Studies on the occurrence of high molecular weight adenovirus type 2 DNA in productively infected cells

VIROLOGY

(1978)

88,186-190

Studies on the Occurrence of High Molecular Weight Adenovirus DNA in Productively Infected Cells’ ELLEN

FANNING,2

JOCHEN

SCHICK,

AND

WALTER

Type 2

DOERFLER3

Institute of Genetics, University of Cologne, Cologne, Germany Accepted March 17,197s Newly synthesized viral DNA in human cells productively infected with adenovirus type 2 (Ad21 occurs in four size classes which can be separated by zone velocity sedimentation in alkaline sucrose density gradients. The 34 S size class represents unit-length Ad2 DNA and the ~20 S size class consists of fragments of viral DNA. The high molecular weight size classes, >lOO S and 50-90 S, contain viral DNA sequences integrated in cellular DNA. The factors which influence the amount and composition of the high molecular weight viral DNA have been investigated. The proportion of Ad2 DNA sequences in 50-90 S DNA varies from one cell line to another and is enhanced in rapidly growing cultures relative to confluent monolayers of KB cells. An increased multiplicity of infection also results in a higher proportion of viral sequences in the 60-90 S size class. KB cells infected with Ad2 inactivated by ultraviolet light and unable to replicate its DNA, however, contain nearly the same amount of high molecular weight viral DNA as cells infected with fully infectious virus. There is much evidence from previous work that the high molecular weight form of viral DNA represents viral sequences linked to cellular DNA. The present data suggest that the synthesis of high molecular weight viral DNA does not depend on the concurrent synthesis of 34 S viral DNA but requires active cell growth, possibly cellular DNA replication.

Newly synthesized Ad2 DNA from productively infected KB cells can be fractionated into four size classesby zone velocity sedimentation in alkaline sucrose density gradients: >lOO S, 50-90 S, 34 S (unit-size Ad2 DNA), and ~20 S DNA (3,7,16). High molecular weight DNA (>lOO S, 50-90 S) from KB cells productively infected with human adenovirus type 2 (Ad2) has been quantitated and characterized by reassociation kinetics (7, 20) and by restriction enzyme analysis (2, 16). This DNA has been shown by several criteria to represent, at least in part, viral DNA integrated into cellular DNA (2, 3, 16). We are investigating this system because the study of the mechanism of integration is favored by the ’ Part of this work was presented at the NATO Advanced Study Institute on “DNA Synthesis, Present and Future,” Palermo, Italy, June ZO-29,1977. * Present address: Fachbereich Biologie, University of Konstanz, D-7750 Konstanz, Germany. 3 Author to whom requests for reprints should be addressed. 186 0042~6822/78/0881-0186$02.00/0 Copyright All rights

0 1978 by Academic Press, of reproduction in any form

Inc. reserved.

large amounts of integrated viral DNA in KB cells infected with Ad2. In the present study we have defined the conditions under which high molecular weight Ad2 DNA occurs and what factors influence the quantity and quality of high molecular weight viral DNA. In particular, we have attempted to determine whether cellular or viral functions, or both, are involved in the generation of high molecular weight viral DNA. Human KB cells (8) were propagated in monolayer or suspension cultures in Eagle’s medium (9) containing 10% calf or fetal calf serum. HeLa cells (15) were grown in monolayer cultures in Dulbecco’s modification of Eagle’s medium (1) and 10% calf serum, and human embryonic kidney (HEK) cells were grown in Dulbecco’s medium supplemented with 20% fetal calf serum. The xeroderma pigmentosum human fibroblast cultures XP4 (ATCC CR1260) and XP25 (ATCC CR1261) were propagated in Dulbecco’s medium and 10% fetal

