun. 76,78-85. 13. Botchan, M., Topp, W. & Sambrook, J. (1976) Cell 9, 269-
287. 14. Kang, C.-Y. & Temin, H. M. (1974) J. Virol. 14, 1179-1188. 15. Tereba, A. & McCarthy, B. J. (1973) Biochemistry 12, 46754679. 16. Laskey, R. A. & Mills, A. D. (1977) FEBS Lett. 82,314-316. 17. Graham, F. L. & Van der Eb, A. J. (1973) Virology 52, 456467. 18. Reed, L. J. & Muench, H. (1938) Am. J. Hyg. 27,493-497. 19. Hirt, B. (1967) J. Mol. Biol. 26,365-369. 20. Cooper, G. M. & Temin, H. M. (1976) J. Virol. 17,422-430.
Sites of integration of reticuloendotheliosis virus DNA in chicken DNA (RNA tumor virus/integrated provirus/restriction endonuclease/blotting/nucleic acid hybridization)
ELI KESHET AND HOWARD M. TEMIN McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706
Contributed by Howard M. Temin, May 5, 1978
ABSTRACT The pattern of integration of spleen necrosis virus (SNV) DNA in DNA from a large population of SNV-infected chicken cells was studied by nucleic acid hybridization with iodinated viral RNA by the blotting technique of Southern. SNV DNA was found to be integrated at multiple sites in acutely infected chicken cells. Concomitant with the transition from acute to chronic infection, a shift in the pattern of integration was observed. The majority of integrated SNV DNA found in acutely infected cells was absent from chronically infected cells. This result is consistent with the hypothesis that the cell death that occurs after infection of avian cells with reticuloendotheliosis viruses is a consequence of the multiple integrations of the provirus. Viral DNA was also integrated at multiple sites in chronically infected cells. However, infectious viral DNA molecules in chronically infected cells migrated in a uniform manner in agarose gel electrophoresis after EcoRI digestion (which does not cut viral DNA), indicating that not all integrated SNV copies are equally infectious.
consistent with the hypothesis that the cell death that occurs after infection of avian cells by reticuloendotheliosis virus is a consequence of these multiple integrations (6, 7). (iii) Viral DNA is also found integrated at multiple sites in chronically infected cells. However, consistent with our previous studies (6, 7), infectious viral DNA is only present at a restricted fraction of these sites.
MATERIALS AND METHODS The general sources and procedures for obtaining and propagating avian cells and avian reticuloendotheliosis viruses have been described (9-11). Chicken cells were infected with SNV at a multiplicity of less than 1 plaque-forming unit per cell. Data on the growth of SNV-infected avian cells have been presented (7, 9, 10). For analysis of acutely infected cells, cells were harvested 2 days after infection. For analysis of chronically infected cells, cells were transferred twice and harvested more than 3 weeks after infection. DNA was extracted from cells as described (11). Restriction endonuclease EcoRI, prepared according to Stimegi et al. (12), was a gift from S. Mizutani. Bgl II restriction endonuclease was a gift from J. Mertz. DNA was digested with EcoRI at 370 in 90 mM Tris-HCl, pH 7.5/10 mM MgCl2/50 mM NaCl, and with Bgl II in 20 mM Tris-HCI (pH 7.2) containing 6 mM MgCl2, 6 mM mercaptoethanol, and 100 jig of bovine serum albumin per ml. DNAs were electrophoresed through horizontal agarose slab gels under conditions specified in each figure legend. Electrophoresis buffer included 0.5 ,ug of ethidium bromide per ml. Blotting of DNA onto nitrocellulose filter paper was performed as described by Southern (8). Nitrocellulose filters were soaked for 16-20 hr at 650 in the buffer solution suggested by Botchan et al. (13) and hybridized in 3.0 ml of 6X SSC (SSC is 0.15 M NaCl/15 mM sodium citrate, pH 7) containing (wt/vol) 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 0.5% sodium dodecyl sulfate, and 1 mM EDTA. Hybridization mixtures included 100 ,ug of carrier yeast RNA per ml and 5-10 X 106 cpm of SNV 125I-labeled RNA. Hybridization was at 