State of viral DNA in BK virus-transformed rabbit cells

Semipermissive rabbit liver, brain, and kidney cells were transformed by BK virus (BKV). All cells of the three resulting cell lines produced BKV T antigen. Viral DNA was detected by DNA-DNA reassociation kinetics and by blot-transfer hybridization. Hybridization patterns were different for the three lines, indicating a different mode of integration for each line. In addition to integrated viral DNA, two of the lines contained also free viral DNA sequences, which in one case were defective viral genomes. Absence or splitting of particular regions of the viral genome was not a necessary condition for the maintenance of the transformed state. HindIII-generated segment A, which contains all the sequences coding for the late viral proteins, was absent (in an intact form) in the only line from which virus could not be rescued.     

Discovery of the sequences of herpes simplex virus type 1 in the genomes of normal and transformed mouse and hamster cell lines

Sequences capable of hybridization with cloned fragments of herpes simplex virus type I (HSVI) DNA were found in genome DNA of Syrian hamster fibroblast lines transformed either by intact HSVI genome (cell line 14.012.81) or this virus DNA fragments (cell line EH/A44). The method of pinpoint hybridization demonstrated the presence in DNA of the cell lines under study of sequences capable of hybridization both with unique areas and with HSVI genome replicas. A correlation was established between the tumorigenic properties of the transformed lines and the number of HSVI sequences present in genomes of these lines. The genome of mouse cell line BaLB/3T3 was found to contain sequences homologous to HSVI replica areas, their number exceeding that of virus sequences present in the genome of nontransformed hamster cells (BHK/21 line).     

Absence of BK virus sequences in transformed hamster cells transfected by human tumor DNA

In an attempt to gain insight into the mechanism of oncogenic transformation by BK virus (BKV), a human papovavirus, we have probed for BKV sequences in transformed hamster cells in which oncogenic transformation had occurred as a result of transfection by human tumor DNA positive for BKV sequences. Even though the sources of the transfecting DNA contained BKV sequences, the transformed hamster cells which arose from the transfection for the most part did not retain BKV sequences. In only one barely detectable case was BKV-specific DNA found associated with chromosomal DNA, and in only a small minority of the transformed cells was BKV DNA detected in the Hirt supernatant, indicating an episomal configuration. Even in these few cases where BKV sequences were present in an episomal form, altered migration on gels of some BKV-positive bands (compared to bands derived from cloned viral DNA) suggested deletions and rearrangements of BKV DNA. We employed several different probe methodologies for these studies, including nick-translation, random primer and a non-isotopic biotinylated probe which gave a sensitivity that could detect better than 0.01 copy of viral genome per diploid cell. We conclude that transformation by transfection with human tumor DNA does not require persistence of the BKV viral genome, suggesting that either BKV virus was irrelevant to original oncogenesis, in analogy with models proposed by others for herpesvirus oncogenesis.     

The polyoma virus 100K large T-antigen is not required for the maintenance of transformation

Thirteen Rat-1 cell lines that were transformed in vitro by polyoma virus (PY) were examined for the following: the state of the viral sequences present (by blot hybridization (E. Southern, 1975, J. Mol. Biol.98, 503–515); the T-antigen species synthesized; the ability to display a transformed phenotype in vitro; and the ability to induce tumors in vivo. Eleven of the lines contained free viral genomes in addition to integrated viral sequences. Two lines (82-Rat and 53-Rat) contained no free viral DNA and only a single insert of integrated viral DNA. The alignment of the integrated viral sequences in these two lines was deduced by restriction enzyme analysis and blot hybridization as previously described by Botchan et al. (M. Botchan, W. Topp, and J. Sambrook. 1976. Cell9, 269–287). The viral sequences were found to be integrated into different regions of the rat cell DNA via linkages at different points on the viral genome. The integrated viral sequences in both lines were present in partial head-to-tail duplications. Neither line contained a complete early region. Discontinuities, either as a result of deletion or fusion to host sequences, affected the region which codes for the C-terminal portion of the 100K large T-antigen and which includes the position where the TS-A mutants have been mapped (L. K. Miller and M. Fried, 1976, J. Virol.18, 824–832). In both lines at least one copy of the region coding for both the 55K middle and 22K small T-antigen species was apparently unaltered. As expected from the maps of the integrated viral sequences neither line synthesized a full-size 100K large T-antigen but the 55K middle and 22K small T-antigens were both produced. Upon fusion to permissive mouse cells virus could be rescued readily from PY transformed lines containing a 100K large T-antigen and free viral genomes. However, with the 53-Rat line virus was rescued in reduced yield and with 82-Rat no rescue was detected. The 82-Rat and 53-Rat lines could not be distinguished from the other PY lines studied in their transformed phenotype in vitro or their ability to form tumors in vivo. Thus we find that the presence of the 100K large T-antigen (A gene product) is not essential for the maintenance of transformation in the 82-Rat and 53-Rat cell lines. Together with our previous studies in which we found that a Rat-1 cell line containing hr-t viral genomes in both integrated and free forms displayed a normal phenotype and synthesized a functional 100K large T-antigen but not the 55K middle nor 22K small T-antigen species (L. Lania, M. Griffiths, B. Cooke, Y. Ito, and M. Fried, 1979, Cell. 18, 793–802) our data strongly suggest that it is the 55K middle and/or 22K small T-antigen species that are required for the maintenance of PY mediated transformation. The role of the 100K large T-antigen (A gene product) in the initiation of transformation and the excision of PY viral sequences is discussed.     