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calf serum, and diploid human fibroblasts (WI-38) were grown in Eagle’s medium containing 10% fetal calf serum. Baby hamster kidney (BHK21) cells (17) were propagated in monolayer cultures in Dulbecco’s medium supplemented with 10% fetal calf serum and 10% tryptose phosphate broth (Difco), and Chinese hamster ovary (CHO) cells were propagated in Dulbecco’s medium or Ham’s F12 medium containing 10% fetal calf serum. All cell cultures were tested for mycoplasma contamination by microbiological techniques ( 12) or by staining the cells with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) (4) according to the method of Russell et al. (14). Growing monolayers of different cell lines were infected with 106 PFU/cell of Ad2 which was grown and purified as outlined previously ( 6). The cells were labeled with [3H]thymidine (25-100 ~Ci/ml) from 16 to 18 hr postinfection (pi.), and the four size classes of newly synthesized DNA were prepared as described (3). The amounts of Ad2 DNA sequences in each size class were determined by DNA-DNA hybridization to Ad2 virion DNA on filters using the method of Denhardt (5). Filters contained 4 pg of Ad2 DNA. Empty filter binding was <2%, and all values were corrected for this background. High molecular weight viral DNA occurs not only in Ada-infected KB cells, but also in other cells productively infected with Ad2 (data not shown). Like KB cells, HeLa and HEK cells contain a large proportion of Ad2 sequences in the newly synthesized 50-90 S and >lOO S size classes. As to the production of unit length Ad2 DNA, HEK cells are by far the most efficient cells. Five other cell lines, WI-38, XP4, XP25, BHK21, and CHO cells also contain viral sequences in high molecular weight DNA, but the proportion of Ad2 DNA sequences in these size classes appears smaller than in KB, HeLa, or HEK cells. The replication of Ad2 DNA in these latter cell lines, however, is strikingly reduced when compared to replication in KB, HeLa, or HEK cells, and therefore it is difficult to quantitate precisely differences in the relative amounts of high molecular weight viral DNA in different cell lines. KB cells were seeded at different densi-

187

ties and mock-infected with PBS (phosphate-buffered saline) or infected with Ad2. The newly synthesized DNA was labeled with [3H]thymidine and fractionated into four size classes as described (3). The amount of viral DNA in each of the size classes was quantitated by DNA-DNA hybridization (Fig. 1). In mock-infected cells the amount of newly synthesized 50-90 S DNA, as judged by incorporation of [3H]thymidine, decreases as the cell density increases (data not shown). Thus KB cell DNA replication appears to be inhibited in dense cultures. With increasing cell density the amount of newly synthesized DNA decreases in each of the four size classes (data not shown). The percentage of viral DNA in each size class was determined by DNA-DNA filter hybridization. The data presented in Fig. 1 demonstrate that the percentage of viral DNA in the 50-90 S size class declines markedly with increasing cell density, whereas the proportion of viral sequences in the other size classes remains constant, although the total amount of newly synthesized DNA is diminished. These data are consistent with the notion that viral DNA replication is in some way related to active cellular DNA replication (10) and that the production of 50-90 S DNA is particularly dependent on cellular DNA synthesis. KB cells infected with Ad2 at various multiplicities of infection (lo-’ to lo4 PFU/ cell) were labeled with [3H]thymidine from 16 to 18 hr p.i., and the newly synthesized DNA was analyzed by zone velocity sedimentation in alkaline sucrose density gradients. The 3H-labeled DNA from the four size classes was pooled and hybridized to Ad2 virion DNA. Over a range of multiplicities of infection from 5 to lo4 PFU/cell, the amount of hybridization of the 50-90 S DNA size class varies widely from 5-108 up to 60-70% (Fig. 2). Over the same range of multiplicities, the hybridization of the t20 S size class to Ad2 DNA filters also varies with multiplicity but over a somewhat narrower range from 5-10% up to 40-456. A similar dependence on the multiplicity of infection is observed for the ~100 S size class of Ad2 DNA. Within a multiplicity range of 5-lo4

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PFU/cell, the amount of 3H-labeled Ad2 DNA sequences in this size class varies from O-5% up to 30-35s (Fig. 2). Within the same range of multiplicities, however, there is very little dependence on the multiplicity of infection with respect to the amount of Ad2 sequences in the 34 S size

ii:;jJy&~

100. g25--r-4 @In

i

1o-2

I

I 5

10

15 20 Growth density

25 30 35 (cells/cm2~10~h)