650 for 20-24 hr in sealed polyethelene bags. Filters were then washed extensively at 650 with 2X SSC containing 0.1% sodium dodecyl sulfate, treated for 1 hr with 20 ,g of RNase A per ml in 2X SSC at 370, rewashed with 2X SSC, and air dried. SNV RNA was extracted from sucrose-banded virions (14). RNA (60-70S) was isolated by centrifugation through 15-30% glycerol gradients, denatured by incubating 1 min at 85°, and further purified by two successive passages through oligo(dT)Abbreviations: SNV, spleen necrosis virus; Ch(SNV), chicken cells infected with SNV; SSC, standard saline-citrate solution (0.15 M sodium chloride/0.015 M sodium citrate, pH 7); Mdal, megadaltons; ID50,
Retrovirus DNA is stably integrated in host cell DNA (1, 2). However, little is known about the sites of integration and the mechanism by which viral DNA integrates into host cell DNA. Early after retrovirus infection of sensitive cells, many complete viral DNA molecules are synthesized and some are also found in the nucleus. Only a portion of these molecules become stably integrated in the host genome (2-5). The reason for the restricted number of integrated viral DNA molecules is unknown. A limited number of sites in host cell DNA that can integrate viral DNA molecules has been suggested (3). Previous studies from our laboratory have shown that infectious reticuloendotheliosis virus DNA is integrated at multiple sites in acutely infected avian cells (soon after infection) and at a unique site in chronically infected avian cells (late after infection) (6, 7). However, since the assay method used in these experiments detected only infectious viral DNA molecules, it is possible that noninfectious viral DNA is also present at additional sites in the host cell. We used nucleic acid hybridization techniques to test this possibility and to obtain more information about the pattern of integration of spleen necrosis virus (SNV) in chicken cell DNA. DNA from a large population of SNV-infected chicken cells was digested with restriction endonucleases. DNA fragments that contain viral nucleotide sequences were specifically detected by nucleic acid hybridization with iodinated viral RNA by the blotting technique of Southern (8) and were visualized by autoradiography. Size analysis and quantification of DNA fragments that contain viral nucleotide sequences yielded the following conclusions: (i) Viral DNA in acutely infected cells is integrated at multiple sites. (ii) The majority of this integrated viral DNA is absent from chronically infected cells, a result 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. §1734 solely to indicate this fact.
mean infective dose.
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cellulose columns. RNA was iodinated according to Teroland McCarthy (15), was further purified by Sephadex G-75 column chromatography and repeated extractions with phenol and chloroform/isoamyl alcohol (24:1), and was concentrated by ethanol precipitation. Specific activities of about 5 X 107 cpm/,gg were obtained. Preparations of SNV 125-Ilabeled RNA were assayed for the presence of contaminating ribosomal RNA by comparison of autoradiograms with autoradiograms obtained with iodinated ribosomal RNA. Contaminating ribosomal RNA was usually undetectable or hardly detectable. Autoradiography was carried out at -70° with Kodak X-Omat x-ray film with the use of Ilford fast-tungstate enhancing screens (16). Exposure time varied from 1 to 20 days in different experiments. Autoradiograms were scanned with a Joyce-Loebel microdensitometer. Preliminary experiments, performed under identical conditions to those reported in this manuscript, showed a linear relationship between densitometry tracings of autoradiograms and amounts of input, hybridizable, Hirt supernatant SNV DNA (data not shown). Infectious DNA was assayed by the calcium phosphate coprecipitation procedure (17) as described (7). Mean infective dose (IDso) was calculated according to Reed and Muench (18).
