Evidence for integrated EBV genomes in Raji cellular DNA.

Human lymphoid cell lines cannot be grown in long-term tissue culture, as a rule, unless the cells have been transformed by Epstein-Barr virus (EBV). The latent EBV DNA in established cell lines, is mainly present as free covalently closed circles but viral DNA sequences with properties of integrated DNA also seem to be present. We have extended the studies on the physical state of the EB viral DNA sequences in the cell line Raji which appear at a lower density than that for free EB viral DNA during fractionation on CsCl density gradients. In such material a novel EcoRI EBV DNA fragment is present, which hybridizes to viral sequences homologous to EcoRI A. This fragment is not present in free covalently closed circular EBV DNA. When this EcoRI fragment is further analysed with HindIII a smaller fragment than expected, which contains BamHI W sequences, is detected. The demonstration of this HindIII fragment and its characteristics as a joint, viral-host chromosome fragment will be discussed.     

Integration Sites of Adenovirus Type 12 DNA in Transformed Hamster Cells and Hamster Tumor Cells

The patterns and sites of integration of adenovirus type 12 (Ad12) DNA were determined in three lines of Ad12-transformed hamster cells and in two lines of Ad12-induced hamster tumor cells. The results of a detailed analysis can be summarized as follows. (i) All cell lines investigated contained multiple copies (3 to 22 genome equivalents per cell in different lines) of the entire Ad12 genome. In addition, fragments of Ad12 DNA also persisted separately in non-stoichiometric amounts. (ii) All Ad12 DNA copies were integrated into cellular DNA. Free viral DNA molecules did not occur. The terminal regions of Ad12 DNA were linked to cellular DNA. The internal parts of the integrated viral genomes, and perhaps the entire viral genome, remained colinear with virion DNA. (iii) Except for line HA12/7, there were fewer sites of integration than Ad12 DNA molecules persisting. This finding suggested either that viral DNA was integrated at identical sites in repetitive DNA or, more likely, that one or a few viral DNA molecules were amplified upon integration together with the adjacent cellular DNA sequences, leading to a serial arrangement of viral DNA molecules separated by cellular DNA sequences. Likewise, in the Ad12-induced hamster tumor lines (CLAC1 and CLAC3), viral DNA was linked to repetitive cellular sequences. Serial arrangement of Ad12 DNA molecules in these lines was not likely. (iv) In general, true tandem integration with integrated viral DNA molecules directly abutting each other was not found. Instead, the data suggested that the integrated viral DNA molecules were separated by cellular or rearranged viral DNA sequences. (v) The results of hybridization experiments, in which a highly specific probe (143-base pair DNA fragment) derived from the termini of Ad12 DNA was used, were not consistent with models of integration involving true tandem integration of Ad12 DNA or covalent circularization of Ad12 DNA before insertion into the cellular genome. (vi) Evidence was presented that a small segment at the termini of the integrated Ad12 DNA in cell lines HA12/7, T637, and A2497-3 was repeated several times. The exact structures of these repeat units remained to be determined. The occurrence of these units might reflect the mechanism of amplification of viral and cellular sequences in transformed cell lines.     

Herpesvirus saimiri DNA in tumor cells--deleted sequences and sequence rearrangements.