LO

FIG. 1. Dependence on ceII density of the amount of viral DNA sequences in the four size classes. KB cells were seeded in 60-mm dishes at different densities: 1 X 106, 3 X 106, and 1 x lo7 cehs per dish. The cehs were infected with Ad2 at multiplicities of 106 PFU/ceII, labeled with [3H]thymidine (25 pCi/mI) from 16 to 18 hr p.i., and the DNA was analyzed by zone velocity sedimentation on alkaline sucrose density gradients (3). Usually lo6 cells in 0.1-0.2 ml were lysed in a 0.4 to 0.5~ml layer of 0.6 N NaOH, 0.05 M EDTA. In some experiments 1 N NaOH, 0.1 M EDTA was used for ceII lysis. Increasing the alkali concentration in the Iysis Iayer to 1.0 N or in the sucrose gradients to 0.9 N NaOH, 0.1 M NaCl, 0.01 M EDTA did not affect the sedimentation profile or the patterns of hybridization of the viral DNA in different size classes. We have aho employed the heparin lysis procedure (20). Briefly, 10’ Ad2-infected or mock-infected KB cells were lysed in 0.4 ml of 1% Sarkosyl, and 1 mg/mI of heparin in TE layered on top of an alkaline sucrose density gradient in 0.3 N NaOH, 0.7 M NaCl, 0.01 M EDTA. Lysis was ahowed to proceed for 30 min at ambient temperature. Subsequently, the lysis layer was made up to 0.5 N NaOH, 0.05 M EDTA; the gradients were kept for 30 min at 4’ and then centrifuged. The distribution of Ad2 DNA sequences in the four size classes was identical to that obtained with the conventional alkaline lysis procedure (3). Fractions from the >lOO S, 50-90 S, 34 S, and t20 S regions of the gradients (16) were pooled and analyzed for the amounts of viral DNA sequences present by DNA-DNA fiber hybridization. The total number of counts per minute per size class of the gradient was computed, and the percentage of viral-specific counts per minute in each fraction was determined. (O), 34 S DNA; (x), 50-90 S DNA; (A), >lOO S DNA; (O), t20 S DNA, The actual counts per minute used as inputs and the percentage values hybridized to Ad2 DNA in

10-l

loo 10' PFU/cell

lo2

xl3

lo4

FIG. 2. The amount of viral DNA sequences in four size cIasses of newly synthesized DNA in AdS-infected KB cehs varies with multiplicity of infection. KB cehs (lo6 cells per 60-mm dish) were infected at different muhiphcities with Ad2, labeled with 100 pCi/rnI of [3H]thymidine from 16 to 18 hr p.i., and then lysed on top of 5-20% aIkaIine sucrose density gradients. The four size classes of newly synthesized DNA were prepared by zone velocity sedimentation as described. The DNA from the different size classes was pooled, and the different POOLS were analyzed by DNA-DNA fiber hybridization using Ad2 DNA. (0), 34 S DNA; (x), 50-90 S DNA; (A), >lOO S DNA, (O), <20 S DNA. A large number of hybridization data are summarized in thii figure. The actual counts per minute used as inputs ranged from 807 to 9817 cpm, (>lOO S); 771 to 12694 cpm, (50-90 S); 4502 to 53286 cpm, (34 S); and 2982 to 15350 cpm (<20 S). Empty filter binding was ~1%. This background was subtracted from ah values.

class; hybridization values vary from 85 to 100% (Fig. 2). As one might expect, at low multiplicities (10-‘-l PFU/cell), the amount of Ad2 DNA sequences in the 34 S size class is relatively low (5-3056) and dependent on the multiplicity of infection. With the use of such low multiplicities, time after infection becomes an important factor, since at 16-18 hr p.i., the time of labelsome of the experiments CelIs seeded per plate 1 x 108 3 x 10S 10 x lo6

were

as follows:

>lWS

50-90s

2,630 (15.1%) 1,023 (15.8%) 1,430 (12.1%)

3,720 (53.7%) 1,428 (28.7%) 1,217 (9.1%)

Empty filter binding was 0.9%. This subtracted from ah values.