RESULTS SNV DNA is integrated at multiple sites in acutely infected chicken cells The general design of the experiments to study the pattern of integration of SNV DNA in chicken cell DNA was as follows: DNA was extracted from a large population of chicken cells either acutely or chronically infected with SNV; the DNA was digested to completion with restriction endonuclease EcoRI, which does not cut viral DNA; and the resulting DNA fragments were separated according to size by electrophoresis through agarose gels. DNA fragments were then denatured in situ and immobilized on nitrocellulose filters by the blotting technique of Southern (8). DNA fragments that contain SNV DNA were specifically detected by nucleic acid hybridization with an iodinated viral RNA probe and were visualized by autoradiography. Information regarding the integration pattern of SNV in avian cells was obtained from the quantification and size analysis of the DNA fragments that contain viral DNA sequences. The predominant form of nonintegrated viral DNA in acutely infected cells is a linear double-stranded DNA of approximately 6 megadaltons (Mdal) (Fig. 1, lane A). Previous studies showing that EcoRI does not inactivate infectivity of SNV DNA were confirmed here by showing that extensive digestion with EcoRI did not change the mobility of nonintegrated SNV DNA (Fig. 1, lane B). Under the conditions used, phage X DNA included in the reaction mixture was completely
digested. No hybridization could be detected to DNA from uninfected chicken cells (Fig. 1, lane C). Since DNA from uninfected chicken cells contains nucleotide sequences homologous to only about 10% of the SNV genome (14), the failure to detect the endogenous SNV nucleotide sequences could be a result either of the limited sensitivity of the detection method or of a noncontiguous arrangement of these nucleotide sequences in the chicken genome. Therefore, when DNA from SNV-infected cells is digested with EcoRI, it is expected that every DNA fragment that is detectable by hybridization should contain all of the viral nucleotide sequences that are integrated in that
Proc. Natl. Acad. Sci. USA 75 (1978)
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FIG. 1. Digestion with restriction endonuclease EcoRI of DNAs from acutely or chronically SNV-infected chicken cells-size of SNV-containing DNA fragments. Ten micrograms of cellular DNA (from uninfected, acutely infected, or chronically infected chicken cells) were digested with 40 units of EcoRI for 2 hr under standard conditions. Preliminary experiments in which phage X DNA was included in the incubation mixture assured complete digestion of cellular DNA under this condition. DNA from the supernatant fraction of SNV-infected chicken cells after extraction by the Hirt procedure (19) was also digested to completion with EcoRI together with phage A DNA. Digestion reactions were terminated by incubation for 5 min at 650, and the complete reaction mixtures were electrophoresed through a 6-mm-thick, 0.4% agarose horizontal slab gel. Electrophoresis was at 70 V for 20 hr. DNA fragments were denatured and transferred to nitrocellulose filter paper by the blotting technique of Southern (8). Hybridization with SNV 1251-labeled RNA and autoradiography were as described in Materials and Methods. Exposure was for 3 days (lanes A-G) or 13 days (lanes H and I). (A) Hirt supernatant DNA from SNV-infected chicken cells [Ch(SNV)]; (B) Hirt supernatant DNA from Ch(SNV) digested with EcoRI; (C) DNA from uninfected chicken cells; (D) DNA from acutely infected Ch(SNV); (E) DNA from chronically infected Ch(SNV); (F) DNA from acutely infected Ch(SNV) digested with EcoRI; (G) DNA from chronically infected Ch(SNV) digested with EcoRI; (H and I) longer exposures of lanes F and G, respectively. Molecular size markers were Ava I and Sma I digests of phage A DNA run in parallel lanes and EcoRI-generated fragments of X DNA included in lane B.