Herpesvirus saimiri DNA in continuous lymphoblastoid cell lines obtained from viral induced tumors in marmosets has been analyzed by gel electrophoresis of restricted DNA. Southern transfer to nitrocellulose filters, and hybridization to 32P-labeled viral DNA or DNA fragments. The viral DNA fragments EcoRI-G, -H, -D, and -I, KpnI-A, and BamHI-D and -E were not detected in Southern transfers of DNA from the nonproducing 1670 cell line. For each restriction endonuclease, a new fragment appeared, consistent with a 13.0-megadalton deletion of viral DNA sequences. This deletion encompassed 35 to 48 +/- 0.6 megadaltons from the left end of the unique DNA region. A sequence arrangement map is presented for the major population of H. saimiri DNA sequences in the 1670 cell line. Although H. saimiri DNA in the nonproducing 70N2 cell line can be distinguished from viral DNA in the 1670 cell line by several criteria, the same sequences were found to be deleted in the major population of viral DNA molecules. Unlike 1670 and 70N2 cells, restricted DNA from the virus-producing cell lines 77/5 and 1926 contained all of the DNA fragments present in the parental virion DNA. DNA from 1670, 70N2, and 77/5 cells contained additional viral DNA fragments that did not comigrate with any virion DNA fragments. Most of these unexplained fragments were confined to or highly enriched in partially purified circular or linear DNA fractions. DNA from tumor cells taken directly from a tumor-bearing animal contained viral DNA indistinguishable from the parental virion DNA by the assay conditions used. These results indicate that viral DNA sequence rearrangements can occur upon cultivation of tumor cells in vitro and that excision of DNA sequences from the viral genome may play a role in establishing the nonproducing state of some tumor cell lines.     

Relationship between integrated and nonintegrated viral DNA in rat cells transformed by polyoma virus

Fischer rat fibroblasts transformed by polyoma virus contain, in addition to viral sequences integrated into the host genome, nonintegrated viral DNA molecules, whose presence is under the control of the viral A gene. To understand the mechanism of production of the "free" viral DNA, we have characterized the DNA species produced by several rat lines transformed by wild-type virus or by ts-a polyoma virus and compared them with the integrated viral sequences. Every cell line tested yielded a characteristic number of discrete species of viral DNA. The presence of defectives was a very common occurrence, and these molecules generally carried deletions mapping in the viral "late" region. The production of multiple species of free viral DNA was not due to heterogeneity of the transformed rat cell population, and its pattern did not change upon fusion with permissive mouse cells. Analysis of the integrated viral DNA sequences in the same cell lines showed, in most cases, a full head-to-tail tandem arrangement of normal-size and defective molecules. The free DNA produced by these lines faithfully reflected the integrated species. This was true also in the case of a cell line which contained a viral insertion corresponding to approximately 1.3 polyoma genomes, with each of the repeated portions of the viral DNA molecule carrying a different-size deletion. These results support the hypothesis that the free DNA derives from the integrated form through a mechanism of homologous recombination leading to excision and limited replication.     

Free viral DNA in BK virus-induced hamster tumor cells

The biological properties of nine clonal lines of BK virus-induced hamster tumor cells were studied. All clonal lines were oncogenic and showed an enhanced ability to form colonies in semisolid medium. The cells of each clonal line contained T antigen; no virus could be rescued from any of the clonal lines. The number of viral DNA copies was determined in three of the clonal lines and varied from 10 to 20 copies per diploid amount of cell DNA. The state of the viral genome was studied in these lines, and the great majority of the viral DNA molecules appeared to be present as free (nonintegrated) molecules. At least six length classes of free defective BK virus DNA molecules, which all lacked a part of the late region of the genome, were detected in these cells. Three of the six length classes of BK virus DNA molecules acquired a TaqI recognition site, which suggested substitution of cellular DNA.     

Patterns of integration of viral dna sequences in the genomes of adenovirus type 12-transformed hamster cells

