Vol. 23, No. 1

JOURNAL OF VIROLOGY, July 1977, p. 205-208 Copyright © 1977 American Society for Microbiology

Printed in U.S.A.

NOTES Uniform Representation of the Human Papovavirus BK Genome in Transformed Hamster Cells PETER M. HOWLEY*- AND MALCOLM A. MARTIN Laboratory ofPathology, National CancerInstitute,* and Laboratory ofBiology of Viruses, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20014 Received for publication 25 February 1977

The DNA of three cloned lines of hamster kidney cells transformed by human papovavirus BK DNA was examined by reassociation kinetics for viral sequences and found to contain 2.7 to 5.3 equivalents of viral DNA per diploid genome. In the one line examined with the four R HindIII fragments of the human papovavirus BK genome, the entire viral genome was uniformly represented. Human papovavirus BK (BKV), like other representatives of the papovavirus group, has oncogenic potential. BKV induces malignant transformation of hamster cells in vitro (9, 12) and induces tumors in inoculated hamsters (2, 11, 13). Purified BKV DNA has also been used to transform hamster embryonic kidney cells in culture (15). The association of BKV with such transformed cells has been established by the demonstration of virus-specific intranuclear tumor (T) antigen or the recovery of BKV after fusion with permissive human cells (2, 12, 13, 15). In this communication we have quantitated the amount of viral nucleic acid in three cloned lines of hamster kidney cells transformed by BKV DNA and have found that all regions of the viral genome are nearly equally represented. The transformed hamster kidney lines examined in this study have been characterized previously (15). Each of the three cloned lines contains intranuclear T antigen, when antisera prepared from hamsters bearing BKV- or simian virus 40 (SV40)-induced tumors are used. Although virus is rescuable from the population of transformed cells from which the lines were cloned (15), no attempt was made to rescue virus from the individual cloned lines studied, BK-HK-2, BK-HK-3, and BK-HK-6. The amount of viral DNA present in each of the lines of cells transformed with BKV DNA was determined by examining the influence of large amounts of unlabeled BKV-transformed cell DNA on the rate of reannealing of radiolabeled BKV DNA (3). Cellular DNA was extracted from each of the cloned lines after ap-

proximately 12 passages in tissue culture as previously described (3). 32P-labeled BKV DNA was prepared from human embryonic kidney cells, infected at an input multiplicity of 0.1 PFU/cell with plaque-purified BKV, by differential salt precipitation (6). Supercoiled DNA was purified by isopycnic centrifugation in CsCl-ethidium bromide, and the viral DNA was further purified by sedimentation in a neutral sucrose gradient. The radiolabeled viral DNA as well as the unlabeled cellular DNAs was mechanically sheared at 50,000 lb/in2 in a Ribi Cell Fractionater (Ivan Sorvall, Inc.) to a molecular size of 3.1 x 105 daltons prior to DNA reassociation analyses (3). The 32P-labeled, fragmented BKV DNA was denatured and allowed to reanneal in the presence of a 0.5 M excess of denatured HK-BK-2, HK-BK-3, HK-BK-6, or salmon sperm DNA (Fig. 1). In the presence of salmon sperm DNA the rate of reassociation of the radiolabeled BKV DNA was unaffected; each of the three transformed cell DNAs, however, accelerated the reassociation of the 32P-labeled BKV DNA, indicating that viral genome sequences were present in each of the transformed cell lines. The calculated amount of viral DNA present in the cloned lines, expressed as copies of whole viral genome per diploid cell, is presented in Table 1. The quantity of viral DNA in these three BKV-transformed lines is similar to the amount reported by others in cells transformed by SV40 and polyoma viruses (10). When restriction enzyme cleavage fragments of adenovirus type 2 are used in reassociation experiments to detect and quantitate virus-spe-

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NOTES

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

I

1.0

I/t 2

FIG. 1. Kinetics of reassociation of radiolabeled BKV DNA in the presence of unlabeled DNAs from virus-transformed lines. Fragmented, 32P-labeled BKV DNA (1.53 X 10-3 pg/mi) was reannealed in reaction mixtures containing 0.0025 M EDTA, 0.6 M sodium phosphate buffer (pH 6.8), and 1.03 mg of HK-BK-2 (A), HK-BK-3 (-), HK-BK-6 (A), or salmon sperm (0) DNA per ml at 680C. Portions of the reaction mixtures were taken at various times and assayed for the fraction of radiolabeled DNA remaining single stranded (f,4) by hydroxyapatite column chromatography. The t1l2 for the 32P-labeled BKV DNA reassociating in the presence of unlabeled salmon sperm DNA (25.6 h) was the average of seven separate determinations. An analysis of these data is presented in Table 1. TABLE 1. Quantitation of BKV DNA sequences present in transformed hamster kidney lines EquivaCellular Accelera- lents of DNA Cellular viral t112 (h) tion fac--DNA/dipDNA concn tor loid ge(mg/ml) nome 10.7 2.7 HK-BK-2 1.03 1.39 HK-BK-3 1.03 7.0 2.66 5.3 HK-BK-6 1.03 9.8 1.57 3.1 Salmon 1.03 25.6 0 0 sperm a Analysis of data shown in Fig. 1. Acceleration factor and equivalents of viral DNA per diploid genome are calculated as previously described (3).