particular site and the adjacent cellular sequences. In addition, the hybridization technique will detect nonintegrated DNA molecules, which contain only viral nucleotide sequences. When total DNA isolated from acutely infected chicken cells was electrophoresed and hybridized, integrated SNV DNA was found associated predominantly with very large DNA (Fig. 1, lane D). In addition, a large amount of nonintegrated SNV DNA was found. After digestion with EcoRI, integrated SNV DNA was found associated with a heterogeneous population of DNA molecules distributed over a wide size range (Fig. 1, lane F). This result confirms the finding of Battula and Temin (6, 7) that, in DNA from acutely infected chicken cells digested with EcoRI, infectious DNA is found in DNA fragments with a wide size distribution. These results indicate that SNV DNA is integrated at multiple sites in chicken DNA soon after infection (acute phase of infection). In addition to DNA fragments of 6 Mdal or larger, a low level of DNA fragments smaller than 6 Mdal that contain SNV DNA was also detected. When such fragments were eluted from the gel and again electrophoresed, they migrated to the same position as after the first electrophoresis (data not shown). The nature of these DNA fragments is not known. The size distribution of SNV-containing fragments in DNA from infected cells prior to EcoRI digestion (Fig. 1, lane D and Fig. 2, lane B) indicates that the DNA fragments of molecular size smaller than 6 Mdal are not degradation products of high molecular weight DNA. Also, the detection of roughly similar levels of these DNA
Genetics: Keshet and Temin
3374 Silrei~dali Origin
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Proc. Natl. Acad. Sci. USA 75 (1978) G
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FIG. 2. Resolution into size classes of DNA fragments that contain SNV DNA from EcoRI-digested DNA from chronically infected Ch(SNV). Fractionation of EcoRI-digested DNA from chronically infected Ch(SNV): 550 ,gg of DNA from chronically infected Ch(SNV) was digested with 1400 units of EcoRI for 2.5 hr under standard conditions, loaded into a 140-mm-long and 6-mm-thick slot of a 0.4% agarose gel, and electrophoresed. DNA fragments generated by Ava I and Sma I digestion of X DNA were run in parallel to serve as molecular size markers. Electrophoresis was at 70 V for 20 hr. Eight gel slices, 5 mm wide each, that collectively covered the size range of 20-S Mdal, were sliced from the gel. DNA was eluted from each of these fractions as described (7). The ethidium bromide was removed by repeated extractions with isoamyl alcohol saturated with water. The DNAs were then concentrated by ethanol precipitation and resuspended in 150 Ml of O.1X SCC. Re-electrophoresis of DNA fractions: 25 ,l of DNA from each fraction (3.7-5.2 Mig) was electrophoresed in an agarose gel along with an EcoRI digest of uninfected chicken DNA (lane A) and total DNA from chronically infected Ch(SNV) undigested (lane B) or digested with EcoRI (lane C). Lanes D-J correspond to DNA fractions of decreasing size. The size of the DNAs after re-electrophoresis, as seen by UV light, was the same as the initial size. Therefore, no significant degradation of DNA occurred. The DNAs were blotted, hybridized, and autoradiographed as described.
fragments in undigested and EcoRI-digested DNAs (compare lanes B and C of Fig. 2) indicates that they are not composed of integrated SNV DNA sequences. Previous studies from our laboratory have shown that acutely infected chicken cells contain more integrated infectious SNV DNA per cell than do chronically infected chicken cells (2). A comparison was made, therefore, of the content of integrated SNV DNA in acutely and chronically infected chicken cells. It was found that in the chronic phase of infection chicken DNA contained less integrated SNV DNA than in the acute phase of infection (compare, in Fig. 1, lanes D and E, F and G, and H and I). This difference in number of integrated copies was quantified by densitometry of appropriate autoradiograms (see Materials and Methods). The results showed about 7-fold more integrated SNV DNA per cell in acutely infected chicken cells than in chronically infected chicken cells (data not shown), indicating a loss of the majority of the integrated SNV DNA present in acutely infected cells. SNV DNA is integrated at multiple sites in chronically infected chicken cells Integration of SNV DNA in chronically infected chicken cells also is at multiple sites. This conclusion is evident from the broad size distribution of DNA fragments that contain SNV DNA sequences that are generated by EcoRI digestion of DNA from chronically infected cells (Fig. 2, lane C and Fig. 4, lane C). When the same experiment was carried out with DNA from chronically infected pheasant or turkey cells, similar results were obtained; that is, after digestion with EcoRI, the resultant DNA fragments that hybridized with SNV RNA were of a broad size range (data not shown). Nonintegrated SNV DNA (migrating as a linear DNA molecule with molecular size of 6 Mdal) was detected in all preparations of DNA extracted from chronically infected cells (Fig.