The patterns of integration of the viral genome have been analyzed in four hamster cell lines transformed by adenovirus type 12 (Ad12). It has previously been shown that in each of the cell lines HA12/7, T637, A2497-2 and A2497-3, the viral genome persists in multiple copies, and that different parts of the viral DNA are represented non-stoichiometrically (Fanning and Doerfler, 1976). All four cell lines are oncogenic when injected into hamsters.The DNA from each of the cell lines was extracted and cleaved in different experiments with restriction endonucleases Bam HI, Bgl II, Eco RI, Hind III, Hpa II or Sma I. The DNA fragments were separated on 1% agarose slab gels and transferred to nitrocellulose filters by the Southern technique. Ad12 DNA sequences were detected by hybridization to Ad12 DNA, which was ³²P-labeled by nick translation, and by subsequent autoradiography. In some experiments, the ³²P-labeled Eco RI restriction endonuclease fragments of Ad12 DNA were used to investigate the distribution of specific segments of the viral genome in the cellular DNA.For each cell line, a distinct and specific pattern of integrated viral DNA sequences is observed for each of the restriction endonucleases used. Moreover, viral sequences complementary to the isolated Eco RI restriction endonuclease fragments are also distributed in patterns specific for each cell line. There are striking differences in integration patterns among the four different lines; there are also similarities. Because the organization of cellular genes in virus-transformed as compared to normal cells has not yet been determined, conclusions about the existence or absence of specific integration sites for adenovirus DNA appear premature. Analysis of the integration patterns of Ad12 DNA in the four hamster lines investigated reveals that some of the viral DNA molecules are fragmented prior to or during integration. Analysis with specific restriction endonuclease fragments demonstrates that the Eco RI B, D and E fragments, comprising a contiguous segment from 0.17–0.62 fractional length units of the viral DNA, remain intact during integration in a portion of the viral DNA molecules. Although each cell line carries multiple copies of Ad12 DNA, the viral DNA sequences are concentrated in a small number of distinct size classes of fragments. This finding is compatible with, but does not prove, the notion that at least a portion of the viral DNA sequences is integrated into repetitive sequences, or else that the integrated viral sequences have been amplified after integration.In the three cell lines which were tested, the integration pattern is stable over many generations, with continuous passage-twice weekly-of cells for 6–7 months. In the three cell lines which were examined, the integration pattern is identical in a number of randomly isolated clones. Hence it can be concluded that the patterns of integration are identical among all cells in a population of a given line of transformed cells.     

Detection of Epstein-Barr Viral Genome in Nonproductive Cells

Hybridization experiments have shown that EB viral DNA comprises between 0.06 and 1.6% of the total DNA of Burkitt lymphoma cells and established leucocyte cell lines.     

Deoxyribonuclease activity found in Epstein--Barr virus producing lymphoblastoid cells.

A deoxyribonuclease activity from Epstein--Barr (EB) virus producer lymphocyte cell lines which is correlated with viral production and which is not present in virus non-producer or negative lymphocyte cell lines has been purified 220-fold with 20% recovery and characterized. This nuclease copurifies through diethylaminoethylcellulose column chromatography with the EB virus induced deoxyribonucleic acid (DNA) polymerase in EB virus producer cells which was recently reported by this laboratory, but elutes as a separate peak of activity upon phosphocellulose chromatography. This nuclease activity has a sedimentation coefficient of 4.0 S, a strong divalent cation requirement, an alkaline pH optimum, and the ability to utilize both native and denatured lymphocyte DNA as substrate, reducing both to monophosphonucleosides.     

Structure of nonintegrated, circular Herpesvirus saimiri and Herpesvirus ateles genomes in tumor cell lines and in vitro-transformed cells.

Nonintegrated, circular DNA molecules of Herpesvirus saimiri and Herpesvirus ateles were found in five lymphoid cell lines originating from tumor tissues or established by in vitro immortalization of T lymphocytes. The arrangement of unique (L) and repetitive (H) DNA sequences in circular viral genomes was analyzed by partial denaturation mapping followed by visualization with an electron microscope. Three types of circular viral DNA structures were found. (i) The virus-producing cell line RLC, which is derived from an H. ateles-induced rabbit lymphoma, contains circular viral genomes which consist of a single L-DNA and a single H-DNA region, both the same length as in virion DNA. (ii) The circular viral genomes of the nonproducer cell lines H1591 and A1601, in vitro transformed by H. saimiri and H. ateles, respectively, have deletions in the unique L-DNA region and larger H-DNA regions. Cell line A1601 lacks about 8% of virion L-DNA, and H1591 cells lack about 40% of viral L-DNA information. (iii) The nonproducing H. saimiri tumor cell lines 1670 and 70N2 harbor viral genomes with two L-DNA and two H-DNA regions, respectively. Both types of circular molecules have a long and a short L-segment. The sequence arrangements of circular DNA molecules from H. saimiri-transformed cell lines were compared with those of linear virion DNA by computer alignment of partial denaturation histograms. The L-DNA deletion in cell line H1591 was found to map in the right half of the virion DNA. Comparison of the denaturation patterns of both L regions of cell lines 1670 and 70N2 identified the short L regions as subsets of the long L regions. Thus, circular viral DNA molecules of all four nonproducer cell lines represent defective genomes.     