Rescue of BKV from BKV-transformed hamster, rat, and mouse cells: correlation with levels of nonintegrated viral DNA

Infectious BKV was rescued from 39 of 40 lines of virus-free, BKV-transformed hamster, rat, and mouse cells, which had been either maintained continuously in culture or reestablished in culture after one or more passage in the appropriate host, by Sendai virus-catalyzed fusion with permissive cells. Striking differences were observed among the 39 lines with respect to the efficiency of virus rescue. Fourteen of the lines were examined for the presence of nonintegrated viral DNA by dot-blot hybridization. The values obtained, which ranged from less than 1 to 2880 viral genome equivalents/cell, reveal a strong correlation between the efficiencies with which BKV can be rescued from these lines and the amounts of free viral DNA that they contain.     

Stable transformation of mouse, rabbit and monkey cells and abortive transformation of human cells by BK virus, a human papovavirus.

Semi-permissive mouse, rabbit and monkey cells were stably transformed by BK virus (BKV). The specificity of transformation was demonstrated by the presence of BKV tumour (T) antigen in nuclei of transformed cells and by virus rescue with Sendai virus-mediated fusion or transfection. Two out of seven BKV-transformed cell lines were oncogenic. Permissive human cells were only abortively transformed by BKV, since morphologically modified cells persisted in culture for a few passages and eventually died.     

Comparison of wild-type BK virus DNA and BK virion DNA rescued from virus-transformed BHK cells

We have examined some biological and biochemical properties of the human papovavirus, BKV, and its DNA which was rescued from baby hamster kidney cells (BHK-21) transformed by the wild-type BKV. A particular clone of transformed BHK-21 cells, BK-BHK-L29, was selected for study since BKV T antigen could not be detected in the nuclei of these cells by histochemical assays but did release infectious virus following fusion with permissive human embryonic kidney cells. Comparison of the genome from the rescued virus with a plaque-purified, prototype BKV DNA was done by restriction endonuclease cleavage analysis and molecular hybridization. In these tests, the rescued virus and the viral DNA were indistinguishable from the wild-type, parental virus and its DNA. Immunoprecipitation experiments performed with transformed BHK cells did not demonstrate a 94K or large T BKV protein when analyzed on SDS-polyacrylamide gels.     

Organization of polyoma virus DNA in mouse tumor cell lines

Summary Restriction mapping of polyoma virus DNA in mouse tumor cell lines gave patterns that varied with the cell line examined. These reflected differences in both the organization and the state of integration of virus genomes in the host chromosomes. (The cell lines were derived from tumors induced by polyoma virusin vivo and were propagated continuously in culture.) Two of the cell lines contained multiple copies of tandemly integrated virus genomes as well as free virus DNA molecules. Two other cell lines appeared to contain only integrated virus genomes arranged as tandem repeats. Based on restriction analysis with eleven different endonucleases, the virus DNA in one of the cell lines containing both free and integrated virus genomes was not detectably defective or hypermethylated. This is in contrast to most previously described polyoma virus transformed mouse cells. These virus genomes may, however, contain point mutations or unobserved rearrangements. The second cell line possessing free virus DNA molecules contained both nondefective and defective virus genomes. Most, if not all, defective virus genomes in this line were integrated. The two other cell lines possessing only detectable integrated virus DNA apparently contained only defective virus genomes. The defect in both cases was a small deletion (0.2 kb) encompassing 0.12 map units on the physical map of polyoma virus DNA, a region coding for the proximal part of the large T antigen. Moreover, in contrast to the cell lines with free and detectably nondefective virus DNA, the virus DNA was extensively methylated in cell lines containing only integrated and defective virus genomes.With 6 Figures     