1, lanes E, G, and I, and Fig. 2). Other experiments showed that infectious nonintegrated SNV DNA persists in chicken cells as late as 6 weeks after infection (data not shown). To demonstrate further that SNV integrates at multiple sites in DNA from chronically infected chicken cells, an attempt was made to resolve the broad band of SNV-specific hybridization into discrete bands of different molecular sizes. DNA from chronically infected chicken cells was digested with EcoRI and electrophoresed through an agarose gel. The gel was sliced into segments, and DNA was eluted from each slice, re-electrophoresed through an agarose gel, transferred to nitrocellulose paper, and hybridized to labeled viral RNA. The results (Fig. 2) show that SNV-containing DNA fragments can be resolved into discrete size classes. Furthermore, each one of the hybridizable fractions shown in Fig. 2 is itself probably composed of more than one DNA species, judged from the broadness of the bands in respect to the broadness of a band composed of a single molecular species of comparable intensity (for example, in lane H of Fig. 2 both a sharp band of nonintegrated SNV DNA and a broader band of integrated SNV DNA molecules are seen). To establish that the hybridizable DNA fragments that are larger than 6 Mdal that are visualized in different fractions of the autoradiogram shown in Fig. 2 are indeed integrated SNV DNA sequences flanked by cellular DNA sequences, we carried out the following experiment: DNA from a large population of chronically infected cells was digested with EcoRI and the DNA fragments were separated into size classes as described in the legend of Fig. 2. Samples of DNA recovered from each of these fractions were then digested with restriction endonuclease Bgl II, and the resulting fragments were assayed by the blotting technique for their content of SNV DNA. Preliminary experiments have shown that Bgl II introduces two cuts in SNV DNA, generating fragments of 3.5, 1.4, and 1.2 Mdal. These fragments were placed in order, and the 3.5Mdal fragment was found to be the middle one (unpublished data). The rationale of the experiment was that if the hybridization in the original DNA fragments from the experiment of Fig. 2 is of SNV DNA that is flanked by cellular DNA sequences of different lengths, a discrete fragment of 3.5 Mdal will appear after Bgl II digestion. The results of this experiment are shown in Fig. 3. A 3.5-Mdal band was observed in the three DNA fractions that were tested. In each of the fractions tested, additional hybridization was also observed over a wide range of molecular sizes. The latter hybridization is most likely the result of that fraction of the SNV genome that corresponds to the two terminal Bgl II fragments. The detection of this hybridization spread over a wide range rather than as discrete hybridization bands is what one expects from integration at multiple sites. Little hybridization was detected in the region between 3.5 and 4.9 Mdal. This result may indicate that in the population of different DNA fragments that contain SNV DNA produced by EcoRI digestion certain molecular species are more abundant than others. For example, from the pattern shown in lane C of Fig. 2 (where DNA fragments of molecular size 9.5-12.0 Mdal were digested with Bgl II), it may be that the provirus is more abundantly integrated at a distance of 3.5-3.7 Mdal from one of the EcoRI sites. SNV DNA molecules integrated at different sites in
chronically infected chicken cells are not equally infectious
Previous experiments from this laboratory have shown that when DNA from chicken cells chronically infected with SNV
Genetics: Keshet and Temin A
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Proc. Natl. Acad. Sci. USA 75 (1978) C
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FIG. 3. Digestion with BgI of EcoRI-generated DNA fragments of different sizes derived from DNA of chronically infected Ch(SNV). Samples of DNA from three different fractions of EcoRI-digested DNA from chronically infected Ch(SNV) were digested with Bgl II. The DNA samples were from the same fractions shown in Fig. 2 (prepared as described in the legend of Fig. 2). The DNAs used in lanes B, C, and D correspond to DNAs from lanes E, F, and G of Fig. 2, respectively. Three micrograms of DNA was digested with 5 units of Bgl II for 4 hr. Electrophoresis, nucleic acid hybridization, and autoradiography were done as before. DNA from Ch(SNV) from the supernatant fraction of a Hirt extraction and digested with Bgl II was electrophoresed in lane A. The DNA fragment of 4.9 Mdal in lane A is a product of incomplete digestion.
is digested with EcoRI, electrophoresed, and assayed for infectivity, the infectious molecules are distributed in a narrow range of molecular size (6, 7). On the other hand, when DNA from chicken cells chronically infected with SNV is digested with EcoRI and assayed for the presence of SNV DNA sequences, SNV DNA sequences are distributed in a wide range of molecular sizes (Fig. 2). This difference indicates that there might exist, in chronically infected chicken cells, integrated SNV copies that are not infectious or that are much less infectious than SNV copies integrated at other sites of the chicken genome.