cific sequences in transformed cells, only portions of the viral genome can be shown to be present (5, 14). As little as 5% of adenovirus type 2 DNA appears necessary for the maintenance of the transformed state (5, 14). This result is compatible with the observation that no virus can be rescued from adenovirus-transformed cell lines. Botchan et al. (1) used a similar approach to examine the viral DNA sequences present in one SV4O-transformed mouse line (1). They observed that segments

encompassing the early region of the viral genome (0.17 to 0.76 SV40 map units) are represented 5 to 8 times per diploid cell equivalent of DNA, whereas the late segments are represented 0.8 to 2 times (1). The viral sequences present in HK-BK-3 were similarly examined to assess the uniformity of representation of the BKV genome in this cell line. It should be pointed out that no significant deviation from a simple second-order reaction was detected when denatured 32P-labeled BKV DNA was reannealed in the presence of HK-BK-3 DNA (Fig. 1), suggesting that the viral genome is uniformly represented. The uniformity of representation was more precisely assessed, however, by reassociating each of the four R-HindIII fragments of BKV DNA (8) in the presence of large excesses of unlabeled HK-BK-3 DNA (Fig. 2). The amount of viral DNA for each fragment expressed in copies per diploid genome equivalent varied from 4.1 for HindIII-B to 5.8 for HindIII-C (Table 2). R *HindIH fragments B and D are located in the early gene region of the BKV genome, whereas fragments A and C contain DNA sequences that are expressed late in the lytic cycle (P. M.

t/t 1,7

FIG. 2. Reassociation ofeach of the R -Hind fragments of BKV DNA in the presence of unlabeled DNA from HK-BK-3 cells. Denatured, 32P-labeled BKV DNA fragments (specific activity, 5.5 x 105 cpmlpg) were allowed to reassociate in the presence of large excesses ofdenatured, fragmented HK-BK-3 DNA (0) or salmon sperm DNA (0) in reaction mixtures containing 0.0025 M EDTA and 0.6 M sodium phosphate buffer (pH 6.8) at 68"C. Portions of the reaction mixtures were taken at various times and assayed for the fraction of radiolabeled DNA remaining single stranded (fm) by hydroxyapatite column chromatography. The dashed lines represent the theoretical curves for the reassociation of the BKV fragments alone, and the solid lines represent the theoretical curves for the reassociation of each of the BKV fragments in the presence of a eukaryotic DNA containing the indicated equivalents of viral DNA per diploid genome. The data presented in this figure are summarized in Table 2.

VOL. 23, 1977

207

NOTES

TABLE 2. Quantitation of BKV DNA sequences present in HK-BK-3 32P-labeled probe

Fraction of viral genome

HindIH-A HindIII-B HindIII-C HindIII-D

0.43 0.35 0.12 0.10

Concn of viral probe (gg/ml)

2.28 2.34 5.46 5.84

x x x x

10-3 10-3 10-4 10-4

Concn of cell Salmon DNA (mg/ sperm DNA ml) t1/2 (hW

2.30 2.89 1.32 1.47

10.5 8.5 11.1 8.5

Equivalents HK-BK-3 Acceleration of viral DNA factor n diploid segment/ (h) DNAHtK2 N 12() fco genome