Genetic heterogeneity of the human papovaviruses BK and JC

We have examined the structure and infectivity of BKV and JCV genomes from prototype strains after cell culture passage and of BKV genomes from primary isolates. Genomic structures were determined by restriction endonuclease analysis of molecularly cloned DNA. Infectivity was determined by transfection of the cloned genomes into urine-derived epithelial cells and assaying for viral proteins and virus production. Prototype BKV DNA, which was cloned after 14 passages in three different cell lines, contained no alterations in restriction enzyme sites and was infectious. In contrast, prototype JCV acquired changes in the late region of the genome during passage in cell culture and the cloned DNA was not infectious. Urine-derived cells were used to isolate virus from the urine of two renal transplant patients and one asymptomatic individual. The genome of the virus isolated from the normal individual was indistinguishable from prototype BKV except for a 60-base pair deletion, which was localized between 0.62 and 0.72 map units. Two isolates from transplant patients differed from each other and from prototype BKV at a number of restriction enzyme cleavage sites located in the early region and were infectious. Genomes containing deletions from 100 to 600 base pairs were also cloned but were not infectious.     

Simian virus 40-Chinese hamster kidney cell interaction II. The semipermissivity of the cell system

Summary Throughoutin vitro passages, Chinese hamster kidney (CHK) cells progressively lost susceptibility to SV 40 virus infection while remaining continuously susceptible to viral DNA infection. Upon infection with SV 40 virus or viral DNA, the CHK cell line supported viral DNA and virus replication at a low level. SV 40-transformed CHK cell lines spontaneously produced small amounts of viral DNA and virions. The percentage of virus-producing cells was low. Various clones derived from each of these lines behaved as the parental cell population, leading to the conclusion that each CHK cell, whether transformed or not with SV 40, is potentially permissive for this virus.With 4 Figures     

Fate and expression of simian virus 40 DNA after introduction into murine cells under nonselective conditions

When SV40 infects mouse cells, it does not replicate but instead causes neoplastic transformation of a small percentage of the cells. It is unknown, however, what happens to the virus in those cells that do not become transformed. We introduced SV40 into mouse cells by nonselective means, either by cotransfection of SV40 DNA with a selectable marker or by random cloning of SV40-infected cells. We analyzed the fate of viral DNA sequences, expression of T antigens, and transformation properties of these cells. We found that, upon infection, viral DNA integration occurs at a frequency that is at least 10-fold higher than the frequency of transformation. The majority of these cells are not transformed due to lack of expression of T antigen. One cell line which expresses a truncated T antigen is not transformed. We have mapped the viral sequences in the genome of these cells and find that integration in the large T intron is probably responsible for the defect. Lack of transformation can therefore be attributed to both cellular and viral factors, namely, introduction of viral DNA into cells that are resistant to transformation or integration of viral DNA in such a way that T antigen expression is prohibited.     

Occurrence of free, defective viral DNA in a hamster tumor induced by human papovavirus BK

We characterized viral DNA present in an osteosarcoma cell line (Os-513), which had been originally induced in a hamster with human papovavirus BK (BKV), by hybridization of ³²P-labeled BKV DNA to DNA extracted from cells, undigested or digested with various restriction endonucleases, fractionated by electrophoresis, and transferred to a nitrocellulose filter. Os-513 was found to contain free viral DNA and high molecular weight DNA with viral sequences. The free viral DNA seemed to be defective, for its size was about two-thirds of the wild type. The deletion, one-third of the entire BKV DNA, extended over a segment between map positions 0.72 and 0 which includes the leader and intervening sequences and part of the body sequences for late mRNAs. The high molecular weight DNA with viral sequences appeared to be composed of tandem, head-to-tail repeats of the same defective BKV DNA as the free DNA.     

Events preceding stable integration of SV40 genomes in a human cell line

We have examined the organization of integrated SV40 sequences in an uncloned population of a transformed human fibroblast cell line. Somatic cell hybrids between mouse B82 cells and human GM847 cells were examined for SV40 T-antigen expression and individual human chromosome presence. This analysis revealed that a functional SV40 genome is located on human chromosome 7. Restriction endonuclease digestion followed by blot hybridization of the parental human cell line revealed that it contains mutliple normal and defective SV40 copies integrated into the host genome in tandem. A similar analysis of several T-ag⁺ hybrid cell lines indicated that the integrated viral sequences in different hybrid cell lines (thus in different cells of the original population) are very closely related but not always identical. Analysis of subclones of GM847 also revealed such differences. Based upon these results, we postulate that following the initial integration event, viral as well as the flanking host DNA sequences become unstable and are subject to deletions and rearrangements. This short-lived structural instability is followed by highly stable integration of SV40 which is maintained in these cells or their hybrid derivatives for at least hundreds of cell generations.     