34s

<20 s

14,721 (86.6%) 10,208 (92.0%) 4,209 (97.5%)

5,984 (55.1%) 3,421 (64.0%) 1,725 (64.7%)

background

was

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ing, viral DNA synthesis has not reached maximal intensity. Thus the proportion of viral sequences in newly synthesized DNA of the high molecular weight (>lOO S and 50-90 S) and of the ~20 S size classes is dependent on the multiplicity of Ad2. Integration of parental Ad2 DNA (11) or of newly synthesized Ad2 DNA would yield this result. In the newly synthesized 34 S unit-length DNA, however, the proportion of viral sequences is independent of multiplicity of infection. KB cells were infected with Ad2 which had been inactivated for different periods of time by exposure to uv light (Table 1). The four size classes of newly synthesized 3H-labeled DNA were prepared on alkaline sucrose density gradients and hybridized to Ad2 virion DNA on filters. In KB cells infected with Ad2 which had been exposed to uv light, the amount of newly synthesized 34 S unit-length viral DNA is reduced compared to that in cells infected with unirradiated virus. The extent of inactivation of Ad2 DNA replication depended on the length of exposure to uv light (Table 1). It has been shown electron microscopically that uv-inactivated Ad2 can penetrate into KB cells (D. T. Brown, personal communication) .

The proportion of the newly synthesized high molecular weight DNA which represents viral sequences was measured by DNA-DNA filter hybridization. Neither the fraction of high molecular weight DNA sequences of viral origin in cells infected with irradiated virus nor the total amount of [“Hlthymidine incorporated into high molecular weight DNA was markedly different from the equivalent values in cells infected with unirradiated Ad2 (Table 1). The length of exposure of the virus to uv light had no striking effect on the proportion of viral sequences in the newly synthesized high molecular weight DNA (Table 1). The amount of viral DNA in the ~20 S size class was not affected by uv irradiation of the inoculum either. Thus, in KB cells infected with uv-inactivated Ad2, synthesis of 34 S viral DNA is reduced or nearly abolished depending on the uv dose, but Ad2 sequences can be found in high molecular weight DNA. These findings are consistent with the idea that parental viral DNA is integrated and that inhibition of viral DNA replication does not abolish integration. Much evidence has been presented (2,3, 7,10,16,18) which documents that the high molecular weight viral DNA in cells pro-

TABLE AMOUNT Time of irradiation (min)

OF VIRAL

DNA

Biological activity* (PFU/mI)

1.4 3.8 3.4 8.6 3.4

x x x x x

10’ 10” lo6 lo5 105

I

SEQUENCES IN THE FOUR SIZE CLASSES OF DNA INFECTED WITH UV-INACTIVATED Ad2” Counts

per minute >109

0 0.5 1.5 3.5 8.5

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13,030 17,880 21,530 18,965 28,815

s (6.9) (7.8) (6.1) (3.3) (3.1)

in pools

of different

50-90 9,795 14,255 20,400 16,635 19,075

s (16.8) (14.6) (10.1) (4.9) (7.2)

ISOLATED

FROM KB CELLS

size classes (percentage

of AdL-specific

34 s

<20 s

cpm)’

60,055 26,855 14,320 10,945 9,275

(89.4) (83.1) (38.9) (12.9) (7.8)

36,485 37,770 32,055 29,065 18,715

(7.7) (4.7) (7.9) (4.1) (5.2)

o KB cells (5.5 x Id ceils per 35-mm-diameter dish) were infected with 1 ml/dish of Ad2 or Ad2 which had been partly inactivated by exposure to uv light (254 nm) for various lengths of time. CsCl-purified Ad2 (10’ PFU/celi) was diluted in 7 ml of cold PBS and was irradiated in a 60-mm-diameter dish. The infected cells were labeled 16-18 hr p.i. with [3H]thymidine (50 &i/ml), and the newly synthesized DNA was fractionated on alkaline sucrose density gradients by zone velocity sedimentation as described. Fractions from the rlO0 S, 50-90 S, 34 S, and <20 S size classes were pooled and analyzed for the presence of viral DNA by DNA-DNA filter hybridization to Ad2 DNA. * Infectivity of each sample was determined by plaque assays as described (13). ’ The total counts per minute in the pools of each size class were computed, and the percentage of Ad2specific counts per minute in DNA as determined by DNA-DNA filter hybridization was calculated. Empty Nter binding values, which were subtracted from all results, were 0.8 (>lOO S), 1.2 (50-90 S), 1.5 (34 S), and 2% (t20 S).