Sequence complexity of circular Epstein-Bar virus DNA in transformed cells

A simplified procedure, based on several methods previously used to isolate circular DNA molecules from bacteria, was derived for the preparation of covalently closed circular viral DNA molecules from large quantities of lymphocytes transformed by Epstein-Barr virus. The protocol can be applied both to virus nonproducer lines and to lines containing cells activated to virus production. Sufficient amounts o highly purified viral DNA of intracellular origin were obtained from B95-8 and Raji cells to allow direct visual analysis of their sequence complexities after cleavage with EcoRI and separation of fragments by gel electrophoresis. No major differences in complexity were observed between circular DNA and linear virion DNA from B95-8 cells. The fragment patterns observed in this fashion agree well with those detected by conventional blotting and hybridization methods. The procedure can also be used as an analytical method to assay for small amounts of circular Epstein-Barr virus DNA molecules in other transformed cells. In this connection, no circular Epstein-Barr virus DNA was detected in Namalva cells.     

Some recent developments in the molecular epidemiology of Epstein-Barr virus infections

We have applied two different recombinant DNA techniques to the study of the epidemiology of Epstein-Barr virus infections. In the first application, cloned subfragments of viral DNA were used as probes to detect EBV DNA in a variety of lymphoproliferative disorders and in lymphoid cell lines. Patients who are epidemiologically unrelated harbor EBV genotypes which can readily be distinguished from each other. Patients who are epidemiologically related (such as mothers and infants) have similar EBV genotypes. Some patients, especially those who are immunocompromised, are infected with two distinct genotypes. In the second application, we have examined the immune response to specific EBV antigens expressed from small cloned viral DNA subfragments. We have identified a group of patients with presumed chronic EBV infection who selectively fail to recognize one subcomponent of the EB nuclear antigen complex.     

State of the viral DNA in rat cells transformed by polyoma virus. I. Virus rescue and the presence of nonintergrated viral DNA molecules.

The interaction of polyoma virus with a continuous line of rat cells was studied. Infection of these cells with polyoma did not cause virus multiplication but induced transformation. Transformed cells did not produce infectious virus, but in all clones tested virus was rescuable upon fusion with permissive mouse cells. Transformed rat cells contained, in addition to integrated viral genomes, 20 to 50 copies of nonintegrated viral DNA equivalents per cell (average). "Free" viral DNA molecules were also found in cells transformed by the ts-a and ts-8 polyoma mutants and kept at 33 C. This was not due to a virus carrier state, since the number of nonintegrated viral DNA molecules was found to be unchanged when cells were grown in the presence of antipolyoma serum. Recloning of the transformed cell lines produced subclones, which also contained free viral DNA. Most of these molecules were supercoiled and were found in the muclei of the transformed cells. The nonintegrated viral DNA is infectious. Its specifici infectivity is, however, about 100-fold lower than that of polyoma DNA extracted from productively infected cells, suggesting that these molecules contain a large proportion of defectives.     

Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function.

Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the "curing" of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for "curing" by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo "curing" with about the same frequency at 33 degrees or 39 degrees C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 degrees than at 39 degrees C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable.     

Mechanism of replication of bacteriophage phi X174 XX. Sensitivity of nascent DNA to single-strand-specific nucleases.