Oncogenity of BK virus for immunosuppressed hamsters

Summary Tumors were induced by BK virus (BKV) inoculated intravenously in 3-week-old Syrian golden hamsters immunosuppressed with anti-lymphocyte serum or methylprednisolone acetate alone or in association with -radiation (⁶⁰Co). The induced neoplasms were ependymoma, carcinoma of pancreatic islets, lymphoma, osteosarcoma, undifferentiated sarcoma, kidney and renal pelvis carcinoma, pheochromocytoma and hemangiosarcoma. High levels of insulin and glucagon and altered concentrations of glucose were detected in blood of animals with tumors of pancreatic islets. No antibodies to BKV tumor antigen (T Ag) and low levels of hemagglutination-inhibiting antibodies to BKV viral coat protein Ag were detected in hamster sera. BKV T Ag was found in tumors by complement fixation. Blot hybridization analysis of tumor DNA showed the presence of both free and integrated BKV genomes in tumor cells. BKV DNA inoculated intravenously and subcutaneously in immunosuppressed or immunocompetent hamsters was not oncogenic, whereas it was weakly oncogenic when inoculated intracerebrally.With 3 Figures     

Nonintegrated viral DNA in rat cells doubly transformed by SV40 and polyoma virus.

Rat F2408 cells transformed by polyoma virus contain, in addition to integrated viral genomes, a small number (an average of 20–50 copies per cell) of nonintegrated viral DNA molecules. On the other hand, SV40-transformed rat cells contain only integrated viral genomes. SV40-transformed rat cells also differ from the polyoma transformants, in that they grow in soft agar medium at a much slower rate. Cells doubly transformed by polyoma and SV40 can be easily isolated following polyoma superinfection of SV40-transformed rat cells, as their rate of growth in agar is enhanced. These doubly transformed cells yield polyoma or SV40, respectively, after fusion with cells permissive for each virus. However, only polyoma-specific DNA sequences can be detected in these cells in a “free” state.     

Excision of polyoma virus DNA from that of a transformed mouse cell: identification of a hybrid molecule with direct and inverted repeat sequences at the viral-cellular joints

Excision (and replication) of the integrated viral DNA is induced when mouse cells transformed by an early thermosensitive mutant of polyoma (Py) virus (Cyp cells) are transferred from the restrictive temperature of 39° at which they are propagated, to that of 33°. In a number of clones derived from the Cyp line, the low-molecular-weight DNA generated after such an induction is represented almost exclusively by nondefective, monomeric, Py genomes (5.3 kb). In one clone, however, this DNA was found to consist predominantly of copies of a cyclic molecule of approximately 7.1 kb (Rm I). This molecule was shown to include sequences colinear with most, if not the whole, of Py DNA, as well as other sequences that presumably originated from those flanking the integrated viral DNA. The data reported here demonstrate that Rm I is a hybrid molecule comprising 1.03 copies of Py DNA and about 1.6 kb of mouse DNA, joined in a unique fashion: whereas the mouse DNA insertion terminates with an imperfect inverted repeat of 7 bp, it is connected with viral by 3273 at one end, and with viral by 3092 at the other end. As Rm I is colinear with Py DNA from by 3092 to 5292/1, and from by 1 to by 3273, it follows that the cellular insertion is flanked by a direct viral repeat of 182 bp. These results strongly suggest that a selective mechanism, necessarily distinct from nonspecific homologous recombination, is involved in the excision of Py DNA in induced Cyp cells.     

Characterization of human papovavirus RFV: use of iodinated viral DNA to detect viral DNA sequences in cellular DNA.

The amount of viral DNA present in a viral transformed cell line has been determined using saturation reassociation of cellular DNA and ¹²⁵I-labeled viral DNA. The virus, RFV, is a new human papovavirus which was used to transform hamster embryo cells in vitro. Only 40% of the viral genome is present in the transformed cells and less than 5% was present in cells from normal hamster livers. The high specific activity of the [¹²⁵I]RFV DNA permitted the quantitative determination of RFV sequences present using less than 260 μg of cellular DNA.     

Characterization of the integration site of the CMV mtr in a tumor cell line

Previous studies have shown that a 558-bp fragment of human cytomegalovirus (CMV) DNA contained within pCM4127 and designated CMV mtr can morphologically transform rodents cells in vitro. By cotransformation with pCM4127 and a plasmid conferring G418 resistance, pSV2neo, morphologically transformed NIH3T3 cell lines were isolated. Dot blot hybridization indicated that approximately 30% of the transformants retained CMV sequences. Two cell lines which retained viral DNA were chosen for further study. They were capable of anchorage independent growth and formed tumors in nude mice. Integrated viral sequences in the transformants and tumor cell lines were analyzed by Southern blotting. A bacteriophage lambda library was constructed using a tumor cell line which retained a single copy of the viral sequences, and a phage was isolated which contained the integrated plasmid and the flanking cellular sequences. A complex rearrangement between pCM4127 and pSV2neo had occurred. DNA sequence analysis showed integration of the plasmid sequences into repetitive mouse DNA and identified an adjacent mouse sequence.