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ductively infected with adenovirus represents, at least in part, viral DNA sequences covalently linked to cellular DNA. This evidence will not be reviewed here in detail. For the purpose of this discussion we assume that the amount of viral specific high molecular weight DNA is a measure of the amount of integrated viral DNA. The analysis presented is restricted to late times (16-18 hr) postinfection. The present results show that the cell does, in fact, play a role in the formation of high molecular weight Ad2 DNA. The amount of high molecular weight viral DNA differs from one cell line to another. The state of growth of the cells also affects the integration of viral DNA (Fig. 1). Actively growing cells contain more high molecular weight Ad2 DNA. Taken together with the fact that 50-90 S DNA is an intermediate in cell DNA replication in uninfected cells (16), these data suggest that cell DNA replication may play a part in integration of viral DNA. In cells infected with avian sarcoma virus, Varmus et al. (19) have shown that cellular functions are essential for the synthesis and integration of viral DNA. It should also be remembered, however, that the larger amount of 34 S DNA found in actively growing cells may lead to increased integration of viral DNA. ACKNOWLEDGMENTS We thank M. Stupp for preparing media and performing plaque assays, and B. Kierspel for typing this manllscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 74) and by the Federal Ministry of Research and Technology (01 VW 015 B13 BCT 112).

REFERENCES

1. BABLANIAN, R., ECGERS, H. J., and TAMM, I., Virology 26, 100-113 (1965). 2. BACZKO, K., NEUMANN, R., and DOERFLER, W., Virology 85, 557-567 (1978). 3. BURGER, H., and DOERFLER, W., J. Viral. 13, 975-992

(1974).

4. DANN, O., BERGEN, G., DEMANT, E., and VOLZ, G., Ann. Chem. 749,68-69 (1971). 5. DENHARDT, D. T., Biochem. Biophys. Res. Commun. 23,641~646 (1966). 6. DOERFLER, W., Virology 38.587-666 (1969). 7. DOERFLER, W., BURGER, H., ORTIN, J., FANNING, E., BROWN, D. T., HOFF, U., WEISER,

WESTPHAL, M., B., and SCHICK,

WINTERJ., Cold

Spring Harbor Symp. Quant. Biol. 39.505-521 (1974).

8. EAGLE, H., Proc. Sot. Exp. Biol. Med. 89,362-364 (1955).

9. EAGLE, H., Science 130,432-437 (1959). 10. FANNING, E., and DOERFLER, W., Virology 81, 433-448 (1977). 11. GRONEBERG, J., BROWN, D. T., and DOERFLER, W., Virology 64, 115-131 (1975). 12. HAYFLICK, L., Texas Rep. Biol. Med. 23,285-303 (1965).

13. LUNDHOLM, U., and DOERFLER, W., Virology 45, 827-829 (1971). 14. RUSSELL, W. C., NEWMAN, C., and WILLIAMSON, D. H., Nature (London) 263, 461-462 (1975). 15. SCHERER, W. F., SYVERTON, J. T., and GEY, G. O., J. Exp. Med. 97, 695-769 (1953). 16. SCHICK, J., BACZKO, K., FANNING, E., GRONEBERG, J., BURGER, H., and DOERFLER, W., Proc. Nat. Acad. Sci. USA 73, 1043-1047 (1976). 17. STOKER, M., and MACPHERSON, I., Nature (London) 203,1355-1357 (1964). 18. TYNDALL, C., YOUNGHUSBAND, H. B., and BELLEW, A. J. D., J. Viral. 25, l-10 (1978). 19. VARMUS, H. E., PADGE’IT, T., HEASLEY, S., SIMON, G., and BISHOP, J. M., Cell 11, 307-319 (1977). R. A., and HILDEBRANDS, C. E., Biophys. Acta 407, 120-124 (1975).

20. WALTERS, B&him.