This hypothesis was tested by a direct comparison of the size distribution of DNA molecules that hybridize with SNV DNA (quantified by a densitometry tracing of an autoradiogram) and of the infectivity of DNA fragments eluted from the corresponding gel slices (quantified by an endpoint dilution assay and determination of ID50 values). DNA from chronically infected chicken cells was first purified in an agarose gel to remove unintegrated SNV DNA; it was then digested with EcoRI and electrophoresed in two parallel lanes. The lane used for infectivity assays was sliced into 30 2-mm-wide slices. DNA was eluted and used in a standard transfection assay. DNA from the parallel lane was blotted and hybridized with labeled SNV RNA. The results are shown in Fig. 4. DNA fragments that contain SNV nucleotide sequences (evident by nucleic acid hybridization) are distributed over a wide size range. However, infectivity is restricted to a narrow size range. This pattern of size distribution of infectious DNA molecules with a peak at 10-11 Mdal was reproducibly obtained in this study and is essentially the same pattern obtained by Battula and Temin in previous studies (6, 7). Thus, these results
8
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FIG. 4. Digestion of DNA from chronically infected Ch(SNV) with EcoRI-comparison of the size distribution of hybridizable fragments with the size distribution of infectious DNA fragments. DNA from chronically infected Ch(SNV) was purified by electrophoresis in a 0.4% agarose gel in order to remove unintegrated SNV DNA. High molecular size DNA (over 50 Mdal) was eluted and recovered from the gel as described in the legend of Fig. 2. DNA was digested with EcoRI under the conditions described in the legend of Fig. 1, and two aliquots of 25 gg of EcoRI-digested DNA were electrophoresed in two parallel lanes of a 0.4% agarose gel. The size distribution of the infectious DNA fragments was determined by slicing one of the gel lanes into 2-mm-wide slices. DNA was eluted from each of the slices and assayed for infectivity as described (6). The results are expressed as ID50 values (0). The size distribution of the hybridizable DNA was determined by transfer of the DNA fragments from the parallel gel lane (lane C) onto a nitrocellulose filter, nucleic acid hybridization, and autoradiography. DNAs from uninfected chicken cells (lane B) and unintegrated SNV DNA (lane A) were also treated the same way. Densitometry tracings of the autoradiograms are shown for the relative quantification of integrated SNV DNA of each molecular size. For direct comparison, infectivity and hybridization profiles were superimposed on the same scale of molecular size (deduced from the mobility of size markers electrophoresed in parallel).
indicate that not all integrated SNV copies are equally infectious, and that the infectious molecules after EcoRI digestion migrate in a uniform manner. DISCUSSION The production of avian reticuloendotheliosis viruses in avian cells.occurs in two phases. In the first phase, acute infection, virus production is accompanied by cell death and cytopathic effects. In the second phase, chronic infection, virus is produced without cell death or cytopathic effects. Concurrent with the transition from the acute to the chronic phase of infection, there is a shift in the pattern of integration of SNV DNA. The results reported here show that the majority of the integrated viral DNA present in acutely infected cells is absent from chronically infected cells. This finding is consistent with the previous finding of a loss in infectivity of the integrated viral DNA during the transition from the acute to the chronic phase of infection (2) and indicates that this loss is not a result of inactivation of integrated viral DNA, but is primarily a loss of integrated DNA molecules from the DNA of the infected cells. Two mechanisms can account for such a loss: excision of viral DNA from the cell genome or a selection process in which those cells that have a large number of integrated viral DNA copies die. Under the latter hypothesis, the cells that survive and es-
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tablish a chronic infection are those that have integrated a smaller number of viral DNA copies. We favor the latter mechanism, since there is a correlation between the decrease in number of integrated viral DNA molecules and cell death, and since this mechanism does not necessitate a specific excision mechanism. The decrease in number of integrated SNV copies per cell during the transition from acute to chronic infection that is reported here (about 7-fold) is roughly the same as the decrease in the amount of infectious DNA molecules per cell that was previously reported (2). The finding of a similar loss of infectivity and of hybridizable DNA combined with the finding that in chronically infected cells not all integrated SNV DNA molecules are infectious indicates that also in acutely infected cells not all integrated SNV copies are equally infectious. Both infectious and noninfectious integrated SNV DNA molecules were found in chronically infected chicken cells. Previous studies have shown that all chronically infected chicken cells produce infectious virus (10). From other experiments using nucleic acid hybridization in liquid it was estimated that in chronically infected chicken cells there is an average of five SNV DNA copies per genome (14). It seems likely, therefore, that most of the chronically infected cells have multiple integrated viral DNA copies and that only one of them leads to production of infectious virus. It also seems likely that the noninfectious molecules are integrated in different sites in different cells. The difference in specific infectivities of integrated SNV DNA molecules may have several causes. The host DNA sequences that are covalently linked to the viral DNA may play a role in determining the infectivity of the fragment. An example of a lack of infectivity of DNA fragments that contain a complete nondefective endogenous provirus is the RAV-0 in DNA from V + tvbr chicken cells (20). It was suggested there that the cellular DNA sequences that flank the provirus and that determine its activity in a transfection assay are acting as a control element for viral expression (20). If the situation in SNV-infected chicken cells reflects an analogous situation for an exogenous virus, then viral DNA integrated at particular sites might have different biological effects from viral DNA integrated in other sites. It is also possible that the occurrence of noninfectious integrated viral copies is a result of integration of defective genomes, either incomplete genomes or rearranged DNA molecules. Another possibility is integration of viral genomes in the wrong orientation. Experiments that can distinguish between these possibilities are now feasible.