We reported earlier that dephosphorylated nascent phi X174 viral strand DNA molecules were less extensively degraded from the 5' end by spleen exonuclease than were non-nascent molecules. Experiments described here revealed that the insensitivity to the 5'-OH end-specific nuclease was more evident among the longer molecules in the population than among the shorter, all of the molecules being less than unit length in size. The smallest molecules in the population were about as sensitive to the enzyme as the control molecules and hence must possess unblocked 5'-terminal nucleotides. Degradation of the nascent DNA with the 3' end-specific snake venom phosphodiesterase revealed only a small enrichment for [3H]thymidine near the 3' end, seemingly insufficient to account completely for the apparent insensitivity of the longer molecules to spleen exonuclease. When the nascent molecules were isolated without the use of proteolytic enzymes, some pronase-sensitive material was found associated with the DNA, particularly the longer molecules. We suggest that the resistance of the longer nascent (pronase-treated) molecules to spleen exonuclease occurs because they have remnants of the viral gene A or A* protein covalently bound to the 5' end.     

Production of non-defective and defective oligomers of viral DNA in mouse 3T3 cells transformed by a thermosensitive mutant of polyoma virus

Cultures of three lines of mouse 3T3 cells transformed independently by the thermosensitive ts-a mutant of polyoma virus yield virus upon lowering their incubation temperature to 31°C. At 31°C, the internal pools of DNA of all three lines contain not only superhelical viral monomers, but also a small proportion of viral oligomers.From one of these three cell lines, several sublines of different clonal morphology were isolated at 38.5°C. The viral DNA synthesized at 31°C by each different subline displayed a unique oligomer pattern which has been stable through many cell passages and further reclonings. In contrast to the parental line, the monomer in most of these sublines is a minor component of the viral DNA pool. In one subline, more than 80% of the viral DNA consists of superhelical molecules about 1.6-times the size of a monomer. The specific infectivity of these molecules is only about one-tenth that of monomers, whereas the efficiency in transforming hamster (BHK21) cells is about twice that of monomers.     

State and organization of polyoma virus DNA sequences in transformed rat cell lines.

Polyoma virus-transformed rat cell lines were isolated as colonies growing in agar after infection of F2408 cells with low multiplicities of wild-type virus. Viral DNA present in the transformed cells was analyzed by fractionating the cellular DNA on agarose gels before and after digestion with various restriction endonucleases, followed by detection of the DNA fragments containing viral sequences using the procedure described by Southern (E. Southern, J. Mol. Biol., 98:503--515, 1975). Five lines, independently derived, were studied in detail. All five lines, when examined after a minimum number of passages in culture, contained both free and apparently integrated viral DNA. The free polyoma DNA in three of the lines was indistinguishable, by restriction enzyme analysis, from wild-type viral DNA, whereas the two other lines also contained smaller free DNA molecules which lacked parts of the wild-type genome. The integrated DNA in the five lines studies existed as head-to-tail tandem repeats of unit-length polyoma DNA covalently attached to nonviral DNA. The same five polyoma-transformed rat lines were examined after further passage in culture. Free viral DNA was then either undetectable or greatly reduced in amounts, whereas the high-molecular-weight, integrated units persisted after passage of the cells. The subclones, derived from one of the five lines selected for detailed analysis, showed some variations in the quantity and size of the free viral DNA as well as minor alterations in the pattern of the apparently integrated sequences.     

The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells.

Epstein-Barr nuclear antigen 1 (EBNA-1) is the only viral protein required to support latent replication of Epstein-Barr virus (EBV). To assess the likelihood that EBNA-1 regulates the amount of EBV DNA in a cell, we measured the average numbers of EBNA-1 molecules and EBV DNA molecules per cell in different clones of cells. The amount of EBNA-1 protein present in recently established lymphoblastoid cell lines was measured with affinity-purified anti-EBNA-1 antibodies, and viral DNA was measured by nucleic acid hybridization. The average levels of EBNA-1 protein varied little between these cell lines, whereas the average amount of viral DNA present varied substantially; consequently, these numbers were not correlated. There is no apparent relationship between amounts of EBNA-1 and viral DNA.