3.6 3.6 5.1 4.1

1.92 1.35 1.18 1.07

5.7 4.1 5.8 5.7

Howley, G. Khoury, and M. A. Martin, manuscript in preparation). We do not interpret the observed variance as significant and conclude that the four BK HindIII fragments are equally represented in HK-BK-3. These data are summarized in Fig. 3. The uniform representation of the various segments of the BKV genome in HK-BK-3 is in striking contrast to the results of Botchan et al. (1), who reported that the SV40 genome is unequally represented in the SVT2 cell line (1). Hin C (4. 1) There are numerous differences, however, be0.6 0.4 tween these two papovavirus-transformed cell 0.5 lines. HK-BK-3 was derived from BHK cells Hin D (5.7) transformed with low multiplicities of BKV FIG. 3. Physical map ofthe BKV genome (7). The DNA prepared from plaque-purified virus (15), whereas SVT2 was established after the infec- equivalents of viral DNA for each of the four R -Hind tion of 3T3 mouse cells with SV40 virus at an fragments in HK-BK-3 as expressed per diploid geunknown multiplicity of infection. The major nome are noted in parentheses. difference is probably related to the different LITERATURE CITED histories of the two transformed cell lines. The 1. Botchan, M., B. Ozanne, B. Sugden, P. A. Sharp, and SVT2 line was derived from BALB/3T3 cells J. Sambrook. 1974. Viral DNA in transformed cells. nearly 9 years ago and has been propagated III. The amounts of different regions of the SV40 genome present in a line of transformed mouse cells. under varying conditions during this period. Proc. Natl. Acad. Sci. U.S.A. 71:4183-4187. HK-BK-3, on the other hand, is a newly estabJ., C. Yee, T. S. Tralka, and A. S. Rabson. 1976. lished transformed cell line that was passaged 2. Costa, Hamster ependymomas produced by intracerebral inno more than 12 times prior to the experiment oculation of a human papovavirus (MMV). J. Natl. Cancer Inst. 56:863-864. shown in Fig. 2. Since it has been shown that L. D., D. E. Kohne, and M. A. Martin. 1971. the SV40 DNA fragment extending from 0.18 to 3. Gelb, Quantitation of simian virus 40 sequences in African 0.74 map unit, which includes the entire early green monkey, mouse and virus transformed cell geregion, is sufficient for the transformation of nomes. J. Mol. Biol. 57:129-145. rat cells (4), the results reported by Botchan et 4. Graham, F. L., P. J. Abrahams, C. Mulder, H. L. Heijneker, S. 0. Warnaar, F. A. J. deVries, W. Fiers, al. (1) could reflect recombinational events ocand A. J. van der Eb. 1974. Studies on in vitro transcurring during the long passage history of the formation by DNA and DNA fragments of human SVT2 cell line and/or the selective amplificaadenoviruses and simian virus 40. Cold Spring Harbor Symp. Quant. Biol. 39:637-650. tion of the polynucleotide sequences necessary 5. Graham, F. L., A. J. van der Eb, and H. L. Heijneker. for the maintenance of the transformed state. 1974. Size and location of the transforming region in Our findings clearly indicate that during inihuman adenovirus type 5 DNA. Nature (London) tial passages, the entire papovavirus genome 251:687-691. becomes stably associated with cell DNA and is 6. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. represented uniformly within transformed 26:365-369. cells. Reexamination of the viral sequences 7. Howley, P. M., G. Khoury, J. C. Byrne, K. K. Takepresent in this line after continued passage will moto, and M. A. Martin. 1975. Physical map of the BK virus genome. J. Virol. 16:959-973. be important in establishing whether selective P. M., M. F. Mullarkey, K. K. Takemoto, and amplification of viral polynucleotide sequences 8. Howley, M. A. Martin. 1975. Characterization of human papooccurs in lines stably transformed by this papovavirus BK DNA. J. Virol. 15:173-181. vavirus.

9. Major, E. O., and G. DiMayorca. 1973. Malignant

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NOTES transformation of BHK-21 clone 13 cells by BK virus, a human papovavirus. Proc. Natl. Acad. Sci. U.S.A. 70:3210-3212.

10. Martin, M. A., and G. Khoury. 1976. Integration of tumor virus genomes. Curr. Top. Microbiol. 73:3565. 11. Nase, L. M., M. Karkkainen, and R. A. Mantyjarvi. 1975. Transplantable hamster tumors induced with

the BK virus. Acta Pathol. Microbiol. Sand. Sect. B 83:347-352. 12. Portolani, M., B. Barbanti-Brodano, and M. LaPlaca. 1975. Malignant transformation of hamster kidney

J. VIROL. cells by BK virus. J. Virol. 15:420422. 13. Shah, K. V., R. W. Daniel, and J. Strandberg. 1975. Sarcoma in a hamster inoculated with a human papovavirus, BK virus. J. Natl. Cancer Inst. 54:945-950. 14. Sharp, P. A., U. Pettersson, and J. Sambrook. 1974. Viral DNA in transformed cells. I. A study of the sequences of adenovirus 2 DNA in a line of transformed rat cells using specific fragments of the viral genome. J. Mol. Biol. 86:709-726. 15. Takemoto, K. K., and M. A. Martin. 1976. Transformation of hamster kidney cells by BK papovavirus DNA. J. Virol. 17:247-253.

Uniform representation of the human papovavirus BK genome in transformed hamster cells.

Vol. 23, No. 1 JOURNAL OF VIROLOGY, July 1977, p. 205-208 Copyright © 1977 American Society for Microbiology Printed in U.S.A. NOTES Uniform Repres...
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