Infectious DNA fragments generated by digestion with EcoRI of chronically infected cell DNA migrate in a uniform manner when electrophoresed in agarose gels. However [as was previously discussed (7)], different DNA fragments of the same length or DNA fragments that differ slightly in length cannot be resolved under these conditions. It is not certain, therefore, that infectious SNV DNA is integrated at one site in chronically infected cells. Moreover, SNV DNA integrated at additional sites might also be slightly infectious, but escape detection as a result of the limited sensitivity of the assay. In any case, it is clear that there is a shift from the complex distribution of infectivity in acutely infected cells to the simple distribution of infectivity found in chronically infected cells. We thank Drs. B. Sugden, L. Estis, and J. O'Rear for comments on the manuscript, Drs. J. Mertz and S. Mizutani for enzymes, and S. Hellenbrand and S. Kuo for excellent technical assistance. This investigation was supported by Research Grant CA-07175 from the National Cancer Institute. H.M.T. is an American Cancer Society Research Professor. 1. Varmus, H. E., Vogt, P. K. & Bishop, J. M. (1973) Proc. Natl. Acad. Sci. USA 70,3067-3071. 2. Fritsch, E. & Temin, H. M. (1977) J. Virol. 21, 119-130. 3. Khoury, A. T. & Hanafusa, H. (1976) J. Virol. 18,383-400. 4. Ali, M. & Baluda, M. A. (1974) J. Virol. 13, 1005-1013. 5. Dastoor, M. N., Shoyab, M. & Baluda, M. A. (1977) J. Virol. 21, 541-547. 6. Battula, N. & Temin, H. M. (1977) Proc. Natl. Acad. Sci. USA 7. 8. 9. 10.
74,281-285. Battula, N. & Temin, H. M. (1978) Cell 13,387-398. Southern, E. M. (1975) J. Mol. Biol. 98,503-517. Temin, H. M. & Kassner, V. K. (1974) J. Virol. 13,291-297. Temin, H. M. & Kassner, V. K. (1975) J. Gen. Virol. 27, 267-
274. 11. Kang, C.-Y. & Temin, H. M. (1973) J. Virol. 12, 1314-1324. 12. Sumegi, J., Breedveld, D., Hossenlopp, P. & Chambon, P. (1977) Biochem. Biophys. Res. Commun. 76,78-85. 13. Botchan, M., Topp, W. & Sambrook, J. (1976) Cell 9, 269-
287. 14. Kang, C.-Y. & Temin, H. M. (1974) J. Virol. 14, 1179-1188. 15. Tereba, A. & McCarthy, B. J. (1973) Biochemistry 12, 46754679. 16. Laskey, R. A. & Mills, A. D. (1977) FEBS Lett. 82,314-316. 17. Graham, F. L. & Van der Eb, A. J. (1973) Virology 52, 456467. 18. Reed, L. J. & Muench, H. (1938) Am. J. Hyg. 27,493-497. 19. Hirt, B. (1967) J. Mol. Biol. 26,365-369. 20. Cooper, G. M. & Temin, H. M. (1976) J. Virol. 17,422-430.
